CN107325161A - A kind of albumen related with high-salt stress to resistance to Low nitrogen stress and its encoding gene and application - Google Patents

Abstract

The invention discloses a protein related to resistance to low nitrogen stress and high salt stress, its coding gene and application. The protein provided by the present invention is a protein according to the following a) or b) or c): a) the amino acid sequence is the protein shown in sequence 1; b) a tag is attached to the N-terminal and/or C-terminal of the protein shown in sequence 1 The resulting fusion protein; c) a protein with the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 1. Experiments have proved that the ZmCCT gene provided by the present invention can improve the ratio of low-nitrogen stress and high-salt stress-resistant plants in the offspring of transgenic ZmCCT gene plants. Fusarium stem rot resistance has been greatly improved.

Description

A protein related to tolerance to low nitrogen stress and high salt stress, its coding gene and application

technical field

The invention belongs to the field of biotechnology, and specifically relates to a protein related to tolerance to low nitrogen stress and high salt stress, its coding gene and application.

Background technique

Poor soil is a major factor affecting the instability of maize production worldwide. Of the 1.4 billion hectares of arable soil in the world, 22.5% of the soil is under severe nutrient stress, and only about 10.0% of the soil has no nutrient stress or mild stress. In maize production, applying nitrogen fertilizer is one of the important measures to increase maize yield, but there are two problems in production practice. First, the excessive application of nitrogen fertilizer in high-yield developed areas not only leads to a decrease in nitrogen fertilizer use efficiency and increased production costs, but also may cause groundwater nitrate to exceed the standard; second, low-yield underdeveloped areas often have low yield levels due to insufficient nitrogen fertilizer application. In view of the further reduction in the space for nitrogen fertilizer application to increase yield, and the many quality and environmental problems caused by nitrogen fertilizer application, breeding nitrogen-efficient maize varieties through genetics and other means is the fundamental solution to food production safety and environmental protection.

Moll et al. (Moll R H, Kamprath E J, Jackson W A. Analysis and interpretation of factors which contribute to efficiency of nitrogen utilization [J]. Agronomy Journal, 1982, 74 (3): 562-564.) made a classic study on nitrogen efficiency The definition of , that is, the crop yield under the condition of unit nitrogen supply. Nitrogen efficiency can be divided into two parts, one is the efficiency of plant roots absorbing nitrogen from the soil, and the other is the efficiency of nitrogen assimilation and utilization in plants. Moll et al. believe that nitrogen use efficiency plays a major role under low nitrogen conditions, while nitrogen uptake efficiency plays a major role under high nitrogen conditions. Ortiz-Monasterio et al. (Ortiz-Monasterio R, Sayre K D, Rajaram S, et al. Genetic progress in wheat yield and nitrogen use efficiency under four nitrogen rates [J]. Crop Science, 1997,37(3):898-904. ) believed that the difference in nitrogen efficiency under low nitrogen conditions was mainly due to absorption efficiency, while utilization efficiency played a major role under high nitrogen conditions. Lafitte and Edmeades (Lafitte H R, Edmeades G O. Improvement for tolerance to low soil nitrogen in tropical maize I. Selection criteria [J]. Field Crops Research, 1994, 39 (1): 1-14.) think that under low nitrogen conditions Nitrogen use efficiency correlates better with yield. Mi Guohua et al. (Mi Guohua, Liu Jianan. Physiological and biochemical basis of nitrogen efficiency in maize and progress in genetic improvement[J]. Maize Science, 1997,5(2):9-13.) The results showed that under high nitrogen conditions, the absorption and utilization of Efficiency is equal to both. And Mi Guohua et al. believed that the different research results that caused the relative importance of nitrogen uptake efficiency and nitrogen use efficiency may be caused by different genotypes in different environments.

Nitrogen efficiency in maize is a complex process. At present, the physiological mechanism of efficient nitrogen absorption in maize includes: (1) good root system architecture (morphology and spatial distribution) and root system characteristics; (2) good physiological and metabolic activities of the root system (respiration, etc.); (3) the aboveground part has Excellent traits; (4) The single cycle in the seedling stage of the plant promotes the absorption of nitrogen by the root system. The physiological mechanism of nitrogen utilization in maize includes: (1) highly active nitrogen metabolism key enzymes; (2) interaction between nutrients and the influence of some substances; (3) feedback of storage capacity; (4) The full utilization of NO3- stored in vacuoles and the reduction of aboveground nitrogen volatilization; (5) The retransfer ability of nitrogen to grain is strong. Most of the important agronomic traits of crops such as yield, quality and stress resistance are quantitative traits controlled by multiple quantitative trait loci (quantitative trait loci, QTL). At present, many research results show that the nitrogen efficiency of crops is also controlled by multiple quantitative genetic loci.

According to statistics from the second national soil census, on the premise of excluding coastal beaches, the area of saline soil in my country is 34.87 million hectares, which is about 500 million mu, and the area that can be developed and utilized reaches 200 million mu. Improving the salt-alkali tolerance of crops is of great significance for developing marginal land and increasing grain production. At present, cultivated corn requires a lot of water in the field, and its adaptability to salinity is relatively low. The seedling stage is more sensitive to salt stress, and its limit salt concentration (referring to the salt concentration at which plant growth is inhibited and yield decline) is only about 1.7dsm-1, about 0.1% NaCl, genetically speaking, the salt tolerance of plants is Quantitative traits controlled by multiple genes have complex genetic mechanisms and are easily affected by environmental conditions. Locating and cloning salt tolerance is of great significance for elucidating the mechanism of plant salt tolerance and breeding applications. At present, significant progress has been made in the research on QTL of salt tolerance in crops, such as wheat, barley, soybean, rice, etc., among which rice is the most systematic research. However, research on QTLs for salt tolerance in maize has progressed slowly.

Contents of the invention

The technical problem to be solved by the invention is how to regulate the stress resistance of plants.

In order to solve the above technical problems, the present invention firstly provides a protein related to plant stress resistance.

The name of the protein related to plant stress resistance provided by the present invention is ZmCCT, which is the protein of a) or b) or c) as follows:

a) the amino acid sequence is the protein shown in Sequence 1;

b) a fusion protein obtained by connecting a tag to the N-terminal and/or C-terminal of the protein shown in Sequence 1;

c) A protein with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in Sequence 1.

Among them, sequence 1 consists of 238 amino acid residues.

In order to make the protein in a) easy to purify, the amino terminus or carboxy terminus of the protein shown in Sequence 1 in the Sequence Listing can be linked with the tags shown in Table 1.

Table 1. Sequence of tags

For the protein in c) above, the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues.

The protein in the above c) can be synthesized artificially, or its coding gene can be synthesized first, and then obtained by biological expression.

The gene encoding the protein in the above c) can be deleted by deleting one or several amino acid residue codons in the DNA sequence shown in Sequence 2, and/or performing missense mutations of one or several base pairs, and/ Or it can be obtained by connecting the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end.

In order to solve the above-mentioned technical problems, another object of the present invention is to provide biological materials related to the above-mentioned proteins.

The biological material related to the above protein provided by the present invention is any one of the following A1) to A12):

A1) a nucleic acid molecule encoding the above-mentioned protein;

A2) an expression cassette containing the nucleic acid molecule of A1);

A3) a recombinant vector containing the nucleic acid molecule of A1);

A4) a recombinant vector containing the expression cassette described in A2);

A5) a recombinant microorganism containing the nucleic acid molecule of A1);

A6) a recombinant microorganism containing the expression cassette described in A2);

A7) A recombinant microorganism containing the recombinant vector described in A3);

A8) a recombinant microorganism containing the recombinant vector described in A4);

A9) a transgenic plant cell line containing the nucleic acid molecule of A1);

A10) a transgenic plant cell line containing the expression cassette described in A2);

A11) a transgenic plant cell line containing the recombinant vector described in A3);

A12) A transgenic plant cell line containing the recombinant vector described in A4).

In the above-mentioned related biological materials, the nucleic acid molecule described in A1) is the gene shown in 1) or 2) or 3) as follows:

1) its coding sequence is the cDNA molecule shown in sequence 2 or the genomic DNA molecule shown in sequence 3;

2) A cDNA molecule or a genomic DNA molecule that has 75% or more identity to the nucleotide sequence defined in 1) and encodes the protein of claim 1;

3) A cDNA molecule or a genomic DNA molecule that hybridizes to the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the protein of claim 1.

Wherein, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

Among them, sequence 2 consists of 717 nucleotides, encoding the amino acid sequence shown in sequence 1.

Those of ordinary skill in the art can easily adopt known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding ZmCCT of the present invention. Those artificially modified nucleotides with 75% or higher identity to the nucleotide sequence of ZmCCT isolated in the present invention, as long as they encode ZmCCT and have the same function, are all derived from the nucleotide sequence of the present invention And is equivalent to the sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher, of the nucleotide sequence of the protein composed of the amino acid sequence shown in the coding sequence 1 of the present invention. Nucleotide sequences of higher identity. Identity can be assessed visually or with computer software. Using computer software, identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

The identity of 75% or more may be 80%, 85%, 90% or more.

In the above-mentioned biological material, the stringent condition is in a solution of 2×SSC, 0.1% SDS, hybridize at 68° C. and wash the membrane twice, each time for 5 minutes, and then in a solution of 0.5×SSC, 0.1% SDS, Hybridize and wash the membrane twice at 68°C, 15 min each time; or, hybridize and wash the membrane at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

Among the above-mentioned biological materials, the expression cassette (ZmCCT gene expression cassette) described in A2) containing a nucleic acid molecule encoding ZmCCT refers to a DNA capable of expressing ZmCCT in a host cell, and the DNA may not only include a promoter that initiates ZmCCT transcription, A terminator that terminates transcription of the ZmCCT may also be included. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to: constitutive promoters; tissue, organ and development specific promoters and inducible promoters. Examples of promoters include, but are not limited to: Cauliflower Mosaic Virus Constitutive Promoter 35S: Wound-Inducible Promoter from Tomato, Leucine Aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato Protease inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoter (US Patent 5,187,267); tetracycline-inducible promoter (US Patent 5,057,422) ; Seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (for example, the promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in their entirety. Suitable transcription terminators include, but are not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine Synthase terminators (see, e.g.: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; ^Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) ) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acids Res., 15:9627).

The existing expression vector can be used to construct the recombinant vector containing the ZmCCT gene expression cassette. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector may also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopaline synthase gene Nos), plant gene (such as soybean The untranslated region transcribed at the 3′ end of the storage protein gene) has similar functions. When using the gene of the present invention to construct plant expression vectors, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG initiation codons or adjacent region initiation codons, etc. The reading frames of the sequences are identical to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) genes, etc.), antibiotic marker genes (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, and the hph gene that confers resistance to the antibiotic hygromycin , and the dhfr gene that confers resistance to methotrexate, the EPSPS gene that confers resistance to glyphosate) or the chemical resistance marker gene (such as the herbicide resistance gene), the mannose-6- that provides the ability to metabolize mannose Phosphate isomerase gene. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

In the above biological materials, the vector can be a plasmid, a cosmid, a phage or a viral vector.

In the above biological materials, the microorganisms can be yeast, bacteria, algae or fungi, such as Agrobacterium.

Among the above biological materials, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs do not include propagation materials.

In order to solve the above-mentioned technical problems, the present invention also provides a new application of the above-mentioned protein or the above-mentioned related biological material.

The present invention provides the application of the above-mentioned protein or the above-mentioned related biological material in at least one of the following (1)-(6):

(1) Regulating plant resistance to Fusarium graminearum stem rot;

(2) Regulating plant stress resistance;

(3) Regulating plant total root length and/or trunk root length and/or main radicle length and/or lateral root length and/or underground dry weight and/or aboveground dry weight and/or plant height;

(4) Regulate the expression level of genes related to plant resistance;

(5) Cultivate transgenic plants resistant to Fusarium graminearum stem rot;

(6) Breeding transgenic plants with improved stress resistance.

In the above application, the regulation is to improve; the stress resistance is low nitrogen resistance and/or salt resistance; the low nitrogen is specifically 0.04mmolL ^-1 NO ₃ ^- ; the high salt is specifically 50mmolL ^-1 NaCl; the regulation of plant stress resistance is specifically under the condition of 0.04mmolL ^-1 NO ₃ ^- and/or 50mmolL ^-1 NaCl, the total root length of the ZmCCT plant is higher than that of the recipient plant and/or the ZmCCT plant The length of the trunk root becomes longer and/or the main radicle length of the ZmCCT plant becomes longer and/or the lateral root length of the ZmCCT plant becomes longer and/or the dry weight of the underground part of the ZmCCT plant increases and/or the shoot of the ZmCCT plant The dry weight is increased and/or the plant height of the ZmCCT-transformed plants is increased and/or the expression level of resistance-related genes of the ZmCCT-transferred plants is increased.

In the above method, the resistance-related genes are ZmABA2 and ZmMPK5.

In order to solve the above technical problems, the present invention also provides a method for cultivating transgenic plants with improved stress resistance.

The method for cultivating transgenic plants with improved stress resistance provided by the present invention includes the step of introducing the coding gene of the above protein into a recipient plant to obtain a transgenic plant; the stress resistance of the transgenic plant is higher than that of the recipient plant.

In the above method,

The stress resistance is low nitrogen resistance and/or salt resistance;

The stress resistance of the transgenic plant is higher than that of the recipient plant in any of the following (D1)-(D7):

(D1) the total root length of the transgenic plant is higher than that of the recipient plant;

(D2) the trunk root length of the transgenic plant is higher than that of the recipient plant;

(D3) The main radicle length of the transgenic plant is higher than that of the recipient plant;

(D4) the lateral root length of the transgenic plant is higher than that of the recipient plant;

(D5) The underground dry weight of the transgenic plant is higher than that of the recipient plant;

(D6) The above-ground dry weight of the transgenic plant is higher than that of the recipient plant;

(D7) The plant height of the transgenic plant is higher than the recipient plant;

(D8) The expression level of resistance-related genes in the transgenic plants is higher than that of the recipient plant; the resistance-related genes are specifically ZmABA2 and ZmMPK5.

The invention also provides a method for cultivating transgenic plants resistant to Fusarium graminearum stem rot.

The method for breeding the transgenic plant resistant to Fusarium graminearum stem rot provided by the invention comprises the step of introducing the coding gene of the above-mentioned protein into a recipient plant to obtain a transgenic plant; the transgenic plant is resistant to Fusarium graminearum stem rot The resistance was higher than that of the recipient plants.

In the above method,

The nucleotide sequence of the protein coding gene is the 1-717th nucleotide molecule of sequence 2.

In an embodiment of the present invention, the gene encoding the protein (ie, the DNA molecule shown in Sequence 3 in the Sequence Listing) is introduced into the recipient plant through a ZmCCT gene recombinant expression vector containing a ZmCCT gene expression cassette. The ZmCCT gene recombinant expression vector containing the ZmCCT gene expression cassette is the recombinant expression vector pCAMBIA3301-ZmCCT; the pCAMBIA3301-ZmCCT is the DNA shown in sequence 3 in the Sac I restriction site of the pCAMBIA3301 vector, which is forward inserted molecule, and keep the other sequences of the pCAMBIA3301 vector unchanged to obtain the vector.

In the above method, the transgenic plant is understood to include not only the first-generation transgenic plant obtained by transforming the target plant with the ZmCCT gene, but also its progeny. For transgenic plants, the gene can be propagated in that species, or transferred into other varieties of the same species, particularly including commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

In the above method, the recipient plant is a monocotyledonous plant or a dicotyledonous plant.

In the above method, the recipient plant is a monocotyledonous plant, a Gramineae plant, and corn, specifically a corn (Zea mays L.) variety HiII.

The primer pair for amplifying the full length of the ZmCCT gene or any fragment thereof also belongs to the protection scope of the present invention.

Experiments have proved that the ZmCCT gene provided by the present invention can improve the ratio of low-nitrogen stress and high-salt stress-resistant plants in the offspring of transgenic ZmCCT gene plants. Fusarium stem rot resistance has been greatly improved.

Description of drawings

Fig. 1 is the electrophoresis pattern of the PCR identification of the ZmCCT gene transgenic maize plants of the T ₀ generation. Lane 1 is D2000Marker, the band sizes from bottom to top are 100bp, 250bp, 500bp, 750bp, 1kb and 2kb; lanes 2, 3, 4, 5, 6, 8, 9, 10, 12, 13, 14, 15, 17, 18, 19, 21, 23, 24, 25, and 26 are all unsuccessful transgenic individuals; lanes 7, 11, 16, 20, and 22 are all positive transgenic individuals (the band size is 518bp).

Figure 2 shows the disease resistance performance of ZmCCT gene positive plants (P) and negative plants (N). Among them, Fig. 2A is the plant surface of ZmCCT gene positive plants (P) and negative plants (N); Fig. 2B is ZmCCT gene positive plants (P) and negative plants (N) after stem splitting.

Fig. 3 is the electrophoretic pattern of the qRT-PCR identification of the offspring of the ZmCCT gene-transferred maize plants of the T ₀ generation. Lane 1 is D2000Marker, the upper and lower bands are 250bp and 100bp respectively; Lanes 2, 3, 4, 5, 6, and 7 are all positive transgenic individuals (P); 8, 9, 10, 11, 12, and 13 are all negative Transgenic individuals (N); 28 and 32 are the number of PCR reaction cycles; GAPDH is the internal reference gene.

Figure 4 shows the statistical results of the disease resistance rate _of T1 generation materials. Among them, P represents ZmCCT gene positive plants, N represents negative plants.

Figure 5 shows the statistical results of the disease resistance rate of T ₂ /T ₃ generation materials. Among them, P represents ZmCCT gene positive plants, and N represents negative plants.

Fig. 6 is a statistical chart of growth indicators of the transgenic T ₄ generation under low-nitrogen and high-salt stress. Figure 6A is the statistics of the total root length; Figure 6B is the statistics of the main radicle length; Figure 6C is the statistics of the main root length; Figure 6D is the statistics of the lateral root length; Figure 6E is the statistics of the dry weight of the underground part; Among them, TL-23(+): ZmCCT gene positive plants, TL-23(-): negative plants, LNS (low nitrogen stress): 0.04mmol/L nitrogen stress, HSS (High salty stress): 50mmol/L NaCl stress, Significant difference: *, P<0.05; **, P<0.01.

Figure 7 shows the growth status of ZmCCT gene positive (TL+) and negative (TL-) plants after normal (Mock) and low nitrogen stress (LNS) treatments. Among them, TL-23(+): ZmCCT gene positive plants, TL-23(-): negative plants.

Figure 8 shows the growth status of ZmCCT gene positive (TL+) and negative (TL-) plants after normal (Mock) and high-salt stress (HSS) treatments. Among them, TL-23(+): ZmCCT gene positive plants, TL-23(-): negative plants.

Figure 9 shows the expression changes of ZmCCT after low nitrogen and high salt stress. Fig. 9A is the root of the control; Fig. 9B is the shoot of the control; Fig. 9C is the root of salt stress; Fig. 9D is the shoot of salt stress; Fig. 9E is the root of nitrogen stress; Fig. 9F is the shoot of nitrogen stress. Among them, TL-23(+): ZmCCT gene positive plants, TL-23(-): negative plants.

Figure 10 shows the expression changes of ZmABA2 after low nitrogen and high salt stress. Figure 10A is the root of the control; Figure 10B is the shoot of the control; Figure 10C is the root of the salt stress; Figure 10D is the shoot of the salt stress; Figure 10E is the root of the nitrogen stress; Figure 10F is the shoot of the nitrogen stress. Among them, TL-23(+): ZmCCT gene positive plants, TL-23(-): negative plants.

Figure 11 shows the expression changes of ZmMPK5 after low nitrogen and high salt stress. Figure 11A is the root of the control; Figure 11B is the shoot of the control; Figure 11C is the root of salt stress; Figure 11D is the shoot of salt stress; Figure 11E is the root of nitrogen stress; Figure 11F is the shoot of nitrogen stress. Among them, TL-23(+): ZmCCT gene positive plants, TL-23(-): negative plants.

detailed description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Maize (Zea mays L.) inbred line 1145 in the following examples: National Agricultural Crop Germplasm Conservation Center, its number is 0L010346, this maize is the kind of high tolerance to low nitrogen stress and salt stress (see " Qin Yang, Guangming Yin, Yanling Guo, et al. A major QTL for resistance to Gibberella stalk rot in maize. Theor Appl Genet, (2010) 121:673-687." article).

Maize (Zea mays L.) variety HiII in the following examples: described in "Lorena Moeller, Qinglei Gan, Kan Wang. Establishment and characterization of a maize Hi-II endosperm culture. In Viro Cellular & Developmental Biology-Plant, 2012 (48 ):283-294." "maize Hi-II" in the article.

Plasmid pCAMBIA3301 in the following examples: described in "Huixia Shou, Reid G. Palmer, Kan Wang. Irreproducibility of the Soybean Pollen-Tube Pathway Transformation Procedure. Plant Molecular Biology Reporter, 2002 (20): 325-334." .

The Agrobacterium tumefaciens LBA4404 Electro-Cells in the following examples are Agrobacterium tumefaciens LBA4404 Electro-Cells from Clontech Company, product number: 9115.

Example 1. Acquisition of the gene ZmCCT related to tolerance to low nitrogen stress and high salt stress

1. Acquisition of the full-length cDNA sequence of the gene ZmCCT related to tolerance to low nitrogen stress and high salt stress

The method of soil burial with wounded roots (see "Song Zuoheng et al. Research on the Control Effect of Healthy Cultivation Measures on Corn Stem Rot, Liaoning Agricultural Science. 1993 No. 05") artificially artificially planted conidia of Fusarium graminearum Schw. Plants of maize (Zea mays L.) inbred line 1145 with high tolerance to low nitrogen stress were inoculated at the tasseling stage. 16h after inoculation, the leaves were taken. Total RNA was extracted using TriZol reagent provided by Invitrogen. Use the BD SMART ^TM RACE cDNA Amplification Kit, using gene-specific primers—5'RACE primer (5'GSPB) and 3'RACE primer (3'GSPA) (see Table 1), as well as the universal primers provided in the kit, and The 5'RACE product and the 3'RACE product were amplified and sequenced according to the kit instructions. The 5' end sequence and the 3' end sequence of the obtained target gene ZmCCT were spliced to obtain the full-length cDNA sequence of the ZmCCT gene, and its nucleotide sequence is shown in sequence 2 in the sequence list.

Table 1. All primer sequence information used for gene function identification

In order to further confirm the correctness of the ZmCCT gene full-length cDNA sequence (Sequence 2) obtained by the above splicing, the following specific primers for amplifying the ZmCCT gene full-length cDNA sequence were designed.

Primer 1: 5'-ATGTCGTCGGGGCCAGCAGC-3' (position 1-20 of sequence 2 in the sequence listing);

Primer 2: 5'-TTGCCAAGGTAACCGAATGA-3' (reverse complementary sequence of positions 698-717 of Sequence 2 in the Sequence Listing).

The cDNA obtained by reverse transcription of the above total RNA was used as a template, amplified by the above primers 1 and 2, and the amplified product was detected by 1% agarose gel electrophoresis. The results showed that a fragment with a length of about 720bp was obtained by PCR amplification. The product was recovered and purified, connected to the pEASY-T1 vector (Beijing Quanshijin Biotechnology Co., Ltd.), and sequenced for identification. Sequencing results show that the PCR amplification product is completely identical to the sequence 2 spliced above, that is, the full-length cDNA sequence of the ZmCCT gene is shown in sequence 2 in the sequence listing, and the ORF is the 1-717th position of sequence 2, and the coding sequence listing The protein shown in Sequence 1 was named ZmCCT.

2. Acquisition of Genomic DNA Sequence of ZmCCT Related to Low Nitrogen Stress and High Salt Stress

The method of soil burial with wounded roots (see "Song Zuoheng et al. Research on the Control Effect of Healthy Cultivation Measures on Corn Stem Rot, Liaoning Agricultural Science. 1993 No. 05") artificially artificially planted conidia of Fusarium graminearum Schw. Plants of maize (Zea mays L.) inbred line 1145 with high tolerance to low nitrogen stress in the tasseling stage were inoculated. 16 hours after inoculation, the leaves were taken, and genomic DNA was extracted by SDS alkaline lysis method.

Using the genomic DNA obtained above as a template, PCR amplification was performed using the above primers 1 and 2, and the amplified product was detected by 1% agarose gel electrophoresis. The results showed that a fragment with a length of about 2600bp was obtained by PCR amplification. The product was recovered and purified, connected to the pEASY-T1 vector (Beijing Quanshijin Biotechnology Co., Ltd.), and sequenced for identification. Sequencing results show that the nucleotide sequence of the product is No. 5136-7682 of Sequence 3 in the Sequence Listing, wherein No. 5136-5609 is the first exon sequence, No. 5610-7439 is the intron sequence, and No. 5610-7439 is the intron sequence. Positions 7440-7682 are the second exon sequence.

3. Obtaining the Genome Segment Containing the ZmCCT Genomic DNA Sequence

Prepare the "genome segment sequence containing the ZmCCT genomic DNA sequence" with restriction endonuclease Sac I recognition sites at both ends, that is, "GAGCTC+sequence 3+GAGCTC", which is denoted as DNA fragment A. The DNA fragment A was connected to the pEASY-T1 vector (Beijing Quanshijin Biotechnology Co., Ltd.), and the resulting recombinant plasmid was named pEASY-ZmCCT. Sequence identification of pEASY-ZmCCT.

The sequencing results showed that the exogenous gene sequence inserted into the pEASY-T1 vector was exactly "GAGCTC+sequence 3+GAGCTC". Among them, the 1-5135th position of sequence 3 is the promoter sequence; the 5136-7682th position is the genome sequence of the ZmCCT gene (the 5136-5609th position is the first exon sequence, and the 7440-7682th position is the second exon sequence subsequence, the 5610-7439 position is the intron sequence); the 7683-8147 position is the untranslated region sequence.

Example 2, the acquisition of transgenic ZmCCT maize and its functional identification

1. Construction of recombinant expression vector pCAMBIA3301-ZmCCT

Digest the recombinant plasmid pEASY-ZmCCT obtained in Step 3 of Example 1 with restriction endonuclease Sac I, recover the target fragment (genome segment containing the ZmCCT genomic DNA sequence, about 8.1K), and combine it with the same Sac I enzyme The backbone fragments of the cut pCAMBIA3301 vector were connected to obtain the recombinant plasmid pCAMBIA3301-ZmCCT.

The resulting recombinant plasmid was identified by Sac I restriction enzyme digestion, and the recombinant plasmid identified by restriction enzyme digestion showed that it contained a target band with a size of about 8.1kb. The DNA molecule shown in Sequence 3 in the Sequence Listing was inserted forwardly between the sites, and the other sequences of the pCAMBIA3301 vector were kept unchanged to obtain the vector. In the recombinant expression vector pCAMBIA3301-ZmCCT, the promoter for initiating the transcription of ZmCCT genomic DNA sequence is the 1-5135 position of sequence 3.

2. Obtaining of transgenic ZmCCT maize

1. Transformation and identification of Agrobacterium

The recombinant expression vector pCAMBIA3301-ZmCCT constructed in step 1 was introduced into Agrobacterium LBA4404. The specific operation is as follows:

(1) Add 5 μl of pCAMBIA3301-ZmCCT plasmid DNA with a concentration of 100 ng/μl into 50 μl of LBA4404 Agrobacterium competent cells, flick and mix well, and place on ice for 30 minutes.

(2) The mixture in (1) was frozen in liquid nitrogen for 1 minute.

(3) Incubate the mixture frozen in (2) in a water bath at 37°C for 5 minutes.

(4) Add 1 ml of YEP liquid medium to the mixture obtained in (3). Under the condition of 28°C, culture at 120rpm for 4 hours.

(5) Centrifuge the culture solution obtained in (4) at 1000 rpm for 30 seconds, discard the supernatant, and obtain the bottom cell suspension.

(6) Add 100ul of YEP liquid medium to the cell suspension obtained in (5), resuspend the cells, and apply the resuspension mixture on the solid medium containing kanamycin and rifampicin.

(7) Cultivate the solid medium coated in (6) at 28° C. for 36-48 hours under dark conditions.

(8) Perform bacterial PCR identification and sequencing identification on the colony obtained in (7) to obtain a positive transformed strain.

The transformed recombinant Agrobacterium was identified by PCR using a primer pair consisting of primer 3 and primer 4. The identified Agrobacterium LBA4404 containing the ZmCCT genome segment shown in Sequence 3 (the PCR target band size is about 8.1 kb) was named LBA4404/pCAMBIA3301-ZmCCT. At the same time, the Agrobacterium control transformed into the pCAMBIA3301 empty vector was set, and the Agrobacterium LBA4404 transformed into the pCAMBIA3301 empty vector was named LBA4404/pCAMBIA3301.

Primer 3: 5'-GAGCTCTTGTTGCGACTTGT-3' (position 1-20 of sequence 3 in the sequence listing);

Primer 4: 5'-GAGCTCGACAAACAGTACAT-3' (reverse complementary sequence of positions 8128-8147 of sequence 3 in the sequence listing).

2. Transformation and identification of recombinant Agrobacterium on maize plants

The recombinant Agrobacterium LBA4404/pCAMBIA3301-ZmCCT obtained above (or the empty vector control LBA4404/pCAMBIA3301) was transformed into maize (Zea mays L.) variety HiII.

Soak corn callus with pre-activated recombinant Agrobacterium to obtain transformed callus. After transformation, use herbicide resistance screening to obtain transgenic seedlings, that is, corn plants transformed into pCAMBIA3301-ZmCCT and pCAMBIA3301 empty vector corn plant.

Further PCR identification was performed on the transgenic maize plants (T ₀ generation) obtained above, and PCR positive lines were screened. The genomic DNA of the transgenic corn plant was extracted, and used as a template to carry out PCR identification on the corn plant transformed into pCAMBIA3301-ZmCCT, with the ZmCCT genome segment shown in sequence 3 and the sequence of the pCAMBIA3301 vector itself as the target gene, and the primer pair (LBCCT F/R ) for PCR amplification. At the same time, the non-transgenic recipient parent maize (Zea mays L.) variety HiII was set as a control.

LBCCT FP: 5'-TAGCTAGCTCCACCACAGCA-3' (positions 7693-7712 of SEQ ID NO: 3);

LBCCT RP: 5'-TGTGGAATTGTGAGCGGATA-3' (this sequence corresponds to the self-contained sequence on the pCAMBIA3301 vector).

The result of PCR identification of maize plants transformed into pCAMBIA3301-ZmCCT (Fig. 1) shows that the transgenic maize that amplifies the expected size band (518bp) is positive, and the 5 T ₀ generation transgenic positive plants obtained are marked as Y3 -1, Y3-14, Y3-18, Y3-23, and Y3-25.

3. Identification of disease resistance of transgenic ZmCCT maize

(1) Experimental method

The 5 T ₀ generations obtained in step 2 were transformed into ZmCCT gene plants Y3-1, Y3-14, Y3-18, Y3-23 and Y3-25, and after selfing, 5 transgenic T ₁ generation populations were obtained, and the T ₁ The _first -generation population was planted in the experimental field of Shangzhuang, Beijing, and each transgenic T1 generation population was planted with 137 plants, which were used to identify the resistance traits of each individual plant to Fusarium graminearum stem rot, and combined with genotype analysis to identify the ZmCCT gene function. The experiment was repeated 3 times, and the results were averaged. The specific operation is as follows:

1. Genotype analysis and ZmCCT gene expression determination:

(1) Genotype analysis

Because the non-homozygous transgenic positive plants will segregate in the offspring, that is, transgenic positive and negative plants will appear in the progeny population, so the above 5 T ₀ generation transgenic ZmCCT gene plant progeny populations can be divided into two genotypes, i.e. positive ( P) and negative (N).

Take the method of PCR genotyping, use pCAMBIA3301-ZmCCT carrier-specific pair of primers (LBCCT F/R, the sequence is the same as above) PCR to identify the genotypes of the above five T ₀ generation transgenic ZmCCT gene plant progeny populations, and the bands can be expanded (518bp) individuals are transgene-positive individuals (P), and individuals who cannot expand the corresponding bands are transgene-negative individuals (N).

(2) Determination of ZmCCT gene expression

The ZmCCT gene-positive plant population (P) and the ZmCCT gene-negative plant population (N) in step (1) were respectively used as experimental materials. Total RNA was extracted from each experimental material. TIANGEN RNAprep pure plant total RNA extraction kit was used for RNA extraction, and the extraction method was the same as the instruction manual.

RNA was reverse-transcribed into cDNA using a reverse transcription kit (Fermentas), and stored at -80°C for later use.

The kit SYBR Premix EX Taq Kit (Bao Biological Engineering) was used to perform qRT-PCR according to the kit instructions to detect the expression level of the ZmCCT gene.

Forward primer: 5'-ATGAGAACGACGACCAGCCT-3' (position 5455-5474 of Sequence 3);

Reverse primer: 5'-GACGACTGATCTACCGGCAT-3' (reverse complementary sequence of positions 7418-7537 of Sequence 3). (Note: primers spanning introns, using cDNA as a template)

Reaction system: 20ul.

Reaction procedure: Step 1: 94°C for 3min;

Step 2: 94°C for 30s;

Step 3: 60°C for 30s;

Step 4: 72°C for 30s;

Step 5: Go to step 2for 28or 32cycles; (here is the number of repeated cycles)

Step 6: 72°C for 10min;

Step 7: 5°C forever.

The internal reference gene was GAPDH. The amplification primers of the internal reference gene are 5'-ATCAACGGCTTCGGAAGGAT-3' and 5'-CCGTGGACGGTGTCGTACTT-3'

At the same time, the maize positive plants transformed into the pCAMBIA3301 empty vector identified in step 2 were set as the empty vector control (CK), and the non-transgenic recipient parent maize (Zea mays L.) variety HiII was set as the parental control (WT).

2. Analysis of resistance traits to Fusarium graminearum stem rot:

Take the method of soil burying and wounding roots (see "Song Zuoheng et al. Research on the Control Effects of Healthy Cultivation Measures on Corn Stem Rot, Liaoning Agricultural Science. 1993 No. 05"), and use Fusarium graminearum to treat non-homozygous transgenic progeny populations. The plants are inoculated, and the specific steps are: after the corn spinneret, cut off part of the capillary roots vertically downward at a distance of 5-10 cm from the corn plant, throw away the soil, and bury 60-80 grams of corn kernels for propagation graminearum Fusarium graminearum, and water to moisturize.

Further, statistics on the proportion of disease-resistant plants were performed on the ZmCCT gene-positive plant population (P) and the ZmCCT-transfer gene-negative plant population (N) identified in step 1, respectively. The details are as follows: About 45 days after the artificial inoculation with Fusarium graminearum, the corn plants were split. The stem base and root were hollow and rotten as the susceptible plant, and the stem base and root were intact and normal as the resistant plant. strain (Figure 2). The ratios of disease-resistant plants in the ZmCCT gene positive plant population (P) and ZmCCT gene negative plant population (N) were calculated respectively.

At the same time, the maize positive plants transformed into the pCAMBIA3301 empty vector identified in step 2 were set as the empty vector control (CK), and the non-transgenic recipient parent maize (Zea mays L.) variety HiII was set as the parental control (WT).

In addition, the T ₁ generation plants identified as transgene positive by the above genotype analysis were selfed to obtain the T ₂ generation population; the T ₂ generation plants identified as the transgene positive by the above genotype analysis were selfed to obtain the T ₃ generation population. Genotype analysis and disease resistance trait analysis _were also performed _on the T2 generation population and the T3 generation population using the above method.

(2) Experimental results

1. Determination of ZmCCT gene expression in offspring of 5 T ₀ generation transgenic ZmCCT gene plants

The results are shown in Figure 3. It can be seen from the figure that among the offspring populations of 5 T ₀ generation ZmCCT gene transgenic plants, the ZmCCT gene expression level of the positive transgenic plants (P) is much higher than that of the negative transgenic materials (N). Compared with the negative transgenic material (N), the expression levels of ZmCCT gene in the two maize plants serving as the parental control (WT) and the empty vector control (CK) were basically the same, and there was no statistical difference.

2. Determination of disease resistance of offspring of 5 T ₀ generation transgenic ZmCCT gene plants

The results show that: on the T1 generation population level (Figure ₄ ), the proportion of disease-resistant plants of positive plants (P) in Y3-1, Y3-23 and Y3-25 has a significant increase relative to negative plants (N) , wherein the proportion of disease-resistant plants of positive plants (P) in Y3-1 increased by 33% compared with negative plants (N), and the proportion of disease-resistant plants of positive plants (P) in Y3-23 increased by 13% compared with negative plants (N). %, the proportion of disease-resistant plants in positive plants (P) in Y3-25 was 10% higher than that in negative plants (N).

At the T2 generation population level (Figure ₅ ), the proportion of disease-resistant plants of positive plants (P) in Y3-1, Y3-18, Y3-23 and Y3-25 was significantly higher than that of negative plants (N) The proportion of disease-resistant plants of positive plants (P) in Y3-1 was increased by 35% compared with that of negative plants (N), and the proportion of disease-resistant plants of positive plants (P) in Y3-18 was increased by 35% compared with negative plants (N). 17%, the proportion of disease-resistant plants of positive plants (P) in Y3-23 increased by 25% compared with negative plants (N), and the proportion of disease-resistant plants of positive plants (P) in Y3-25 increased by 10% compared with negative plants (N) 9%.

The Y3-23 that was positive for the transgene in the T ₂ generation was selected for selfing to obtain the T ₃ generation population. The results showed that on the T3 generation population level ( _Fig . 5), the proportion of disease-resistant plants of positive plants (P) in Y3-23 was still significantly improved relative to negative plants (N), and positive plants in Y3-23 ( The proportion of disease-resistant plants in P) was up to 7% higher than that in negative plants (N).

3. Determination of disease resistance of parental control and empty vector control

Compared with the identification results of the above five ZmCCT gene-transferred offspring of the T ₀ generation (Figure 4 and Figure 5), the proportion of disease-resistant plants in the non-transgenic maize (Zea mays L.) variety HiⅡ(WT) is only 5% , much lower than the proportion of disease-resistant plants in the progeny of ZmCCT gene-transferred plants of the above five T ₀ generations. The experimental results of the empty vector control were basically consistent with those of the parental control, and there was no statistical difference.

Based on the results of 1-3 above, it can be seen that relative to the non-transgenic parental control and the empty vector control, the proportion of disease-resistant plants in the offspring of the above 5 T ₀ generation transgenic ZmCCT gene plants is greatly improved, and the positive plants (P) in the offspring The proportion of disease-resistant plants was much higher than that of negative plants (N), and the expression level of ZmCCT gene in positive plants (P) was also much higher than that of negative plants (N).

4. Analysis of tolerance to low nitrogen and high salt stress of transgenic ZmCCT maize

(1) Measurement of root traits and dry weight of ZmCCT transgenic maize at seedling stage

(1) Experimental method

The T ₄ generation transgenic ZmCCT maize seeds identified as positive by the above genotype analysis were subjected to low nitrogen and high salt stress at the seedling stage under hydroponic planting conditions in an artificial climate chamber. After one week of stress treatment, the root traits, plant height, Measure the SPAD value, etc., and measure the dry weight after drying to constant weight. Statistical analysis was performed by t test method.

(2) Experimental steps

The ZmCCT gene transgenic maize seeds of the T ₄ generation were sterilized with 10% (v/v) H ₂ O ₂ for 30 minutes, washed with deionized water, soaked in saturated CaSO ₄ for 6 hours, and grown in an artificial climate chamber in the dark for 2 days. When the germination is about 1-2cm, select the seeds with the same growth, roll the seedlings with filter paper, roll them into a cylindrical paper roll from one side, move them into a small bucket to grow to the stage of 1 leaf and 1 heart, select the seedlings with the same growth, and transfer them into 1L of nutrient solution (Hoagland's nutrient solution: 0.75mmolL ^-1 K ₂ SO ₄ , 0.1mmolL ^-1 KCl, 0.25mmolL ^-1 KH ₂ PO ₄ , 0.65mmolL ^-1 MgSO ₄ , 0.13mmolL ^-1 EDTA-Fe, 1.0μmolL ^-1 MnSO ₄ , 1.0 μmolL ^-1 ZnSO ₄ , 0.1 μmolL ^-1 CuSO ₄ , 0.005 μmolL ^-1 (NH ₄ ) ₆ Mo ₇ O ₂₄ ), 4 strains per tank. The growth conditions were controlled as 28°C/22°C, 16/8h light/dark, daily cycle. The luminous flux density was 250–300μmolm ^-2 s ^-1 during the 16h illumination period. After the seedlings were moved into the culture tank, they were pre-cultured with 1/2 (c/c) culture solution for two days, and then cultured with complete nutrient solution to the stage of ₃ leaves ^{and 1} heart. After high-salt (50mmolL ^-1 NaCl) stress treatment, culture for another 7 days. N element was provided by Ca(NO ₃ ) ₂ , and Ca ²⁺ was supplemented to the level of 4mmolL ^-1 NO ₃ ^- in the form of CaCl ₂ under low nitrogen stress treatment. The pH value was adjusted to 6.0 with 1mmol/L NaOH. Oxygen was provided by an electric air pump, the nutrient solution was changed every two days, and each treatment was performed in triplicate.

(3) Measurement indicators

Corn seedlings after 7 days of stress treatment were washed with deionized water, divided into two parts, the aboveground part and the underground part, and the root scanning pictures were measured with Image J software. The measured characters include: total root length (TRL, Total root length), main Radical length (PRL, total primary radicle length), main root length (MRL, main root length) and lateral root length (LRL, lateral root length); 70°C/48h to dry the aboveground and underground parts to constant weight, weigh Measure the dry weight of the aboveground part (SDW, shoot dry weight) and the dry weight of the underground part (RDW, root dry weight); measure the plant height (PH, Plant height) with a ruler. 10 seedlings were measured for each trait, and the average value was taken.

(4) Experimental results

T test statistical measurement results showed that compared with the negative plants, the total root length, lateral root length and plant height of the T ₄ generation transgenic ZmCCT maize plants had significant differences in the normal control environment (Mock), the main root length and underground dry weight There are extremely significant differences; after low nitrogen stress treatment (LNS), there are extremely significant differences in total root length, lateral root length, dry weight of underground parts, dry weight of aboveground parts and plant height, and there are significant differences in main root length _. The total root length, lateral root length, dry weight of underground part, dry weight of aboveground part, plant height and trunk root length of transgenic ZmCCT maize plants were significantly higher than negative plants; after high-salt treatment (HSS), all the measured traits, transgenic The positive plants were significantly different from the negative plants, and the total root length, main root length, main radicle length, lateral root length, dry weight of the underground part, dry weight of the aboveground part and plant height of the ZmCCT gene transgenic maize plants in the T4 generation _were significantly different. The total root length, trunk root length, lateral root length, underground dry weight, aboveground dry weight and plant height of T4 transgenic _ZmCCT maize plants were significantly higher than negative plants. The ZmCCT transgenic maize plants of the T ₄ generation showed strong growth adaptability under low nitrogen and salt stress conditions (Fig. 6 and Table 2).

Table 2. Statistical table of growth indicators of T4 generation transgenic _ZmCCT maize under low-nitrogen and high-salt stress

Since the growth vigor of the ZmCCT transgenic maize plants of the T ₄ generation was quite different from that of the negative plants, a multivariate analysis of variance was performed, and the analysis results are shown in Table 3. There were significant differences in total root length, trunk root length and underground dry weight under low nitrogen stress, and there were significant differences in total root length, main radicle length, lateral root length and plant height under salt stress.

Table 3. Multivariate analysis of variance table for growth indicators of T4 generation _ZmCCT gene-transferred maize under low-nitrogen and high-salt stress

In summary, the ZmCCT gene-positive plants had more developed root growth and strong adaptability under low-nitrogen and salt stress conditions at the seedling stage, and ZmCCT could improve the resistance of maize seedlings to low-nitrogen and high-salt stress (Fig. 7, Figure 8).

(2) Complementary test expression of transgenic materials under low-nitrogen and high-salt stress at seedling stage

(1) Experimental content

The roots and leaves of the T4 generation _ZmCCT transgenic maize plants and negative plants at the three-leaf and one-heart stage were tested at different time points (0, 3, 6 and 9 hours) after 50mmol NaCl high-salt stress treatment and 0.04mmol low nitrogen stress. For expression analysis, 5 strains were randomly selected from each sample at each time point and mixed together for expression research. Three replicates were set for each sample, and the experiment was repeated 3 times.

(2) Experimental operation process

TIANGEN RNAprep pure plant total RNA extraction kit was used for RNA extraction, and the extraction method was the same as the instruction manual. Transscript First-Strand cDNA SuperMix (AT301-03) of Quanshijin Company was used for RNA reverse transcription, and the operation method was the same as the manual. The synthesized cDNA was diluted to an appropriate concentration with DEPC-ddH ₂ O, and the product was stored at -20°C for future use.

SYBR Premix Ex Taq ^TM II (RR820A) from TaKaRa Company was used for qRT-PCR, and RoterGene6000 was used for detection. Relative quantification of gene expression was performed using the comparative CT value method (2 ^{- ΔΔ Ct} ) (Livak and Schmittgen 2001). GAPDH was selected as the internal reference gene and the primers for expression level determination are shown in Table 4.

qRT-PCR program: 95°C, 5min; 95°C, 10S, 60°C, 20s, 40cycles; mealting curve analysis.

qRT-PCR reaction system (20 μl): SYBR Primix Ex Taq ^TM II (2×) 10 μl, Forward Primer (10 μM) 0.4 μl, Reverse Primer (10 μM) 0.4 μl, cDNA 2 μl, ddH ₂ O 7.2 μl.

Table 4. Selection of internal reference gene GAPDH and primers for expression determination

(3) Experimental results

The ZmCCT gene transgenic maize plants of the T ₄ generation were induced to express under the conditions of low nitrogen and salt stress (Fig. 9), and the ZmCCT gene transgenic maize plants of the T ₄ generation (TL-23(+)) Under stress conditions, the expression levels in roots and shoots increased, reaching the peak at 3 hours, and the expression levels were about 5.86 times and 9.71 times that of the ZmCCT gene-transferred maize plants in the T ₄ generation treated at 0 hours, and then declined, and the negative plants were There is no such phenomenon.

ZmABA2 and ZmMPK5 were reported to be involved in ABA-induced antioxidant defense responses in maize. The increased expression of ZmABA2 and ZmMPK5 can enhance the ability of maize to resist abiotic stress such as salt stress, drought stress and oxidative stress, and enhance the adaptability of maize to the environment (Ma, Ni et al.2015).

Under low-nitrogen and high-salt stress conditions, the expression level of ZmABA2 was increased in the roots and shoots of the T ₄ generation ZmCCT transgenic maize plants (TL-23(+)) (Fig. 10), except for the roots under low nitrogen stress, 3 reached the peak every hour, and the expression level was about 2-4 times that at the beginning of the stress treatment, and then declined. The roots of negative plants (TL-23(-)) were induced by salt stress, roots and shoots of low nitrogen stress expression, but the increase in expression was much smaller than that of ZmCCT gene-positive plants.

Under the conditions of low nitrogen and high salt stress, the expression level of ZmMPK5 was increased in the roots and shoots of the ZmCCT gene-positive plants (TL-23(+)) of the T ₄ generation (Figure 11), and the expression level was high after three hours of stress treatment 4-6 times higher than negative plants (TL-23(-)).

In conclusion, the expression level of ZmCCT in maize plants transgenic for ZmCCT increased after low-nitrogen and high-salt stress, and reached a peak within 3 hours. After low-nitrogen and high-salt stress treatments, the expression levels of genes ZmABA2 and ZmMPK5 related to the improvement of maize resistance in ZmCCT gene-positive plants were also much higher than those in negative plants. It shows that the ZmCCT gene has the function of improving resistance to low-nitrogen and high-salt stress in maize seedling stage.

Claims (10)

1. protein, is following protein a) or b) or c)：

A) amino acid sequence is the protein shown in sequence 1；

B) fused protein obtained in N-terminal and/or C-terminal the connection label of the protein shown in sequence 1；

C) by the amino acid sequence shown in sequence 1 by one or several amino acid residues substitution and/or missing and/or Add the obtained protein with identical function.

2. the biomaterial with the albumen qualitative correlation described in claim 1, is following A 1) to A12) in any Kind：

A1) the nucleic acid molecules of the protein described in coding claim 1；

A2 A1) is contained) expression cassettes of the nucleic acid molecules；

A3 A1) is contained) recombinant vectors of the nucleic acid molecules；

A4 A2) is contained) recombinant vector of the expression cassette；

A5 A1) is contained) recombinant microorganisms of the nucleic acid molecules；

A6 A2) is contained) recombinant microorganism of the expression cassette；

A7 A3) is contained) recombinant microorganism of the recombinant vector；

A8 A4) is contained) recombinant microorganism of the recombinant vector；

A9 A1) is contained) the transgenic plant cells systems of the nucleic acid molecules；

A10 A2) is contained) the transgenic plant cells system of the expression cassette；

A11 A3) is contained) the transgenic plant cells system of the recombinant vector；

A12 A4) is contained) the transgenic plant cells system of the recombinant vector.

3. relevant biological material according to claim 2, it is characterised in that：A1) nucleic acid molecules is such as It is lower 1) or 2) or 3) shown in gene：

1) its coded sequence is the genomic DNA molecule shown in the cDNA molecules or sequence 3 shown in sequence 2；

2) there is 75% or more than 75% homogeneity, and coding claim 1 institute with the nucleotide sequence of 1) restriction The cDNA molecules or genomic DNA molecule for the protein stated；

3) under strict conditions with 1) or 2) nucleotide sequence hybridization limited, and described in coding claim 1 The cDNA molecules or genomic DNA molecule of protein.

4. the relevant biological material described in protein or Claims 2 or 3 described in claim 1 following (1)- At least one of (6) application in：

(1) resistance of the regulation and control plant to Fusarium graminearum stem rot；

(2) stress resistance of plant is regulated and controled；

(3) the regulation and control total root length of plant and/or backbone root length and/or main radicle length and/or lateral root length and/or underground part dry weight And/or overground part dry weight and/or plant height；

(4) regulation and control and the expression of plant resistance to environment stress related gene；

(5) genetically modified plants of anti-Fusarium graminearum stem rot are cultivated；

(6) genetically modified plants that resistance is improved are cultivated.

5. application according to claim 4, it is characterised in that：

It is described to be regulated to improve；

The resistance is resistance to low nitrogen and/or salt-resistance.

6. a kind of method for cultivating the genetically modified plants that resistance is improved, including by the protein described in claim 1 Encoding gene is imported in recipient plant, the step of obtaining genetically modified plants；The resistance of the genetically modified plants is higher than institute State recipient plant.

7. method according to claim 6, it is characterised in that：

The resistance is resistance to low nitrogen and/or salt-resistance；

And/or, the resistance of the genetically modified plants is embodied in (D1)-(D7) as follows higher than the recipient plant It is any：

(D1) total root length of genetically modified plants is higher than the recipient plant；

(D2) the backbone root length of genetically modified plants is higher than the recipient plant；

(D3) the main radicle length of genetically modified plants is higher than the recipient plant；

(D4) the lateral root length of genetically modified plants is higher than the recipient plant；

(D5) the underground part dry weight of genetically modified plants is higher than the recipient plant；

(D6) the overground part dry weight of genetically modified plants is higher than the recipient plant；

(D7) plant height of genetically modified plants is higher than the recipient plant；

(D8) expression of the ZmABA2 and ZmMPK5 genes of genetically modified plants is higher than the recipient plant.

8. a kind of method for the genetically modified plants for cultivating anti-Fusarium graminearum stem rot, including by described in claim 1 The encoding gene of protein is imported in recipient plant, the step of obtaining genetically modified plants；The genetically modified plants are to cereal The resistance of corn stalk rot caused by Fusarium is higher than the recipient plant.

9. the method according to claim 6 or 8, it is characterised in that：

The nucleotide sequence of the encoding gene of the protein is 1-717 nucleic acid molecules of sequence 2.

10. according to any described method in claim 6-9, it is characterised in that：The recipient plant is unifacial leaf Plant or dicotyledon.