CN102517326B - Method for improving content of lysine in maize by using zein gene ribonucleic acid interference (RNAi) vector - Google Patents

Abstract

The invention discloses a method for improving the content of lysine in maize by using a zein gene ribonucleic acid interference (RNAi) vector, and belongs to the technical field of plant genetic engineering. According to the method, the zein gene RNAi vector is introduced into a receptor maize variety by a gene gun method and comprises an endosperm specific expression promoter P-zp22/6, forward and reverse target genes 22-KD zein gene part complementary deoxyribonucleic acid (cDNA) fragments 22-KD-P, a glucuronidase (GUS) gene intron and a terminator poly(A). The other zein gene RNAi vector pUC19-RNAi-19KD is also provided and comprises forward and reverse target genes 19-KD zein gene part cDNA fragments 19-KD-P. The two RNAi vectors are introduced into the maize variety respectively, so that a maize transgenic plant with high lysine content is successfully obtained, the content of the lysine is improved by 15 to 60 percent, and the transfer period is only 1 to 2 years.

Description

A kind of method that improves the corn lysine content with zein spirit-soluble gene RNAi carrier

Technical field

The invention belongs to the plant gene engineering technology field, be specifically related to the application of a kind of zein spirit-soluble gene RNAi carrier in the corn quality improvement.

Background technology

China is a large agricultural country, and grain security is the Vital Strategic Problems of overall importance that concerns Chinese national economy development, social stability and ability for the nation to stand on its own all the time.Corn is not only important grain source, but also is important feed, industrial raw material as the second largest crop of China, and the Maize Production situation has very important effect in the grain safety system.In the fodder industry of China take corn as mainstay industry, because the lysine content of corn kernel is low, joining, must add fodder additives Methionin in the mixed fodder, could satisfy the needs of animal growth and development, but these fodder additivess mainly rely on import, thus the not high particularly lysine content of feed quality on the low side be one of the key constraints of present China animal husbandry development.On the other hand, China part province also has sizable a part of crowd take corn as staple food, its nutritive substance is mainly derived from corn, but the corn of producing at present upper plantation is conventional corn mostly, nutritional quality is relatively poor, lacks human body and monogastric animal grow essential Methionin, tryptophane etc.

The protein content of corn kernel is about 10%, wherein the 50-60% prolamine that can not absorb for people and animals.International corn wheat improvement center (CIMMYT) begins to utilize the recessive mutation gene at 20 century 70s Opaque-2( O2) and modifying factor content of prolamine is down to 22.9% by 55.1%, make the lysine content level reach 2 times of conventional corn, and then seed selection large quantities of high-lysine corns be high-quality protein maize and whole world widespread use (Tan waits quietly. Current Situation of QPM Breeding. Maize Sciences 2006,14 (5): 15-19), but the method transformation cycle is grown (7-10), efficient is low, foresight is poor, the high-lysine corn endosperm that in addition selects is soft and the problem such as disease resistance is poor, more and more is not suitable with the needs of modern breeding target.The nutritive value of high-quality protein maize is equivalent to 90% of milk proteins, many countries and regions take corn as staple food all can be used as and substitute nutritive food in the whole world, and particularly school-ager's malnutritive problem is significant to improving crowd take corn as staple food.The feed that this external application high-quality protein maize is main body is raised pigs, and, feeds chicken and can improve egg productivity more than 15% more than 30% than conventional corn feed day weight gain.Studies show that, because the prolamine particularly expression of 22-KD and 19-KD alcohol soluble protein gene has significant negative effect to lysine content, yet traditional breeding method is difficult to find desirable high-quality germplasm, obtain that lysine content is high and the low corn variety difficulty of expressing of prolamine is very large, major cause is that its phenotypic evaluation is that lysine content is measured and need to use high pressure liquid chromatography (HPLC) be analyzed, because HPLC running cost is very high, be difficult to widespread use in the progeny selection in enormous quantities in extensive germplasm screening and breeding of new variety process.

RNA disturbs (RNA interference, RNAi) be a kind of important tool of research biological development and gene function, by introducing the double-stranded RNA (adopted RNA and sense-rna are arranged) that has identical sequence with endogenous target gene, thereby induce the mRNA degraded of endogenous target gene, and then suppress its corresponding genetic expression.Therefore, if can pass through the RNA perturbation technique, by the application of the RNAi carrier that makes up, obtain that lysine content is high and the low transgenic corns of expressing of prolamine will be a kind of easy, quick and effective means.

Summary of the invention

The technology that the present invention will solve is utilized the recessive mutation gene Opaque-2( O2) the transformation high lysine corn varieties cycle is long, efficient is low, foresight is poor and select high lysine corn varieties because O2Defective and the deficiencies such as the soft endosperm that the chain effect of gene causes and disease resistance are poor, a kind of method that improves the corn lysine content with zein spirit-soluble gene RNAi carrier that provides is provided, the method is by importing corn variety with zein spirit-soluble gene RNAi carrier, obtain transfer-gen plant, make the lysine content of corn kernel obtain larger raising, reach the improved purpose of corn quality.

Because the prolamine particularly expression of 22-KD and 19-KD alcohol soluble protein gene all has significant negative effect to lysine content, the inventive method provides two kinds of technical schemes, a kind of is that zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD is imported corn variety, obtain high-lysine content corn germplasm, realize improving BeautifulThe purpose of rice seed lysine content.Another kind of scheme is that zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD is imported corn variety, obtains high-lysine content corn germplasm, reaches the purpose that improves the corn kernel lysine content.Its concrete technical scheme is as follows:

A kind of method that improves the corn lysine content with zein spirit-soluble gene RNAi carrier provided by the present invention, comprise with particle bombardment zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD is imported the acceptor corn variety, described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD contains endosperm specific expression promoter P-zp22/6 at skeleton carrier pUC19, is positioned between the restriction enzyme site Sac I and Sma I of RNAi carrier pUC19-RNAi-22KD; Forward goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P is positioned between the restriction enzyme site Sma I and Xba I of RNAi carrier pUC19-RNAi-22KD; Oppositely goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P is positioned between the restriction enzyme site Xba I and Xho I of RNAi carrier pUC19-RNAi-22KD; In the middle of forward goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P and reverse goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P, contain the gus gene intron, be positioned between the Xba I restriction enzyme site of RNAi carrier pUC19-RNAi-22KD; Terminator poly (A) is positioned between RNAi carrier pUC19-RNAi-22KD restriction enzyme site Hind III and Xho I; The nucleotide sequence of described endosperm specific expression promoter zp22/6 is shown in SEQ ID NO:3, the nucleotide sequence of described 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P is shown in SEQ ID NO:8, the nucleotide sequence of described gus gene intron is shown in SEQ ID NO:14, and the nucleotide sequence of described terminator poly (A) is shown in SEQ ID NO:11; The nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD is shown in SEQ ID NO:15.The collection of illustrative plates of constructed zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD as shown in Figure 8.

Described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD can also make up acquisition by the following method:

(1) makes up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6

1. the acquisition of endosperm specificity expression promoter P-zp22/6

Take the genomic dna of corn variety as template, take sequence shown in SEQ ID NO:1 and the SEQ ID NO:2 as primer pair, carry out pcr amplification, obtain the endosperm specificity expression promoter P-zp22/6 of sequence shown in SEQ ID NO:3;

2. make up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6 sequence

Cut the endosperm specificity expression promoter with corresponding restriction enzyme site that step (1) 1. obtains with Sac I and Xba I enzyme, then be connected with the skeleton carrier pUC19 that is connected with Xba I enzyme through the Sac I equally, obtain the recombinant vectors pUC19-p22/6 with endosperm specificity expression promoter P-zp22/6;

(2) make up the recombinant vectors pUC19-p22/6-22KD1 that inserts forward purpose fragment 22-KD-P and endosperm specificity expression promoter P-zp22/6

1. the acquisition of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

Take the total RNA of corn kernel as template, with sequence shown in SEQ ID NO:4 and the SEQ ID NO:5, carry out the RT-PCR amplification, then take this RT-PCR product as template, take sequence shown in SEQ ID NO:6 and the SEQ ID NO:7 as primer pair, carry out again RT-PCR, obtain the 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P shown in SEQ ID NO:8;

2. make up the recombinant vectors pUC19-p22/6-22KD1 that inserts forward purpose fragment 22-KD-P and endosperm specificity expression promoter P-zp22/6

1. the purpose fragment 22-KD-P with corresponding restriction enzyme site that obtains with Sma I and Xba I double digestion step (2), then with equally be connected the recombinant vectors pUC19-p22/6 that step (1) 2. makes up with Xba I double digestion through the Sma I and be connected, obtained inserting the recombinant vectors pUC19-p22/6-22KD1 of forward goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P and endosperm specificity expression promoter P-zp22/6;

(3) make up the recombinant vectors pUC19-22KD2 that inserts reverse purpose fragment 22-KD-P

Cut the purpose fragment 22-KD-P with corresponding restriction enzyme site that step (2) 1. obtains with Hind III and Xba1 enzyme, then be connected with the pUC19 carrier of being connected with Xba I enzyme through the Hind III equally, obtain inserting the recombinant vectors pUC19-22KD2 of reverse goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P;

(4) make up the recombinant vectors pUC19-22KD2-poly (A) that contains terminator poly (A)

Take expression vector p3301 DNA as template, carry out pcr amplification with the terminator primer of sequence shown in SEQ ID NO:9 and the SEQ ID NO:10, obtain the terminator poly (A) of sequence shown in SEQ ID NO:11; Further use Xho I and Hind III double digestion with the terminator poly (A) of corresponding restriction enzyme site, then with equally be connected the recombinant vectors pUC19-22KD2 that step (3) obtains with Hind III double digestion through the Xho I and be connected, obtain containing the recombinant vectors pUC19-22KD2-poly (A) of terminator poly (A);

(5) structure contains endosperm specificity expression promoter P-zp22/6, the recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) of forward and reverse purpose fragment 22-KD-P and terminator poly (A)

The recombinant vectors pUC19-p22/6-22KD1 of step (2) structure and the recombinant vectors pUC19-22KD2-poly (A) of step (4) structure are used respectively Xba I and Hind III double digestion, the enzyme that then will contain reverse purpose fragment 22-KD-P and terminator poly (A) is cut product 22KD2-poly (A) and is connected on the recombinant vectors pUC19-p22/6-22KD1 that step (2) makes up, obtain containing endosperm specificity expression promoter P-zp22/6, just, the recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) of reverse goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P and terminator poly (A);

(6) structure of the connection of gus gene intron and zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD

Take plasmid vector pGreen-0229 Backbone DNA as template, carry out pcr amplification with the primer of the gus gene intron of sequence shown in SEQ ID NO:12 and the SEQ ID NO:13, obtain the gus gene intron of sequence shown in SEQ ID NO:14; Further use Xba I single endonuclease digestion with the gus gene intron of corresponding restriction enzyme site, then be connected with the same recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) that the step (5) of phosphatizing treatment makes up behind Xba I single endonuclease digestion, obtain described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD, the nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD is shown in SEQ ID NO:15.

The another kind of method that improves the corn lysine content with zein spirit-soluble gene RNAi carrier provided by the present invention, comprise with particle bombardment zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD is imported the acceptor corn variety, described zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD contains endosperm specific expression promoter P-zp22/6 at skeleton carrier pUC19, is positioned between the restriction enzyme site Sac I and Sma I of RNAi carrier pUC19-RNAi-19KD; Forward goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P is positioned between the restriction enzyme site Sma I and Xba I of RNAi carrier pUC19-RNAi-19KD; Oppositely goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P is positioned between the restriction enzyme site Xba I and Xho I of RNAi carrier pUC19-RNAi-19KD; Between forward goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P and reverse goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P, contain the gus gene intron, be positioned between the Xba I restriction enzyme site of RNAi carrier pUC19-RNAi-19KD; Terminator poly (A) is positioned between RNAi carrier pUC19-RNAi-19KD restriction enzyme site Hind III and Xho I; The nucleotide sequence of described endosperm specific expression promoter zp22/6 is shown in SEQ ID NO:3, the nucleotide sequence of described 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P is shown in SEQ ID NO:20, the nucleotide sequence of described gus gene intron is shown in SEQ ID NO:14, the nucleotide sequence of described terminator poly (A) is shown in SEQ ID NO:11, and the nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD is shown in SEQ ID NO:21.The collection of illustrative plates of constructed zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD as shown in Figure 9.

Described zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD can also make up acquisition by the following method:

(1) makes up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6

1. the acquisition of endosperm specificity expression promoter P-zp22/6

Take the genomic dna of corn variety as template, take sequence shown in SEQ ID NO:1 and the SEQ ID NO:2 as primer pair, carry out pcr amplification, obtain the endosperm specificity expression promoter P-zp22/6 of sequence shown in SEQ ID NO:3;

2. make up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6 sequence

Cut the endosperm specificity expression promoter with corresponding restriction enzyme site that step (1) 1. obtains with Sac I and Xba I enzyme, then be connected with the skeleton carrier pUC19 that is connected with Xba I enzyme through the Sac I equally, obtain the recombinant vectors pUC19-p22/6 with endosperm specificity expression promoter P-zp22/6;

(2) make up the recombinant vectors pUC19-p22/6-19KD1 that inserts forward purpose fragment 19-KD-P and endosperm specificity expression promoter P-zp22/6

1. the acquisition of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

Take the total RNA of corn kernel as template, with sequence shown in SEQ ID NO:16 and the SEQ ID NO:17, carry out the RT-PCR amplification, then take this RT-PCR product as template, take sequence shown in SEQ ID NO:18 and the SEQ ID NO:19 as primer pair, carry out again RT-PCR, obtain the 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P shown in SEQ ID NO:20;

2. make up the recombinant vectors pUC19-p22/6-19KD1 that inserts forward purpose fragment 19-KD-P and endosperm specificity expression promoter P-zp22/6

1. the purpose fragment 19-KD-P with corresponding restriction enzyme site that obtains with Sma I and Xba I double digestion step (2), then with equally be connected the recombinant vectors pUC19-p22/6 that step (1) 2. makes up with Xba I double digestion through the Sma I and be connected, obtained inserting the recombinant vectors pUC19-p22/6-19KD1 of forward goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P and endosperm specificity expression promoter P-zp22/6;

(3) make up the recombinant vectors pUC19-19KD2 that inserts reverse purpose fragment 19-KD-P

Cut the purpose fragment 19-KD-P with corresponding restriction enzyme site that step (2) 1. obtains with Hind III and Xba I enzyme, then be connected with the pUC19 carrier of being connected with Xba I enzyme through the Hind III equally, obtain inserting the recombinant vectors pUC19-19KD2 of reverse goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P;

(4) make up the recombinant vectors pUC19-19KD2-poly (A) that contains terminator poly (A)

Take expression vector p3301 DNA as template, carry out pcr amplification with the terminator primer of sequence shown in SEQ ID NO:9 and the SEQ ID NO:10, obtain the terminator poly (A) of sequence shown in SEQ ID NO:11; Further use Xho I and Hind III double digestion with the terminator poly (A) of corresponding restriction enzyme site, then with equally be connected the recombinant vectors pUC19-19KD2 that step (3) obtains with Hind III double digestion through the Xho I and be connected, obtain containing the recombinant vectors pUC19-19KD2-poly (A) of terminator poly (A);

(5) structure contains endosperm specificity expression promoter P-zp22/6, the recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) of forward and reverse purpose fragment 19-KD-P and terminator poly (A)

The recombinant vectors pUC19-p22/6-22KD1 of step (2) structure and the recombinant vectors pUC19-19KD2-poly (A) of step (4) structure are used respectively Xba I and Hind III double digestion, the enzyme that then will contain reverse purpose fragment 19-KD-P and terminator poly (A) is cut product 19KD2-poly (A) and is connected on the recombinant vectors pUC19-p22/6-19KD1 that step (2) makes up, obtain containing endosperm specificity expression promoter P-zp22/6, just, the recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) of reverse goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P and terminator poly (A);

(6) structure of the connection of gus gene intron and zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD

Take plasmid vector pGreen-0229 Backbone DNA as template, carry out pcr amplification with the primer of the gus gene intron of sequence shown in SEQ ID NO:12 and the SEQ ID NO:13, obtain the gus gene intron of sequence shown in SEQ ID NO:14; Further use Xba I single endonuclease digestion with the gus gene intron of corresponding restriction enzyme site, then be connected with the same recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) that the step (5) of phosphatizing treatment makes up behind Xba I single endonuclease digestion, obtain described zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD, the nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD is shown in SEQ ID NO:21.

Compared with prior art, the beneficial effect of the inventive method is:

The present invention uses BeautifulRice alcohol soluble protein gene RNAi carrier pUC19-RNAi-22KD or pUC19-RNAi-19KD import corn variety, obtained high-lysine content transfer-gen plant, compare with the non-transgenic plant, the seed lysine content of transgenic corns has improved 15-60%, compare with the high lysine corn varieties that existing traditional breeding method is cultivated, lysine content has the raising of peer-level or the high lysine corn varieties that a little higher than traditional breeding method is cultivated, and (5-7) compares with conventional breeding, significantly shorten with the inventive method transformation cycle, only need 1-2.The corn gene technology of zein spirit-soluble gene RNAi carrier of the present invention has preferably repeatability and stability.The present invention has solved in the conventional breeding and has utilized by the contents level of the synthetic of RNA perturbation technique adjusting corn prolamine and then raising Methionin in addition O2The high lysine corn varieties endosperm of gene seed selection is soft and the key issue such as disease resistance is poor.Realized utilizing the RNA interference principle to formulate the target of the corn novel material of high-lysine content, significant for the corn quality improvement.

In the sequence table shown in the SEQ ID NO:1 is the upstream primer sequence of the specific promoter p primer of zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD of the present invention or RNAi carrier pUC19-RNAi-19KD.

In the sequence table shown in the SEQ ID NO:2 is the downstream primer sequence of the specific promoter p primer of zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD of the present invention or RNAi carrier pUC19-RNAi-19KD.

In the sequence table shown in the SEQ ID NO:3 is the nucleotide sequence of endosperm specificity expression promoter P-zp22/6.

In the sequence table shown in the SEQ ID NO:4 is the upstream primer sequence of the R22-6 primer of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P.

In the sequence table shown in the SEQ ID NO:5 is the downstream primer sequence of the R22-6 primer of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P.

In the sequence table shown in the SEQ ID NO:6 is the upstream primer sequence of the R22-7 primer of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P.

In the sequence table shown in the SEQ ID NO:7 is the downstream primer sequence of the R22-7 primer of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P.

In the sequence table shown in the SEQ ID NO:8 is the nucleotide sequence of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P.

In the sequence table shown in the SEQ ID NO:9 is the upstream primer sequence of terminator poly (A).

In the sequence table shown in the SEQ ID NO:10 is terminator poly (A) downstream primer sequence.

In the sequence table shown in the SEQ ID NO:11 is the nucleotide sequence of terminator poly (A).

In the sequence table shown in the SEQ ID NO:12 is the upstream primer sequence of gus gene intron.

In the sequence table shown in the SEQ ID NO:13 is the downstream primer sequence of gus gene intron.

In the sequence table shown in the SEQ ID NO:14 is the nucleotide sequence of gus gene intron.

In the sequence table shown in the SEQ ID NO:15 is the nucleotide sequence of zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD of the present invention.

In the sequence table shown in the SEQ ID NO:16 is the upstream primer sequence of the R19-4 primer of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P.

In the sequence table shown in the SEQ ID NO:17 is the downstream primer sequence of the R19-4 primer of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P.

In the sequence table shown in the SEQ ID NO:18 is the upstream primer sequence of the R19-5 primer of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P.

In the sequence table shown in the SEQ ID NO:19 is the downstream primer sequence of the R19-5 primer of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P.

In the sequence table shown in the SEQ ID NO:20 is the nucleotide sequence of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P.

In the sequence table shown in the SEQ ID NO:21 is the nucleotide sequence of zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD of the present invention.

In the sequence table shown in the SEQ ID NO:22 is that RNAi carrier pUC19-RNAi-22KD transforms T ₀Detect the upstream primer sequence of 22Y3 primer for the PCR of regeneration plant.

In the sequence table shown in the SEQ ID NO:23 is that RNAi carrier pUC19-RNAi-22KD transforms T ₀Detect the downstream primer sequence of 22Y3 primer for the PCR of regeneration plant.

In the sequence table shown in the SEQ ID NO:24 is that RNAi carrier pUC19-RNAi-19KD transforms T ₀Detect the upstream primer sequence of 19Y5 primer for the PCR of regeneration plant.

In the sequence table shown in the SEQ ID NO:25 is that RNAi carrier pUC19-RNAi-19KD transforms T ₀Detect the downstream primer sequence of 19Y5 primer for the PCR of regeneration plant.

Description of drawings

Fig. 1 is the structure schematic diagram of RNAi carrier pUC19-RNAi-22KD or pUC19-RNAi-19KD.

Fig. 2 is the PCR qualification result of the promotor P-zp22/6 of endosperm specific expression,

Swimming lane M. DNA marker, 1. the PCR product of the promotor P-zp22/6 of endosperm specific expression.

Fig. 3 is the PCR qualification result of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P,

Swimming lane M. DNA Marker, 1. the PCR product of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P.

Fig. 4 is the PCR qualification result of terminator poly (A), swimming lane M. DNA Marker, 1. the PCR product of terminator poly (A).

Fig. 5 is the PCR qualification result of gus gene intron,

Swimming lane M. DNA marker, 1. the PCR product of gus gene intron.

Fig. 6 is the PCR qualification result of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P,

Swimming lane M. DNA Marker, 1. the PCR product of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P.

Fig. 7 is that the enzyme of RNAi carrier pUC19-RNAi-22KD and pUC19-RNAi-19KD is cut detection,

A is the pUC19-RNAi-22KD restriction analysis, and B is the pUC19-RNAi-19KD restriction analysis; Swimming lane M. DNA Marker, 1 and 5:Xba I and Hind III double digestion, 2 and 6:Xba I single endonuclease digestion, 3 and 7:Sac I and Xba I double digestion, 4 and 8:Sac I and Hind III double digestion.

Fig. 8 is pUC19-RNAi-22KD carrier collection of illustrative plates.

Fig. 9 is pUC19-RNAi-19KD carrier collection of illustrative plates.

Figure 10 is the maize callus.

Figure 11 is the callus after explanation zein spirit-soluble gene RNAi vector gene rifle transforms as an example of the pUC19-RNAi-19KD carrier example.

Figure 12 is the regeneration of callus after explanation zein spirit-soluble gene RNAi vector gene rifle transforms as an example of the pUC19-RNAi-19KD carrier example.

Figure 13 is the strong seedling culture of differentiation seedling after explanation zein spirit-soluble gene RNAi carrier transforms as an example of the pUC19-RNAi-19KD carrier example.

Figure 14 is that explanation zein spirit-soluble gene RNAi carrier transforms the T that obtains as an example of the pUC19-RNAi-19KD carrier example ₀Field performance for transfer-gen plant.

Figure 15 is that explanation zein spirit-soluble gene RNAi carrier transforms the T that obtains as an example of the pUC19-RNAi-19KD carrier example ₀Solid situation for transfer-gen plant.

Figure 16 is the T that the pUC19-RNAi-22KD carrier transforms ₀For the PCR evaluation of transfer-gen plant,

Swimming lane M. DNA Marker, CK+: positive control (amplification of pUC19-RNAi-22KD interference carrier), CK-: negative control (amplification of B73 non-transgenic plant), CK: blank (water is template), 1-9:T ₀Increase for transfer-gen plant.

Figure 17 is the T that the pUC19-RNAi-19KD carrier transforms ₀For the PCR evaluation of transfer-gen plant,

Swimming lane M. DNA Marker, CK+: positive control (amplification of pUC19-RNAi-19KD interference carrier), CK-: negative control (amplification of B73 non-transgenic plant), CK: blank (water is template), 1-7:T ₀Increase for transfer-gen plant.

Embodiment

All ingredients used in the experiment all can be bought from the commercial channel, and the skeleton carrier pUC19 that uses in the experiment, plasmid vector pGreen-0229 Backbone, expression vector P3301, T carrier and plasmid extraction kit, glue reclaim test kit, Marker, high-fidelity DNA polymerase, dNTPs, bacillus coli DH 5 alpha competent cell and bar gene all available from the Beijing Quanshijin Biotechnology Co., Ltd; Restriction enzyme, T4-DNA ligase enzyme are available from U.S. NEB company; The reagent such as penbritin, IPTG, X-gal are all available from the precious biotechnology in Dalian company limited; RQ DNA enzyme, reversed transcriptive enzyme, 5 * reaction buffer, dNTP, RNA enzyme inhibitors are available from Promega company; Other chemical reagent are domestic analytical pure; Particle gun PDS-1000/He and running stores thereof are available from Bio-Rad company.Nucleotide sequencing is finished by the large genome company of China.Used primer sequence is synthetic by Beijing AudioCodes biotech company.Method therefor is ordinary method if no special instructions in the experiment.

The structure of embodiment 1 zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD

One, for the examination material: corn ( Zea maysL.) self-mating system B73 is the corn germplasm that is widely used at present maize genetic research and breeding practice, can from seeds company (such as auspicious the kinds industry in field, Yunnan company limited, address: tap street, the north suburb, Kunming, Yunnan Province) directly purchase.

Two, experimental technique:

1, the extraction of B73 corn gene group DNA:

1) gets maize leaf 300 mg, add liquid nitrogen and fully grind;

2) the 2%CTAB extracting solution of 65 ℃ of preheatings of adding 700 μ l, gently mixing;

3) place 65 ℃ of water-bath 30-60 min;

4) room temperature is placed 2 min, adds 300 μ l chloroform-primary isoamyl alcohol (24:1), fully shakes 1-2 h;

5) centrifugal 10 min of 10,000 rpm;

6) get the upper strata water to new centrifuge tube, add 600 μ l Virahols, room temperature is placed 10 min;

7) centrifugal 1 min of 10,000 rpm abandons supernatant;

8) the ethanol 500 μ l of adding 75%, room temperature is placed 30 min;

9) repeating step 7 and 8;

10) centrifugal 30 s of 10,000 rpm abandon supernatant, seasoning DNA;

11) add 100 μ l TE damping fluid dissolving DNAs;

12) detect rear packing ,-70 ℃ of preservations.

2) reaction conditions: 95 ℃ of denaturation 5 min, (72 ℃ are extended 1 min for 95 ℃ of sex change 50 s, 57 ℃ of annealing 50 s) 35 circulations, 72 ℃ are extended 10 min.The PCR product is electrophoresis detection on 1% sepharose.

3, the extraction of the total RNA of B73 corn kernel:

1) takes from the corn kernel 50-100 mg that hands over pollination rear 18 days, fully grind with liquid nitrogen;

2) add 1 ml Trizol reagent, concuss mixing;

3) centrifugal 10 min of 12,000 rpm get supernatant;

4) add 200 μ l chloroforms, concuss mixing, ice bath 3 min;

5) centrifugal 15 min(4 of 12,000 rpm ℃);

6) get the upper strata water to new centrifuge tube, add the Virahol of equivalent volumes, place 10 min for-20 ℃;

7) centrifugal 15 min(4 of 12,000 rpm ℃), abandon supernatant, add 700 μ l, 70% washing with alcohol precipitation;

8) centrifugal 10 min(4 of 8,000 rpm ℃), abandon supernatant;

9) precipitate with 30 μ l DEPC-H ₂The O dissolving;

10) 1% agarose gel electrophoresis detects RNA.

4, RNA reverse transcription system

Reaction system: the total RNA 8 μ l of corn kernel, add 1 μ l RQ DNA enzyme and 1 μ l RQ damping fluid, 37 ℃ of water-bath 30 min, taking-up is placed on ice, adds 1 μ l RQ stop buffer, 65 ℃ of water-bath 10 min; Then take out 9 μ l as the RNA template, add OligodT 1 μ l, mixing is placed on 70 ℃ of water-bath 5 min, then cooled on ice 2 min; Add again 5 times of RT MLV damping fluid 4 μ l, 10 mM dNTP, 4 μ l, RNA enzyme inhibitors 1 μ l(20U), M-MLV ThermoScript II 1 μ l(200U), 42 ℃ of water-bath 1 h; 72 ℃ of deactivation 10 min, ice bath 10 min are in-20 ℃ of preservations.

Reaction conditions: 94 ℃ of denaturation 5 min, (72 ℃ are extended 1 min for 94 ℃ of sex change 40 s, 59 ℃ of annealing 45 s) 35 circulations, 72 ℃ are extended 10 min.The PCR product is electrophoresis detection on 1% sepharose.

7, connect product to colibacillary conversion

1) gets competent cell DH5 α 100 μ l, add T carrier connector 2 μ l, ice bath 30 min;

2) 42 ℃ of water-bath heat shock 90 s place 2 min on ice immediately;

3) add 500 μ l LB substratum, 200 * g shakes 1 h under 37 ℃ of conditions;

4) centrifugal 1 min of 3500 * g abandons the part supernatant after leaving standstill 1 h;

5) add 5 μ l ZPTG and 50 μ l X-Gal;

6) coated plate, sealing;

7) cultivate 16 h for 37 ℃;

8) choose white single bacterium colony, 37 ℃ are shaken bacterium.

8, the extraction of recombinant vectors DNA

Extract with Beijing Quanshijin Biotechnology Co., Ltd's plasmid extraction kit, step is as follows:

1) gets centrifugal 1 min of bacterium 10,000 * g of 4 ml incubated overnight;

2) add the colourless RB(of 250 μ l and contain RNase A), concussion suspension bacterial precipitation;

3) add the blue LB of 250 μ l, be no more than 5 min;

4) add the yellow NB of 350 μ l, room temperature leaves standstill 2 min;

5) centrifugal 5 min of 15,000 * g get supernatant and add in the adsorption column;

6) centrifugal 1 min of 15,000 * g, the abandoned stream fluid;

7) add 650 μ l solution W B, centrifugal 1 min of 15,000 * g, abandoned stream fluid;

8) centrifugal 2 min of 15,000 * g thoroughly remove residual WB;

9) adsorption column is placed clean centrifuge tube, in post, add the EB of 70 ℃ of preheatings of 50 μ l or deionized water (pH〉7.0) room temperature and leave standstill 1 min;

10) centrifugal 1 min of 10,000 * g, eluted dna is in-20 ℃ of preservations.

10, endonuclease bamhi reclaims and is connected

Reclaim test kit (Beijing Quanshijin Biotechnology Co., Ltd) with glue and reclaim endonuclease bamhi, concrete operation step is as follows:

1) cuts target DNA band in the sepharose, weigh;

2) add 3 times of volume solution GSB, in 55 ℃ of water-bath 10 min;

3) treat that gelating soln is down to room temperature, add in the adsorption column and leave standstill 1 min, centrifugal 1 min of 10,000 * g, abandoned stream fluid;

4) add 650 μ l solution W B, centrifugal 1 min of 10,000 * g, abandoned stream fluid;

5) centrifugal 2 min of 10,000 * g remove residual WB;

6) adsorption column is placed centrifuge tube, add 70 ℃ of preheating EB of 50 μ l or deionized water in post, room temperature leaves standstill 1 min;

7) centrifugal 1 min of 10,000 * g, eluted dna;

8) enzyme cuts back to close being connected of product and pUC19 carrier segments, and reaction system and condition are as follows:

Reaction conditions: mix gently rear 65 ℃ of water-bath 5 min, place immediately 1 min on ice, then add 1.5 μ l and connect damping fluid and 0.5 μ l T4 ligase enzyme, 16 ℃ of connections of spending the night.

11, the evaluation of recombinant vectors

1) PCR identifies

Reaction system and condition see two, 2; According to the PCR detected result, get the positive colony adding and contain antibiotic LB liquid nutrient medium, 200 * g shakes 1 h under 37 ℃ of conditions; Then get 1 ml bacterium liquid and send the order-checking of the large Genetic Biotechnologies limited liability company of Beijing China, sequencing result carries out sequential analysis with seqMan software; In ncbi database, carry out sequence analysis with the sequence of zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD or pUC19-RNAi-19KD respectively to transforming colibacillary target fragment.

2) enzyme is cut evaluation

Method is with embodiment 1 two steps 9.

Following steps relate to carries out pcr amplification to the genome of corn variety B73 and recombinant vectors DNA, the total RNA of B73 corn kernel is carried out the RT-PCR amplification, the T carrier of PCR product connects, and the ligation system and the reaction conditions that make up each carrier are all undertaken by the aforesaid operations step.

Three, the acquisition of endosperm specificity expression promoter P-zp22/6

1, the p primer of design endosperm specificity expression promoter P-zp22/6

According to (A new opaque variant of maize by a single dominant RNA-interference-inducing transgene. Genetics 2003 such as Segal, 165:387-397) report, P-zp22/6 is the promotor of endosperm specificity expression gene zp22/6.The mRNA of zp22/6 gene is at first found in this experiment in MaizeGDB database (http://www.maizegdb.org/), be AC144719.1 take this mRNA as motif in the Genbank(accession number) in find corresponding zp22/6 genomic dna sequence and corresponding coding region, the sequence of front 1.3 kb in coding region is the promotor of this gene.Select the long sequence of 1.5 kb before the coding region as target sequence for finding complete promotor, and according to the multiple clone site analysis that exists on the pUC19 carrier, utilize Sac I and Xba I endosperm specificity expression promoter P-zp22/6 can be connected to the pUC19 carrier, therefore with the Primer5 software design primer sequence (abbreviation specific promoter p primer) of following P-zp22/6 with restriction enzyme site:

2, the acquisition of endosperm specific expression promoter P-zp22/6 sequence

Take the B73 corn gene group DNA as template, take SEQ ID NO:1(upstream primer) and SEQ ID NO:2(downstream primer) as primer pair, carry out pcr amplification (amplification is seen Fig. 2), obtain the endosperm specificity expression promoter P-zp22/6 fragment that the 5' end is introduced Sac I restriction enzyme site, 3' end introducing Xba I and Sma I restriction enzyme site; Then this P-zp22/6 fragment is connected with the T carrier and to its order-checking, sequencing result show 5' end and 3' end the restriction enzyme site of introducing really be Sac I restriction enzyme site, Xba I and Sma I restriction enzyme site;

3, checking: in the NCBI website (on the http://www.ncbi.nlm.nih.gov/BALST, accession number is AC144719.1) to the checking of comparing of step 3,2 sequencing result, discovery is introduced the restriction enzyme site except 5' end, 3' end, the similarity of corresponding sequence reaches 100% among sequencing result and the GeneBank, shows that institute's cloned sequence really is endosperm specific expression promoter P-zp22/6 sequence.Described endosperm specific expression promoter P-zp22/6 sequence is shown in SEQ ID NO:3.

Four, make up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6

Step 3 P-zp22/6 fragment is connected the order-checking plasmid that obtains cuts with Sac I and Xba I enzyme with the T carrier, then be connected with the carrier pUC19 that is connected with Xba I enzyme through the Sac I equally, obtain containing the recombinant vectors pUC19-p22/6 of endosperm specificity expression promoter zp22/6.

Five, the acquisition of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

1, the primer of design 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

According to (A new opaque variant of maize by a single dominant RNA-interference-inducing transgene. Genetics 2003 such as Segal, the multiple clone site analysis of the 22-KD primer sequence of the 22-KD alcohol soluble protein gene of 165:387-397) delivering and recombinant vectors pUC19-p22/6 and skeleton carrier pUC19, utilize Sma I and Xba I the forward fragment of goal gene can be connected to the pUC19-p22/6 carrier, and utilize Hind III and Xba I the reverse fragment of goal gene can be connected to the pUC19 carrier, therefore designed the primer sequence (being called for short R22-6 primer and R22-7 primer) of following 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P with restriction enzyme site:

2, the acquisition of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

Take the total RNA of B73 corn kernel as template, take SEQ ID NO:4(upstream primer) and SEQ ID NO:5(downstream primer) as primer pair, carry out the RT-PCR amplification, obtain holding the 22-KD-P fragment of introducing respectively Sma I and Xba I restriction enzyme site at 5' end and 3'; Take this fragment as template, take SEQ ID NO:6(upstream primer) and SEQ ID NO:7(downstream primer) as primer pair, carry out RT-PCR amplification (amplification is seen Fig. 3), obtain the 22-KD-P fragment about 300 bp of 3' end further introducing Hind III and Xho I restriction enzyme site; This 22-KD-P fragment of introducing restriction enzyme site is connected to T carrier and order-checking, and sequencing result shows successfully introduces Hind III and Xho I restriction enzyme site at 22-KD-P fragment 3' end, introduces Sma I and Xba I restriction enzyme site at the 5' end;

3, checking: in the NCBI website (on the http://www.ncbi.nlm.nih.gov/BALST, accession number is FL392908.1) sequencing result of this 22-KD-P fragment is compared, discovery is introduced the restriction enzyme site except 5' end, 3' end, the similarity of corresponding sequence reaches 99% among sequencing result and the GeneBank, prove that this sequence really is the 22-KD-P fragment of 22-KD alcohol soluble protein gene, can be used for the RNAi vector construction.The 22-KD-P fragment sequence of described 22-KD alcohol soluble protein gene is shown in SEQ ID NO:8.

Six, forward inserts the 22-KD-P fragment in containing the recombinant vectors pUC19-p22/6 of endosperm specificity expression promoter P-zp22/6

Step 5 22-KD-P fragment is connected the order-checking plasmid that obtains cuts with Sma I and Xba I enzyme with the T carrier, then be connected with the pUC19-p22/6 recombinant vectors that the step 4 of being connected with Xba I enzyme through the Sma I equally obtains, obtain containing the recombinant vectors pUC19-p22/6-22KD1 of endosperm specificity expression promoter P-zp22/6 and forward insertion 22-KD-P fragment.

Seven, make up the recombinant vectors pUC19-22KD2 that oppositely inserts the 22-KD-P fragment

Step 5 22-KD-P fragment is connected the order-checking plasmid that obtains cuts with Hind III and Xba I enzyme with the T carrier, then be connected with the pUC19 carrier of being connected with Xba I enzyme through the Hind III equally, oppositely inserted the recombinant vectors pUC19-22KD2 of 22-KD-P fragment.

Eight, make up the recombinant vectors pUC19-22KD2-poly (A) that contains terminator poly (A)

1, the primer of design terminator poly (A)

Terminator sequence (http://www.cambia.org/daisy/cambia/home.html) and the upper multiple clone site analysis that exists of recombinant vectors pUC19-22KD2 according to expression vector p3301, utilize Xho I and Hind III terminator poly (A) can be connected to the pUC19-22KD2 carrier, therefore with the Primer5 software design following terminator poly (A) primer sequence with restriction enzyme site:

2, the acquisition of terminator poly (A)

Take the p3301 expression vector dna as template, take SEQ ID NO:9(upstream primer) and SEQ ID NO:10(downstream primer) as primer pair, carry out pcr amplification (amplification is seen Fig. 4), obtain the terminator poly (A) that the 5' end is introduced Xho I restriction enzyme site, 3' end introducing Hind III restriction enzyme site; This fragment is connected to T carrier and order-checking, sequencing result show 5' end and 3' end the restriction enzyme site of introducing really be Xho I and Hind III restriction enzyme site;

3, checking: corresponding sequence among the sequencing result of terminator poly (A) and the p3301 is compared, and the two similarity reaches 100%, proves that institute's calling sequence is the terminator sequence in the p3301 expression vector.Described terminator poly (A) sequence is shown in SEQ ID NO:11.

4, step 82 terminator poly (A) being connected the order-checking plasmid that obtains with the T carrier cuts with Xho I and Hind III enzyme, then is connected with the recombinant vectors pUC19-22KD2 that the step 7 of being connected with Hind III enzyme through the Xho I equally obtains, obtains inserting and oppositely insert the 22-KD-P fragment and with the recombinant vectors pUC19-22KD2-poly (A) of terminator poly (A).

Nine, structure contains endosperm specificity expression promoter P-zp22/6, the recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) of forward 22-KD-P fragment, reverse 22-KD-P fragment and terminator poly (A)

The recombinant vectors pUC19-22KD2-poly (A) that the recombinant vectors pUC19-p22/6-22KD1 that step 6 is obtained and step 8 obtain uses respectively Xba I and Hind III double digestion, the enzyme that then will contain reverse 22-KD-P fragment and terminator poly (A) is cut product 22KD2-poly (A) and is connected to recombinant vectors pUC19-p22/6-22KD1, obtains containing endosperm specificity expression promoter P-zp22/6, forward 22-KD-P fragment, the recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) of reverse 22-KD-P fragment and terminator poly (A).

Ten, the acquisition of gus gene intron

1, the primer of design gus gene intron

Gus gene sequence (http://www.pgreen.ac.uk/JIT/pG0229.htm) and the upper multiple clone site analysis that exists of recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) according to pGreen-0229 Backbone carrier, utilize Xba I restriction enzyme site the gus gene intron can be connected to pUC19-p22/6-22KD1-22KD2-poly (A) carrier, therefore with the Primer5 software design following gus gene intron primer sequence with restriction enzyme site:

2, the acquisition of gus gene intron

Take pGreen-0229 Backbone carrier DNA as template, take SEQ ID NO:12(upstream primer) and SEQ ID NO:13(downstream primer) as primer pair, carry out pcr amplification (amplification is seen Fig. 5), obtain 5' and 3' and all hold the gus gene intron of introducing Xba I restriction enzyme site; This fragment is connected to T carrier and order-checking, sequencing result show 5' end and 3' end the restriction enzyme site of introducing really be Xba I restriction enzyme site;

3, checking: the corresponding sequence to pGreen-0229 Backbone carrier on the sequencing result of gus gene intron and the http://www.pgreen.ac.uk/JIT/pG0229.htm website is compared, find that the two similarity reaches 100%, prove that institute's calling sequence is the gus gene intron sequences.Described gus gene intron sequences is shown in SEQ ID NO:14;

11, make up zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD of the present invention

1, makes up zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD

Step 10 gus gene intron is connected the order-checking plasmid that obtains Xba I single endonuclease digestion with the T carrier, then with same behind Xba I single endonuclease digestion the recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) of phosphatizing treatment be connected, obtain zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD of the present invention;

2, the checking of zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD

The bacterium liquid that will contain zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD is delivered to the large genome company of China and is carried out the nucleotide sequence order-checking, sequencing result shows and has successfully made up zein spirit-soluble gene RNAi carrier, called after pUC19-RNAi-22KD; Simultaneously according to the vector construction flow process, carry out enzyme with the corresponding enzyme of restriction enzyme site and cut detection (shown in Fig. 7 A) result and show that the pUC19-RNAi-22KD vector construction is complete.The nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD shown in SEQ ID NO:15, structure as shown in Figure 8.

By between the restriction enzyme site Sac I of skeleton carrier pUC19 and Sma I, inserting endosperm specific expression promoter P-zp22/6, forward inserts purpose fragment 22-KD-P between Sma I and Xba I restriction enzyme site, between restriction enzyme site Xba I and Xho I, oppositely insert purpose fragment 22-KD-P, between forward 22-KD-P fragment and reverse 22-KD-P fragment, insert simultaneously the gus gene intron, between between restriction enzyme site Hind III and Xho I, insert terminator poly (A), successfully made up the described zein spirit-soluble gene RNAi of the present embodiment carrier pUC19-RNAi-22KD.The nucleotide sequence of described endosperm specific expression promoter P-zp22/6 is shown in SEQ ID NO:3, the nucleotide sequence of described 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P is shown in SEQ ID NO:8, shown in the nucleotide sequence SEQ ID NO:14 of described gus gene intron, shown in the nucleotide sequence SEQ ID NO:11 of described terminator poly (A); The nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD shown in SEQ ID NO:15, collection of illustrative plates as shown in Figure 8.

The structure of embodiment 2, zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD

The structure of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD is except following step difference, and all the other steps are identical with embodiment 1, repeat no more.

One, the acquisition of endosperm specificity expression promoter P-zp22/6, method is identical with embodiment 1.

Two, make up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6, method is identical with embodiment 1.

Three, the acquisition of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

1, the primer of design 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

In database MaizeGDB, find the mRNA sequence of 19-KD gene, be AC196717.3 take this mRNA as motif in the Genebank(accession number) compare and find corresponding 19-KD genomic dna sequence, multiple clone site analysis to this sequence and recombinant vectors pUC19-p22/6 and skeleton carrier pUC19, utilize Sma I and Xba I the forward fragment of goal gene can be connected to the pUC19-p22/6 carrier, and utilize Hind III and Xba I the reverse fragment of goal gene can be connected to the pUC19 carrier, therefore designed the primer (being called for short R19-4 primer and R19-5 primer) of following 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P with restriction enzyme site:

2, the acquisition of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

Take the total RNA of B73 corn kernel as template, take SEQ ID NO:16(upstream primer) and SEQ ID NO:17(downstream primer) as primer pair, carry out the RT-PCR amplification, obtain holding the 19-KD-P fragment of introducing respectively Sma I and Xba I restriction enzyme site at 5' end and 3'; Take this fragment as template, take SEQ ID NO:18(upstream primer) and SEQ ID NO:19(downstream primer) as primer pair, carry out RT-PCR amplification (amplification is seen Fig. 6), obtain the 19-KD-P fragment about 300 bp of 3' end further introducing Hind III and Xho I restriction enzyme site; This 19-KD-P fragment of introducing restriction enzyme site is connected to T carrier and order-checking, and sequencing result shows successfully introduces Hind III and Xho I restriction enzyme site at 19-KD-P fragment 3' end, introduces Sma I and Xba I restriction enzyme site at the 5' end;

3, checking: in the NCBI website (on the http://www.ncbi.nlm.nih.gov/BALST, accession number is FM187060.1) sequencing result of this 19-KD-P fragment is compared, discovery is introduced the restriction enzyme site except 5' end, 3' end, the similarity of corresponding sequence reaches 99% among sequencing result and the GeneBank, prove that this sequence really is the 19-KD-P fragment of 19-KD alcohol soluble protein gene, can be used for the RNAi vector construction.The 19-KD-P fragment sequence of described 19-KD alcohol soluble protein gene is shown in SEQ ID NO:20.

Four, forward inserts the 19-KD-P fragment in containing the recombinant vectors pUC19-p22/6 of endosperm specificity expression promoter P-zp22/6

The present embodiment step 3 19-KD-P fragment is connected the order-checking plasmid that obtains cuts with Sma I and Xba I enzyme with the T carrier, then be connected with the pUC19-p22/6 recombinant vectors that the present embodiment step 2 of being connected with Xba I enzyme through the Sma I equally obtains, obtain containing the recombinant vectors pUC19-p22/6-19KD1 of endosperm specificity expression promoter P-zp22/6 and forward insertion 19-KD-P fragment.

Five, make up the recombinant vectors pUC19-19KD2 that oppositely inserts the 19-KD-P fragment

The present embodiment step 3 19-KD-P fragment is connected the order-checking plasmid that obtains cuts with Hind III and Xba I enzyme with the T carrier, then be connected with the pUC19 carrier of being connected with Xba I enzyme through the Hind III equally, oppositely inserted the recombinant vectors pUC19-19KD2 of 19-KD-P fragment.

Six, make up the recombinant vectors pUC19-19KD2-poly (A) that contains terminator poly (A)

1, the design of primers of terminator poly (A), acquisition and the checking of terminator poly (A), method is all identical with embodiment 1;

2, the terminator poly (A) that the present embodiment step 61 is obtained is connected the order-checking plasmid that obtains and cuts with Xho I and Hind III enzyme with the T carrier, then be connected the recombinant vectors pUC19-19KD2-poly (A) that obtains inserting reverse insertion 19-KD-P fragment and contain terminator poly (A) with the recombinant vectors pUC19-19KD2 that the present embodiment step 5 of being connected with Hind III enzyme through the Xho I equally obtains.

Seven, structure contains endosperm specificity expression promoter P-zp22/6, the recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) of forward 19-KD-P fragment, reverse 19-KD-P fragment and terminator poly (A)

The recombinant vectors pUC19-19KD2-poly (A) that the recombinant vectors pUC19-p22/6-19KD1 that the present embodiment step 4 is obtained and step 6 obtain uses respectively Xba I and Hind III double digestion, then will then the enzyme that contains reverse 19-KD-P fragment and terminator poly (A) be cut product 19KD2-poly (A) and be connected to recombinant vectors pUC19-p22/6-19KD1, obtain containing endosperm specificity expression promoter P-zp22/6, forward 19-KD-P fragment, the recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) of reverse 19-KD-P fragment and terminator poly (A).

Eight, the acquisition of gus gene intron, method is identical with embodiment 1.

Nine, make up zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD of the present invention

1, makes up zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD

The present embodiment step 8 gus gene intron is connected the order-checking plasmid that obtains Xba I single endonuclease digestion with the T carrier, then with same behind Xba I single endonuclease digestion the recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) of phosphatizing treatment be connected, obtain zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD of the present invention;

2, the checking of zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD

The bacterium liquid that will contain zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD is delivered to the large genome company of China and is carried out the nucleotide sequence order-checking, sequencing result shows and has successfully made up zein spirit-soluble gene RNAi carrier, called after pUC19-RNAi-19KD; Simultaneously according to the vector construction flow process, carry out enzyme with the corresponding enzyme of restriction enzyme site and cut detection (shown in Fig. 7 B) result and show that the pUC19-RNAi-19KD vector construction is complete.The nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD shown in SEQ ID NO:21, structure as shown in Figure 9.

By between the restriction enzyme site Sac I of skeleton carrier pUC19 and Sma I, inserting endosperm specific expression promoter P-zp22/6, forward inserts purpose fragment 19-KD-P between Sma I and Xba I restriction enzyme site, between restriction enzyme site Xba I and Xho I, oppositely insert purpose fragment 19-KD-P, between forward 19-KD-P fragment and reverse 19-KD-P fragment, insert the gus gene intron simultaneously, between restriction enzyme site Hind III and Xho I, insert terminator poly (A), successfully made up the described zein spirit-soluble gene RNAi of the present embodiment carrier pUC19-RNAi-19KD.The nucleotide sequence of described endosperm specific expression promoter P-zp22/6 is shown in SEQ ID NO:3, the nucleotide sequence of described 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P is shown in SEQ ID NO:20, shown in the nucleotide sequence SEQ ID NO:14 of described gus gene intron, shown in the nucleotide sequence SEQ ID NO:11 of described terminator poly (A); The nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD shown in SEQ ID NO:21, collection of illustrative plates as shown in Figure 9.

The RNAi carrier of the present invention that make up to obtain with aforesaid method is as object take the prolamine that the corn lysine content had important decisive action, adopt first segmentation to make up intermediate carrier, again each intermediate carrier is connected, insert at last the mode of intron, be about to specificity promoter and forward purpose fragment and be building up on the same skeleton carrier pUC19; Insert reverse purpose fragment and terminator poly (A) at another pUC19 simultaneously; Again reverse purpose fragment and terminator poly (A) are inserted on the recombinant vectors that contains promotor and forward purpose fragment; Insert at last the gus gene intron and consist of RNAi carrier of the present invention; Because 22-KD and 19-KD alcohol soluble protein gene all are endosperm specificity expressions, so have cloned a specificity promoter among the present invention, then insert on the RNAi carrier.Its advantage of RNAi carrier that makes up with the method mainly comprises: 1) by adding the gus gene intron, improve the silence efficiency of RNAi carrier; 2) conventional RNAi Vector construction method be by promotor, forward purpose fragment, one section not expressed sequence, oppositely the order of purpose fragment, terminator poly (A) is connected on the skeleton carrier one by one, this RNAi Vector construction is connected to promotor and forward purpose fragment, reverse purpose fragment and terminator poly (A) on the skeleton carrier pUC19, and then two carriers are carried out enzyme cut and be connected, and then reverse purpose fragment and terminator poly (A) are inserted on the recombinant vectors that contains promotor and forward purpose fragment.Because two intermediate carriers can make up simultaneously, therefore shortened the time of vector construction, improved efficient.

3 one kinds of methods that improve the corn lysine content with zein spirit-soluble gene RNAi carrier of embodiment

With zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD or pUC19-RNAi-19KD difference maize transformation kind, all make its seed lysine content level obtain larger raising, reached the purpose of improvement corn quality, its step is as follows:

One, for the examination material

Corn ( Zea maysL.) cross-fertilize seed H99 * Hi II B(is purchased from the greatly healthy scientific and technological development of Beijing middle peasant company limited).18 d after the artificial autocopulation pollinates get fruit ear successively with 70% ethanol, 2.5% clorox and aqua sterilisa sterilization, the about rataria of 10-1.2 mm of picking length under aseptic condition.

Two, substratum

The used medium such as the screening of callus and regeneration plant differentiation is as shown in table 1 after the inducing of maize callus, subculture, via Particle Bombardment Transformation, the conversion, in 121 ℃ of autoclaving 20 min.

Annotate: macroelement, trace element, molysite, organic prescription by the N6 basic recipe prepare (Zhu Zhiqing etc. set up a kind of preferably Rice Anther substratum by the nitrogenous source comparison test. Chinese science 1975, (5): 484-490), PH transfers to 5.8.

Three, rataria is cultivated

1, rataria is placed on the culture medium A (table 1), the dark cultivation 3-4 week forms callus, as shown in figure 10 under 27-28 ℃ of condition;

2, get that growing way is good, tissue looseness, the callus that faint yellow, embryo is good, succeeding transfer culture under 27-28 ℃ of condition, every 2-3 week subculture once, the gained callus is as the acceptor material of via Particle Bombardment Transformation;

3, at front 4 h of particle gun bombardment, callus is cut into 2 mm size and transfers to substratum B(table 1) upper stand-by.

Four, via Particle Bombardment Transformation

1, the preparation of the little bullet of particle gun and conversion

1) according to Klein etc. (High velocity microprojectiles for delivering nucleic acids into living cells. Nature 1987, method 327:70-73) prepares bronze suspension (bronze diameter 1.0 μ m);

2) get bronze suspension (60 mg/ml) the 33 μ l of 4 ℃ of preservations, add successively 4 μ l plasmid DNA (1 μ g/ μ l), 50 μ l CaCl ₂(2.5 mol/L), 20 μ l spermidines (0.1 mol/L), 5 min that vibrate, room temperature leaves standstill 10 min, and DNA is deposited on the particulate carrier;

3) 12, centrifugal 3 s of 000 rpm, abandon supernatant, add 200 μ l, 100% ethanol rinsing precipitation, 12, centrifugal 3 s of 000 rpm, abandon supernatant, add 50 μ l, 100% ethanol, the low speed concussion keeps suspended state, every rifle is got the DNA bronze suspension that 12.5 μ l prepare and is evenly coated on little missile-borne body, dries for subsequent use;

4) utilize Sac I and Hind III that the interference goal gene that makes up enzyme on pUC19-RNAi-22KD or the pUC19-RNAi-19KD carrier is scaled off rear collection, then mix in the ratio of 3:1 with the bar gene, adopt the method for particle gun bombardment that goal gene is transferred in the acceptor, the transgenic event statistics sees Table 2.

2, the screening of callus and plant regeneration

1) after the particle gun bombardment, the callus of via Particle Bombardment Transformation is placed on the substratum B, dark overnight incubation under 28 ℃ of conditions, then transfer on the culture medium A 28 ℃ renewal cultivation 5-7 days;

2) with on the callus transport medium C, succeeding transfer culture under 28 ℃ of conditions, 2 all subcultures are once selected normal, the WD callus of color and luster (as shown in figure 11) succeeding transfer culture, subculture 3-4 time;

3) callus is transferred on the division culture medium D, under 16 h illumination/8 h dark conditions, under 28 ℃ of conditions, cultivated for 2 weeks, have green point to occur;

4) when the bud of Calli Differentiation grows to approximately 1-2 cm (as shown in figure 12), be transferred to and continue among the substratum E to cultivate;

5) select the seedling (as shown in figure 13) of well developed root system in the 3-4 leaf phase, with warm water flush away substratum, transplant in the greenhouse and to bury, be transplanted to field planting after 2 weeks, as until blossom and bear fruit (as shown in figure 14).

Five, T ₀PCR for regeneration plant detects

1, design T ₀Detect primer for plant PCR

Classify target sequence as with the nucleotides sequence of the RNAi carrier pUC19-RNAi-19KD shown in the zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD shown in the SEQ ID NO:15 and the SEQ ID NO:21 respectively, utilize Primer5 software to design respectively the T shown in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and the SEQ ID NO:25 ₀Detect 22Y3 primer pair and 19Y5 primer pair for plant PCR;

2, pUC19-RNAi-22KD carrier T ₀PCR for regeneration plant detects

Extract the pUC19-RNAi-22KD carrier and transform the T that obtains ₀(method is with embodiment 1 for the genomic dna of regeneration plant (90), two, 1), extract the DNA(method of pUC19-RNAi-22KD carrier with embodiment 1, two, 9), respectively with transfer-gen plant seed DNA, the DNA(CK+ of pUC19-RNAi-22KD carrier), corn variety B73 non-transgenic plant seed DNA(CK-), blank water (CK) is as template, with SEQ ID NO:22(22Y3 upstream primer) and SEQ ID NO:23(22Y3 downstream primer) shown in the 22Y3 primer pair carry out pcr amplification (as shown in figure 16), the result shows 90 T that obtained by the pUC19-RNAi-22KD carrier ₀In plant, there are 10 strains to amplify the purpose band (comprising goal gene and intron sequences) of 1 kb, positive rate is 11.1%, illustrates that the 22KD alcohol soluble protein gene has been integrated into Maize genome.

3, pUC19-RNAi-19KD carrier T ₀PCR for regeneration plant detects

Extract the pUC19-RNAi-19KD carrier and transform the T that obtains ₀(method is with embodiment 1 for the genomic dna of regeneration plant (120), two, 1), extract the DNA(method of pUC19-RNAi-19KD carrier with embodiment 1, two, 9), respectively with transfer-gen plant seed DNA, the DNA(CK+ of pUC19-RNAi-19KD carrier), corn variety B73 non-transgenic plant seed DNA(CK-), blank water (CK) is as template, with SEQ ID NO:24(19Y5 upstream primer) and SEQ ID NO:25(19Y5 downstream primer) shown in the 19Y5 primer pair of sequence carry out pcr amplification (as shown in figure 17), the result shows 120 T that obtained by the pUC19-RNAi-19KD carrier ₀In plant, there are 12 strains to amplify purpose band (comprising goal gene and intron sequences) about 800 bp, positive rate is 10.0%, illustrates that the 19KD alcohol soluble protein gene has been integrated into Maize genome.

Six, the Methionin acid content of transfer-gen plant is measured

Results transgenosis T ₀Seed (as shown in figure 15) for plant, send agricultural product quality supervision and inspection center of the Ministry of Agriculture (Kunming) to analyze, according to the standard method at international corn wheat improvement center (CIMMYT), analyzed T with the Perten-DA7200 near infrared grain quality analysis meter ₀T for plant ₁For the lysine content of seed, the result shows:

Transform in 10 transgenic corns materials that obtain with zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD of the present invention, there is 1 strain to improve about 52% than the lysine content of non-transgenic plant, have 3 strain lysine contents to improve 15-20%, other 6 strains are without considerable change.

In 12 transgenic lines that zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD maize transformation kind of the present invention obtains, there are 2 strains to improve 50-60% than the lysine content of non-transgenic plant, have 4 strain lysine contents to improve 15-20%, other 7 strains are without considerable change.

Above-mentioned experiment shows, with the inventive method zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD or pUC19-RNAi-19KD maize transformation kind, obtained high-lysine content transfer-gen plant, compare with the non-transgenic plant, the seed lysine content of transgenic corns has improved 15-60%, compare with the high lysine corn varieties that existing traditional breeding method is cultivated, lysine content has the raising of peer-level or the high lysine corn varieties that a little higher than traditional breeding method is cultivated.Simultaneously, the parallel laboratory test explanation has preferably repeatability and stability based on the corn gene technology of this RNAi carrier.The present invention has solved in the conventional breeding and has utilized by the contents level of the synthetic of RNA perturbation technique adjusting corn prolamine and then raising Methionin in addition O2The high lysine corn varieties endosperm of gene seed selection is soft and the key issue such as disease resistance is poor.

＜110〉Cereal Crops Inst., Yunnan Prov. Agricultural Academy

Field, Yunnan auspicious kind industry company limited

＜120〉a kind of method that improves the corn lysine content with zein spirit-soluble gene RNAi carrier

<130> -

<160> 25

<170> PatentIn version 3.3

<210> 1

<211> 26

<212> DNA

＜213〉artificial sequence

<220>

＜223〉upstream primer of specific promoter p primer

<400> 1

gagctcggca aagggactgg caaagg 26

<210> 2

<211> 37

<212> DNA

＜213〉artificial sequence

<220>

＜223〉downstream primer of specific promoter p primer

<400> 2

tctagactag cccgggcgct tcgcttggtg tgtttct 37

<210> 3

<211> 1367

<212> DNA

＜213〉corn (Zea mays L.)

<400> 3

ggcaaaggga ctggcaaagg ggcccactag agggttcttt gccgagtgcc agttcaccag 60

acactcggca aataaacctc ctttgccgag tgtcgccgtg ggcactcggc gaaggatccg 120

tcaccgtcac ttggcgccgt gacggtgact tttctttgcc gagtgccaaa tggcactcgg 180

caaagtcttt gccgagtgcc cgacaaaaag tactcggcaa agatgctgtt gccgatgtac 240

agttcgccga gcgttctttg ccgagtgtta cactcggcaa agcatttgcc gagtgtaaaa 300

tagcctttgc cgtgtgtctc agacacacgg caaagaagct gattccggta gtgtttgggg 360

ggcgactaac acaaatcgat gacctttgat gaagatggat gtatttctcc cacaaaatct 420

tggatccatc ctctactatg aaaagtcatg aattcacaag acgttttcga aaacaatttc 480

agcttcactt gtaaagttgc ttgtgcggct gaagccaaga aaacattaga ggacaaacaa 540

taacttacta tgcttttcgt gaactgtttc agaccagcag taataattat tggtcctaca 600

tgcattaatg gataagcggc tagcatgcca agacggagca ttagttagga gataagacct 660

agcgtttcat tgcatcccca tgaaaggaat actatcacta ctgcatgaat aattttcaaa 720

tgtattaaaa actgatattg aaatgtaggt aatctgtccg cctatgtgaa atgtttaaca 780

gaggcagcca ctctaggatg ttgcctctat taattactca tagatgcagc catccaactc 840

taggatttca attagtctca atctatgcat tcgtctatgc attctaggat tacaattagt 900

ctcaatcttg tagtatttgt tccttccttg tcaaatcact tctcatctaa ctactatgct 960

tgtttaacca tcagaacata actacaacaa catccattta taaaggcttc gatagcaaac 1020

tttacatatt catatcatgt taaggttgtc acatgtgtaa aggtgaagag atcatgcatg 1080

tcattccacg tagataaaaa gaatgcctat ataaaaatgg cacattttct tgtaggtagt 1140

ggaaagtatc tttccagcaa agaccatata atccgataaa gctgataact aaatgtcgaa 1200

atcgagtaga tgccatatca tctatacctt atctgttgtt tggaaaaaaa aaagacaaaa 1260

tccaaaaaaa aatataagag atctcaccta tataaatagc tctgaaatca gtagttaatc 1320

catcacccat aatattttga gcattcagaa acacaccaag cgaagcg 1367

<210> 4

<211> 26

<212> DNA

＜213〉artificial sequence

<220>

＜223〉upstream primer of the R22-6 primer of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

<400> 4

cccgggggcg agcgtctaca acaacc 26

<210> 5

<211> 30

<212> DNA

＜213〉artificial sequence

<220>

＜223〉downstream primer of the R22-6 primer of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

<400> 5

tctagacaca acgagagggc tagatgaaag 30

<210> 6

<211> 36

<212> DNA

＜213〉artificial sequence

<220>

＜223〉upstream primer of the R22-7 primer of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

<400> 6

aagcttctag ctcgagggcg agcgtctaca acaacc 36

<210> 7

<211> 30

<212> DNA

＜213〉artificial sequence

<220>

＜223〉downstream primer of the R22-7 primer of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

<400> 7

tctagacaca acgagagggc tagatgaaag 30

<210> 8

<211> 312

<212> DNA

＜213〉corn (Zea mays L.)

<400> 8

cacaacgaga gggctagatg aaagcagttg ttgttgttgt acgtaggcgg cagggttcac 60

catggctagt tgactgagcg ttggcagaaa ctgttgtagc tgttgttgtt gctggtttgc 120

aactacgttc gccagagcaa gtgggttgga tgcaagcagc tgctcttgca agtaggcggc 180

agggttcacc atggctaggt ggctcagtgc tggtaggaac tgttgttgct gttgcgttgt 240

gatggcttgt attgttagat gtggcaagga ttgttgttgc aattgggcaa ttggttgttg 300

tagacgctcg cc 312

<210> 9

<211> 32

<212> DNA

＜213〉artificial sequence

<220>

＜223〉upstream primer of terminator poly (A)

<400> 9

ctcgagcgag tttctccata ataatgtgtg ag 32

<210> 10

<211> 27

<212> DNA

＜213〉artificial sequence

<220>

＜223〉terminator poly (A) downstream primer

<400> 10

aagctttcgg gggatctgga ttttagt 27

<210> 11

<211> 185

<212> DNA

<213> Plasmid p3301

<400> 11

tcgggggatc tggattttag tactggattt tggttttagg aattagaaat tttattgata 60

gaagtatttt acaaatacaa atacatacta agggtttctt atatgctcaa cacatgagcg 120

aaaccctata ggaaccctaa ttcccttatc tgggaactac tcacacatta ttatggagaa 180

actcg 185

<210> 12

<211> 24

<212> DNA

＜213〉artificial sequence

<220>

＜223〉upstream primer of gus gene intron

<400> 12

tctagagttg cctccctgct gcgg 24

<210> 13

<211> 26

<212> DNA

＜213〉artificial sequence

<220>

＜223〉downstream primer of gus gene intron

<400> 13

tctagacggt cagtggcagt gaaggg 26

<210> 14

<211> 1023

<212> DNA

<213> Plasmid pGreen-0229 Backbone

<400> 14

cattgttgcc tccctgctgc ggtttttcac cgaagttcat gccagtccag cgtttttgca 60

gcagaaaagc cgccgacttc ggtttgcggt cgcgagtgaa gatccctttc ttgttaccgc 120

caacgcgcaa tatgccttgc gaggtcgcaa aatcggcgaa attccatacc tgttcaccga 180

cgacggcgct gacgcgatca aagacgcggt gatacatatc cagccatgca cactgatact 240

cttcactcca catgtcggtg tacattgagt gcagcccggc taacgtatcc acgccgtatt 300

cggtgatgat aatcggctga tgcagtttct cctgccaggc cagaagttct ttttccagta 360

ccttctctgc cgtttccaaa tcgccgcttt ggacatacca tccgtaataa cggttcaggc 420

acagcacatc aaagagatcg ctgatggtat cggtgtgagc gtcgcagaac attacattga 480

cgcaggtgat cggacgcgtc gggtcgagtt tacgcgttgc ttccgccagt ggcgcgaaat 540

attcccgtgc accttgcgga cgggtatccg gttcgttggc aatactccac atcaccacgc 600

ttgggtggtt tttgtcacgc gctatcagct ctttaatcgc ctgtaagtgc gcttgctgag 660

tttccccgtt gactgcctct tcgctgtaca gttctttcgg cttgttgccc gcttcgaaac 720

caatgcctaa agagaggtta aagccgacag cagcagtttc atcaatcacc acgatgccat 780

gttcatctgc ccagtcgagc atctcttcag cgtaagggta atgcgaggta cggtaggagt 840

tggccccaat ccagtccatt aatgcgtggt cgtgcaccat cagcacgtta tcgaatcctt 900

tgccacgcaa gtccgcatct tcatgacgac caaagccagt aaagtagaac ggtttgtggt 960

taatcaggaa ctgttcgccc ttcactgcca ctgaccggat gccgacgcga agcgggtaga 1020

tat 1023

<210> 15

<211> 5870

<212> DNA

＜213〉artificial sequence

<220>

＜223〉zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD

<400> 15

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggc aaagggactg 420

gcaaaggggc ccactagagg gttctttgcc gagtgccagt tcaccagaca ctcggcaaat 480

aaacctcctt tgccgagtgt cgccgtgggc actcggcgaa ggatccgtca ccgtcacttg 540

gcgccgtgac ggtgactttt ctttgccgag tgccaaatgg cactcggcaa agtctttgcc 600

gagtgcccga caaaaagtac tcggcaaaga tgctgttgcc gatgtacagt tcgccgagcg 660

ttctttgccg agtgttacac tcggcaaagc atttgccgag tgtaaaatag cctttgccgt 720

gtgtctcaga cacacggcaa agaagctgat tccggtagtg tttggggggc gactaacaca 780

aatcgatgac ctttgatgaa gatggatgta tttctcccac aaaatcttgg atccatcctc 840

tactatgaaa agtcatgaat tcacaagacg ttttcgaaaa caatttcagc ttcacttgta 900

aagttgcttg tgcggctgaa gccaagaaaa cattagagga caaacaataa cttactatgc 960

ttttcgtgaa ctgtttcaga ccagcagtaa taattattgg tcctacatgc attaatggat 1020

aagcggctag catgccaaga cggagcatta gttaggagat aagacctagc gtttcattgc 1080

atccccatga aaggaatact atcactactg catgaataat tttcaaatgt attaaaaact 1140

gatattgaaa tgtaggtaat ctgtccgcct atgtgaaatg tttaacagag gcagccactc 1200

taggatgttg cctctattaa ttactcatag atgcagccat ccaactctag gatttcaatt 1260

agtctcaatc tatgcattcg tctatgcatt ctaggattac aattagtctc aatcttgtag 1320

tatttgttcc ttccttgtca aatcacttct catctaacta ctatgcttgt ttaaccatca 1380

gaacataact acaacaacat ccatttataa aggcttcgat agcaaacttt acatattcat 1440

atcatgttaa ggttgtcaca tgtgtaaagg tgaagagatc atgcatgtca ttccacgtag 1500

ataaaaagaa tgcctatata aaaatggcac attttcttgt aggtagtgga aagtatcttt 1560

ccagcaaaga ccatataatc cgataaagct gataactaaa tgtcgaaatc gagtagatgc 1620

catatcatct ataccttatc tgttgtttgg aaaaaaaaaa gacaaaatcc aaaaaaaaat 1680

ataagagatc tcacctatat aaatagctct gaaatcagta gttaatccat cacccataat 1740

attttgagca ttcagaaaca caccaagcga agcgcccggg ggcgagcgtc tacaacaacc 1800

aattgcccaa ttgcaacaac aatccttgcc acatctaaca atacaagcca tcacaacgca 1860

acagcaacaa cagttcctac cagcactgag ccacctagcc atggtgaacc ctgccgccta 1920

cttgcaagag cagctgcttg catccaaccc acttgctctg gcgaacgtag ttgcaaacca 1980

gcaacaacaa cagctacaac agtttctgcc aacgctcagt caactagcca tggtgaaccc 2040

tgccgcctac gtacaacaac aacaactgct ttcatctagc cctctcgttg tgtctagaat 2100

atctacccgc ttcgcgtcgg catccggtca gtggcagtga agggcgaaca gttcctgatt 2160

aaccacaaac cgttctactt tactggcttt ggtcgtcatg aagatgcgga cttgcgtggc 2220

aaaggattcg ataacgtgct gatggtgcac gaccacgcat taatggactg gattggggcc 2280

aactcctacc gtacctcgca ttacccttac gctgaagaga tgctcgactg ggcagatgaa 2340

catggcatcg tggtgattga tgaaactgct gctgtcggct ttaacctctc tttaggcatt 2400

ggtttcgaag cgggcaacaa gccgaaagaa ctgtacagcg aagaggcagt caacggggaa 2460

actcagcaag cgcacttaca ggcgattaaa gagctgatag cgcgtgacaa aaaccaccca 2520

agcgtggtga tgtggagtat tgccaacgaa ccggataccc gtccgcaagg tgcacgggaa 2580

tatttcgcgc cactggcgga agcaacgcgt aaactcgacc cgacgcgtcc gatcacctgc 2640

gtcaatgtaa tgttctgcga cgctcacacc gataccatca gcgatctctt tgatgtgctg 2700

tgcctgaacc gttattacgg atggtatgtc caaagcggcg atttggaaac ggcagagaag 2760

gtactggaaa aagaacttct ggcctggcag gagaaactgc atcagccgat tatcatcacc 2820

gaatacggcg tggatacgtt agccgggctg cactcaatgt acaccgacat gtggagtgaa 2880

gagtatcagt gtgcatggct ggatatgtat caccgcgtct ttgatcgcgt cagcgccgtc 2940

gtcggtgaac aggtatggaa tttcgccgat tttgcgacct cgcaaggcat attgcgcgtt 3000

ggcggtaaca agaaagggat cttcactcgc gaccgcaaac cgaagtcggc ggcttttctg 3060

ctgcaaaaac gctggactgg catgaacttc ggtgaaaaac cgcagcaggg aggcaacaat 3120

gtctagacac aacgagaggg ctagatgaaa gcagttgttg ttgttgtacg taggcggcag 3180

ggttcaccat ggctagttga ctgagcgttg gcagaaactg ttgtagctgt tgttgttgct 3240

ggtttgcaac tacgttcgcc agagcaagtg ggttggatgc aagcagctgc tcttgcaagt 3300

aggcggcagg gttcaccatg gctaggtggc tcagtgctgg taggaactgt tgttgctgtt 3360

gcgttgtgat ggcttgtatt gttagatgtg gcaaggattg ttgttgcaat tgggcaattg 3420

gttgttgtag acgctcgccc tcgagcgagt ttctccataa taatgtgtga gtagttccca 3480

gataagggaa ttagggttcc tatagggttt cgctcatgtg ttgagcatat aagaaaccct 3540

tagtatgtat ttgtatttgt aaaatacttc tatcaataaa atttctaatt cctaaaacca 3600

aaatccagta ctaaaatcca gatcccccga aagcttggcg taatcatggt catagctgtt 3660

tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 3720

gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 3780

gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 3840

ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 3900

ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 3960

cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 4020

gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 4080

tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 4140

ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 4200

atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag 4260

gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 4320

tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 4380

cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 4440

cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt 4500

tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 4560

cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 4620

cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 4680

gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 4740

gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 4800

gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 4860

ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 4920

atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 4980

agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 5040

ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 5100

tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 5160

ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 5220

caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 5280

gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 5340

atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 5400

accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 5460

aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 5520

gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 5580

tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 5640

aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 5700

ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 5760

aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat 5820

tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 5870

<210> 16

<211> 29

<212> DNA

＜213〉artificial sequence

<220>

＜223〉upstream primer of the R19-4 primer of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

<400> 16

cccgggccat ccaagcaagc atcttacgg 29

<210> 17

<211> 26

<212> DNA

＜213〉artificial sequence

<220>

＜223〉downstream primer of the R19-4 primer of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

<400> 17

tctagaggca taagggctcc cgacaa 26

<210> 18

<211> 39

<212> DNA

＜213〉artificial sequence

<220>

＜223〉upstream primer of the R19-5 primer of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

<400> 18

aagcttctag ctcgagccat ccaagcaagc atcttacgg 39

<210> 19

<211> 26

<212> DNA

＜213〉artificial sequence

<220>

＜223〉downstream primer of the R19-5 primer of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

<400> 19

tctagaggca taagggctcc cgacaa 26

<210> 20

<211> 286

<212> DNA

＜213〉corn (Zea mays L.)

<400> 20

ccatccaagc aagcatttac ggtcattagc attaaccctc caacaaccat atgctctatt 60

gcaacagcca tccttagtgc atctgtatct ccaaagaatc gcggcacaac aactacaaca 120

acagttgcta ccaacaatca atcaagtagt tgcagcgaac cttgctgctt acctccagca 180

acaacagttt cttccattca atcaactagc tggggtgaac cctgctatct acttgcaggc 240

acaacagcta ctaccattta accaacttgt cgggagccct tatgcc 286

<210> 21

<211> 5819

<212> DNA

＜213〉artificial sequence

<220>

＜223〉zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD

<400> 21

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggc aaagggactg 420

gcaaaggggc ccactagagg gttctttgcc gagtgccagt tcaccagaca ctcggcaaat 480

aaacctcctt tgccgagtgt cgccgtgggc actcggcgaa ggatccgtca ccgtcacttg 540

gcgccgtgac ggtgactttt ctttgccgag tgccaaatgg cactcggcaa agtctttgcc 600

gagtgcccga caaaaagtac tcggcaaaga tgctgttgcc gatgtacagt tcgccgagcg 660

ttctttgccg agtgttacac tcggcaaagc atttgccgag tgtaaaatag cctttgccgt 720

gtgtctcaga cacacggcaa agaagctgat tccggtagtg tttggggggc gactaacaca 780

aatcgatgac ctttgatgaa gatggatgta tttctcccac aaaatcttgg atccatcctc 840

tactatgaaa agtcatgaat tcacaagacg ttttcgaaaa caatttcagc ttcacttgta 900

aagttgcttg tgcggctgaa gccaagaaaa cattagagga caaacaataa cttactatgc 960

ttttcgtgaa ctgtttcaga ccagcagtaa taattattgg tcctacatgc attaatggat 1020

aagcggctag catgccaaga cggagcatta gttaggagat aagacctagc gtttcattgc 1080

atccccatga aaggaatact atcactactg catgaataat tttcaaatgt attaaaaact 1140

gatattgaaa tgtaggtaat ctgtccgcct atgtgaaatg tttaacagag gcagccactc 1200

taggatgttg cctctattaa ttactcatag atgcagccat ccaactctag gatttcaatt 1260

agtctcaatc tatgcattcg tctatgcatt ctaggattac aattagtctc aatcttgtag 1320

tatttgttcc ttccttgtca aatcacttct catctaacta ctatgcttgt ttaaccatca 1380

gaacataact acaacaacat ccatttataa aggcttcgat agcaaacttt acatattcat 1440

atcatgttaa ggttgtcaca tgtgtaaagg tgaagagatc atgcatgtca ttccacgtag 1500

ataaaaagaa tgcctatata aaaatggcac attttcttgt aggtagtgga aagtatcttt 1560

ccagcaaaga ccatataatc cgataaagct gataactaaa tgtcgaaatc gagtagatgc 1620

catatcatct ataccttatc tgttgtttgg aaaaaaaaaa gacaaaatcc aaaaaaaaat 1680

ataagagatc tcacctatat aaatagctct gaaatcagta gttaatccat cacccataat 1740

attttgagca ttcagaaaca caccaagcga agcgcccggg ccatccaagc aagcatttac 1800

ggtcattagc attaaccctc caacaaccat atgctctatt gcaacagcca tccttagtgc 1860

atctgtatct ccaaagaatc gcggcacaac aactacaaca acagttgcta ccaacaatca 1920

atcaagtagt tgcagcgaac cttgctgctt acctccagca acaacagttt cttccattca 1980

atcaactagc tggggtgaac cctgctatct acttgcaggc acaacagcta ctaccattta 2040

accaacttgt cgggagccct tatgcctcta gaatatctac ccgcttcgcg tcggcatccg 2100

gtcagtggca gtgaagggcg aacagttcct gattaaccac aaaccgttct actttactgg 2160

ctttggtcgt catgaagatg cggacttgcg tggcaaagga ttcgataacg tgctgatggt 2220

gcacgaccac gcattaatgg actggattgg ggccaactcc taccgtacct cgcattaccc 2280

ttacgctgaa gagatgctcg actgggcaga tgaacatggc atcgtggtga ttgatgaaac 2340

tgctgctgtc ggctttaacc tctctttagg cattggtttc gaagcgggca acaagccgaa 2400

agaactgtac agcgaagagg cagtcaacgg ggaaactcag caagcgcact tacaggcgat 2460

taaagagctg atagcgcgtg acaaaaacca cccaagcgtg gtgatgtgga gtattgccaa 2520

cgaaccggat acccgtccgc aaggtgcacg ggaatatttc gcgccactgg cggaagcaac 2580

gcgtaaactc gacccgacgc gtccgatcac ctgcgtcaat gtaatgttct gcgacgctca 2640

caccgatacc atcagcgatc tctttgatgt gctgtgcctg aaccgttatt acggatggta 2700

tgtccaaagc ggcgatttgg aaacggcaga gaaggtactg gaaaaagaac ttctggcctg 2760

gcaggagaaa ctgcatcagc cgattatcat caccgaatac ggcgtggata cgttagccgg 2820

gctgcactca atgtacaccg acatgtggag tgaagagtat cagtgtgcat ggctggatat 2880

gtatcaccgc gtctttgatc gcgtcagcgc cgtcgtcggt gaacaggtat ggaatttcgc 2940

cgattttgcg acctcgcaag gcatattgcg cgttggcggt aacaagaaag ggatcttcac 3000

tcgcgaccgc aaaccgaagt cggcggcttt tctgctgcaa aaacgctgga ctggcatgaa 3060

cttcggtgaa aaaccgcagc agggaggcaa caatgtctag aggcataagg gctcccgaca 3120

agttggttaa atggtagtag ctgttgtgcc tgcaagtaga tagcagggtt caccccagct 3180

agttgattga atggaagaaa ctgttgttgc tggaggtaag cagcaaggtt cgctgcaact 3240

acttgattga ttgttggtag caactgttgt tgtagttgtt gtgccgcgat tctttggaga 3300

tacagatgca ctaaggatgg ctgttgcaat agagcatatg gttgttggag ggttaatgct 3360

aatgaccgta agatgcttgc ttggatggct cgagcgagtt tctccataat aatgtgtgag 3420

tagttcccag ataagggaat tagggttcct atagggtttc gctcatgtgt tgagcatata 3480

agaaaccctt agtatgtatt tgtatttgta aaatacttct atcaataaaa tttctaattc 3540

ctaaaaccaa aatccagtac taaaatccag atcccccgaa agcttggcgt aatcatggtc 3600

atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 3660

aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 3720

gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 3780

ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 3840

ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 3900

acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 3960

aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 4020

tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 4080

aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 4140

gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 4200

acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 4260

accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 4320

ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 4380

gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 4440

aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 4500

ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 4560

gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 4620

cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 4680

cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 4740

gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 4800

tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 4860

gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 4920

agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 4980

tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 5040

agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 5100

gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 5160

catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 5220

ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 5280

atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 5340

tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 5400

cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 5460

cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 5520

atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 5580

aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 5640

ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 5700

aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga 5760

aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc 5819

<210> 22

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉RNAi carrier pUC19-RNAi-22KD transforms T0 detects the 22Y3 primer for the PCR of regeneration plant upstream primer

<400> 22

caagccatca caacgcaac 19

<210> 23

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉RNAi carrier pUC19-RNAi-22KD transforms T0 detects the 22Y3 primer for the PCR of regeneration plant downstream primer

<400> 23

cgtatccacg ccgtattcg 19

<210> 24

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉RNAi carrier pUC19-RNAi-19KD transforms T0 detects the 19Y5 primer for the PCR of regeneration plant upstream primer

<400> 24

cagcgaagag gcagtcaac 19

<210> 25

<211> 19

<212> DNA

＜213〉artificial sequence

<220>

＜223〉RNAi carrier pUC19-RNAi-19KD transforms T0 detects the 19Y5 primer for the PCR of regeneration plant downstream primer

<400> 25

cgaaccttgc tgcttacct 19

Claims (4)

1. method that improves the corn lysine content with zein spirit-soluble gene RNAi carrier, comprise with particle bombardment zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD is imported the acceptor corn variety, described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD contains endosperm specific expression promoter P-zp22/6 at skeleton carrier pUC19, is positioned between the restriction enzyme site Sac I and Sma I of RNAi carrier pUC19-RNAi-22KD; Forward goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P is positioned between the restriction enzyme site Sma I and Xba I of RNAi carrier pUC19-RNAi-22KD; Oppositely goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P is positioned between the restriction enzyme site Xba I and Xho I of RNAi carrier pUC19-RNAi-22KD; In the middle of forward goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P and reverse goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P, contain the gus gene intron, be positioned between the Xba I restriction enzyme site of RNAi carrier pUC19-RNAi-22KD; Terminator poly (A) is positioned between RNAi carrier pUC19-RNAi-22KD restriction enzyme site Hind III and Xho I; The nucleotide sequence of described endosperm specific expression promoter P-zp22/6 is shown in SEQ ID NO:3, the nucleotide sequence of described 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P is shown in SEQ ID NO:8, the nucleotide sequence of described gus gene intron is shown in SEQ ID NO:14, the nucleotide sequence of described terminator poly (A) is shown in SEQ ID NO:11, and the nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD is shown in SEQ ID NO:15.

2. usefulness zein spirit-soluble gene RNAi carrier according to claim 1 improves the method for corn lysine content, and it is characterized in that: described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD makes up acquisition by the following method:

(1) makes up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6

1. the acquisition of endosperm specificity expression promoter P-zp22/6

Take the genomic dna of corn variety as template, take sequence shown in SEQ ID NO:1 and the SEQ ID NO:2 as primer pair, carry out pcr amplification, obtain the endosperm specificity expression promoter P-zp22/6 of sequence shown in SEQ ID NO:3;

2. make up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6 sequence

Cut the endosperm specificity expression promoter with corresponding restriction enzyme site that step (1) 1. obtains with Sac I and Xba I enzyme, then be connected with the skeleton carrier pUC19 that is connected with Xba I enzyme through the Sac I equally, obtain the recombinant vectors pUC19-p22/6 with endosperm specificity expression promoter P-zp22/6;

(2) make up the recombinant vectors pUC19-p22/6-22KD1 that inserts forward purpose fragment 22-KD-P and endosperm specificity expression promoter P-zp22/6

1. the acquisition of 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P

Take the total RNA of corn kernel as template, with sequence shown in SEQ ID NO:4 and the SEQ ID NO:5, carry out the RT-PCR amplification, then take this RT-PCR product as template, take sequence shown in SEQ ID NO:6 and the SEQ ID NO:7 as primer pair, carry out again RT-PCR, obtain the 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P shown in SEQ ID NO:8;

2. make up the recombinant vectors pUC19-p22/6-22KD1 that inserts forward purpose fragment 22-KD-P and endosperm specificity expression promoter P-zp22/6

1. the purpose fragment 22-KD-P with corresponding restriction enzyme site that obtains with Sma I and Xba I double digestion step (2), then with equally be connected the recombinant vectors pUC19-p22/6 that step (1) 2. makes up with Xba I double digestion through the Sma I and be connected, obtained inserting the recombinant vectors pUC19-p22/6-22KD1 of forward goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P and endosperm specificity expression promoter P-zp22/6;

(3) make up the recombinant vectors pUC19-22KD2 that inserts reverse purpose fragment 22-KD-P

Cut the purpose fragment 22-KD-P with corresponding restriction enzyme site that step (2) 1. obtains with Hind III and Xba1 enzyme, then be connected with the pUC19 carrier of being connected with Xba I enzyme through the Hind III equally, obtain inserting the recombinant vectors pUC19-22KD2 of reverse goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P;

(4) make up the recombinant vectors pUC19-22KD2-poly (A) that contains terminator poly (A)

Take expression vector p3301 DNA as template, carry out pcr amplification with the terminator primer of sequence shown in SEQ ID NO:9 and the SEQ ID NO:10, obtain the terminator poly (A) of sequence shown in SEQ ID NO:11; Further use Xho I and Hind III double digestion with the terminator poly (A) of corresponding restriction enzyme site, then with equally be connected the recombinant vectors pUC19-22KD2 that step (3) obtains with Hind III double digestion through the Xho I and be connected, obtain containing the recombinant vectors pUC19-22KD2-poly (A) of terminator poly (A);

(5) structure contains endosperm specificity expression promoter P-zp22/6, the recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) of forward and reverse purpose fragment 22-KD-P and terminator poly (A)

The recombinant vectors pUC19-p22/6-22KD1 of step (2) structure and the recombinant vectors pUC19-22KD2-poly (A) of step (4) structure are used respectively Xba I and Hind III double digestion, the enzyme that then will contain reverse purpose fragment 22-KD-P and terminator poly (A) is cut product 22KD2-poly (A) and is connected on the recombinant vectors pUC19-p22/6-22KD1 that step (2) makes up, obtain containing endosperm specificity expression promoter P-zp22/6, just, the recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) of reverse goal gene 22-KD alcohol soluble protein gene Partial cDNA fragment 22-KD-P and terminator poly (A);

(6) structure of the connection of gus gene intron and zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD

Take plasmid vector pGreen-0229 Backbone DNA as template, carry out pcr amplification with the primer of the gus gene intron of sequence shown in SEQ ID NO:12 and the SEQ ID NO:13, obtain the gus gene intron of sequence shown in SEQ ID NO:14; Further use Xba I single endonuclease digestion with the gus gene intron of corresponding restriction enzyme site, then be connected with the same recombinant vectors pUC19-p22/6-22KD1-22KD2-poly (A) that the step (5) of phosphatizing treatment makes up behind Xba I single endonuclease digestion, obtain described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD, the nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-22KD is shown in SEQ ID NO:15.

3. method that improves the corn lysine content with zein spirit-soluble gene RNAi carrier, comprise with particle bombardment zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD is imported the acceptor corn variety, described zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD contains endosperm specific expression promoter P-zp22/6 at skeleton carrier pUC19, is positioned between the restriction enzyme site Sac I and Sma I of RNAi carrier pUC19-RNAi-19KD; Forward goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P is positioned between the restriction enzyme site Sma I and Xba I of RNAi carrier pUC19-RNAi-19KD; Oppositely goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P is positioned between the restriction enzyme site Xba I and Xho I of RNAi carrier pUC19-RNAi-19KD; Between forward goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P and reverse goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P, contain the gus gene intron, be positioned between the Xba I restriction enzyme site of RNAi carrier pUC19-RNAi-19KD; Terminator poly (A) is positioned between RNAi carrier pUC19-RNAi-19KD restriction enzyme site Hind III and Xho I; The nucleotide sequence of described endosperm specific expression promoter P-zp22/6 is shown in SEQ ID NO:3, the nucleotide sequence of described 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P is shown in SEQ ID NO:20, the nucleotide sequence of described gus gene intron is shown in SEQ ID NO:14, the nucleotide sequence of described terminator poly (A) is shown in SEQ ID NO:11, and the nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD is shown in SEQ ID NO:21.

4. usefulness zein spirit-soluble gene RNAi carrier according to claim 3 improves the method for corn lysine content, and it is characterized in that: described zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD makes up acquisition by the following method:

(1) makes up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6

1. the acquisition of endosperm specificity expression promoter P-zp22/6

Take the genomic dna of corn variety as template, take sequence shown in SEQ ID NO:1 and the SEQ ID NO:2 as primer pair, carry out pcr amplification, obtain the endosperm specificity expression promoter P-zp22/6 of sequence shown in SEQ ID NO:3;

2. make up the recombinant vectors pUC19-p22/6 that contains endosperm specificity expression promoter P-zp22/6 sequence

Cut the endosperm specificity expression promoter with corresponding restriction enzyme site that step (1) 1. obtains with Sac I and Xba I enzyme, then be connected with the skeleton carrier pUC19 that is connected with Xba I enzyme through the Sac I equally, obtain the recombinant vectors pUC19-p22/6 with endosperm specificity expression promoter P-zp22/6;

(2) make up the recombinant vectors pUC19-p22/6-19KD1 that inserts forward purpose fragment 19-KD-P and endosperm specificity expression promoter P-zp22/6

1. the acquisition of 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P

Take the total RNA of corn kernel as template, with sequence shown in SEQ ID NO:16 and the SEQ ID NO:17, carry out the RT-PCR amplification, then take this RT-PCR product as template, take sequence shown in SEQ ID NO:18 and the SEQ ID NO:19 as primer pair, carry out again RT-PCR, obtain the 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P shown in SEQ ID NO:20;

2. make up the recombinant vectors pUC19-p22/6-19KD1 that inserts forward purpose fragment 19-KD-P and endosperm specificity expression promoter P-zp22/6

1. the purpose fragment 19-KD-P with corresponding restriction enzyme site that obtains with Sma I and Xba I double digestion step (2), then with equally be connected the recombinant vectors pUC19-p22/6 that step (1) 2. makes up with Xba I double digestion through the Sma I and be connected, obtained inserting the recombinant vectors pUC19-p22/6-19KD1 of forward goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P and endosperm specificity expression promoter P-zp22/6;

(3) make up the recombinant vectors pUC19-19KD2 that inserts reverse purpose fragment 19-KD-P

Cut the purpose fragment 19-KD-P with corresponding restriction enzyme site that step (2) 1. obtains with Hind III and Xba I enzyme, then be connected with the pUC19 carrier of being connected with Xba I enzyme through the Hind III equally, obtain inserting the recombinant vectors pUC19-19KD2 of reverse goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P;

(4) make up the recombinant vectors pUC19-19KD2-poly (A) that contains terminator poly (A)

Take expression vector p3301 DNA as template, carry out pcr amplification with the terminator primer of sequence shown in SEQ ID NO:9 and the SEQ ID NO:10, obtain the terminator poly (A) of sequence shown in SEQ ID NO:11; Further use Xho I and Hind III double digestion with the terminator poly (A) of corresponding restriction enzyme site, then with equally be connected the recombinant vectors pUC19-19KD2 that step (3) obtains with Hind III double digestion through the Xho I and be connected, obtain containing the recombinant vectors pUC19-19KD2-poly (A) of terminator poly (A);

(5) structure contains endosperm specificity expression promoter P-zp22/6, the recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) of forward and reverse purpose fragment 19-KD-P and terminator poly (A)

The recombinant vectors pUC19-p22/6-22KD1 of step (2) structure and the recombinant vectors pUC19-19KD2-poly (A) of step (4) structure are used respectively Xba I and Hind III double digestion, the enzyme that then will contain reverse purpose fragment 19-KD-P and terminator poly (A) is cut product 19KD2-poly (A) and is connected on the recombinant vectors pUC19-p22/6-19KD1 that step (2) makes up, obtain containing endosperm specificity expression promoter P-zp22/6, just, the recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) of reverse goal gene 19-KD alcohol soluble protein gene Partial cDNA fragment 19-KD-P and terminator poly (A);

(6) structure of the connection of gus gene intron and zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD

Take plasmid vector pGreen-0229 Backbone DNA as template, carry out pcr amplification with the primer of the gus gene intron of sequence shown in SEQ ID NO:12 and the SEQ ID NO:13, obtain the gus gene intron of sequence shown in SEQ ID NO:14; Further use Xba I single endonuclease digestion with the gus gene intron of corresponding restriction enzyme site, then be connected with the same recombinant vectors pUC19-p22/6-19KD1-19KD2-poly (A) that the step (5) of phosphatizing treatment makes up behind Xba I single endonuclease digestion, obtain described zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD, the nucleotide sequence of described zein spirit-soluble gene RNAi carrier pUC19-RNAi-19KD is shown in SEQ ID NO:21.

Images