CN102382804B - Soybean cyst nematode RNA helicase CGH-1, and coding gene and application thereof - Google Patents

Abstract

The invention discloses a soybean cyst nematode RNA helicase CGH-1 and its coding gene and application. The protein provided by the present invention is the following (a) or (b): (a) a protein consisting of the amino acid sequence shown in Sequence 1 in the Sequence Listing; (b) the amino acid sequence of Sequence 1 after one or more Substitution and/or deletion and/or addition of a group and a protein derived from Sequence 1 associated with soybean cyst nematode development. The present invention provides soybean cyst nematode RNA helicase CGH-1 and its encoding gene, and an RNAi expression vector is designed based on the encoding gene, and the RNAi expression vector is introduced into a plant, and the dsRNA of the gene in the plant can be used as After ingesting the transgenic plants, the pests will have serious obstacles to reproduction or development, thereby inhibiting the infection and spread of the pests. Through the method of the invention, transgenic plants with strong insect-resistant activity can be obtained, which lays the foundation for further cultivating new soybean varieties with broad-spectrum insect-resistant properties.

Description

Soybean cyst nematode RNA helicase CGH-1 and its coding gene and application

technical field

The invention relates to a soybean cyst nematode RNA helicase CGH-1 and its coding gene and application.

Background technique

Soybean originated in China and is an important source of human protein and edible oil. 31% of the world's edible oil comes from soybean. my country is a big consumer of soybeans, with a total annual consumption of more than 40 million tons, while the total annual domestic soybean output is less than 17 million tons. Therefore, it is necessary to rely on a large number of imports to fill the gap. The main problem of my country's soybean production is the low yield per unit area. The average yield per unit area in 2008 was only 114 kg/mu, which was only 70% of the world's average yield. Compared with the United States and other advanced countries, the gap was even greater. In addition to problems such as varieties and planting, diseases and insect pests are important reasons for the low yield of soybeans in my country.

Soybean cyst nematode is one of the most serious pests and diseases to soybean production in the world. In my country, soybean cyst nematode is the second most serious disease after soybean mosaic virus disease, and it is mainly distributed in the two main soybean producing areas of Northeast China and Huanghuai River. Among them, the multi-year continuous cropping area and the drought and sand alkali old bean area are the most serious. After soybeans are attacked by cyst nematodes, the mild cases will reduce the yield by 20% to 30%, and the serious cases will reduce the yield by 70% to 80%. What's more, large-scale planting land will fail to harvest. Soybean cyst nematode disease is caused by the cyst nematode (Heterodera glycines, Soybean cyst nematode, SCN), which can occur throughout the soybean growth process. Cyst nematodes parasitize on soybean roots, resulting in poor development of main roots and lateral roots, increased fibrous roots, and the entire root system is hair-like. There are white to yellow-white protrusions about 0.5 mm in size on the fibrous roots, which are nematode cysts. There are few or no nodules on the diseased roots of the affected plants; the growth of the aboveground parts is slow, and the cotyledons and true leaves turn yellow and large at the seedling stage; the growth of severely diseased seedlings stops or even dies. Affected plants generally show short stature, yellow leaves, clustered flower buds, shortened internodes, delayed flowering, few or no pods.

In view of this, the control of cyst nematode disease is a long-term arduous task faced by soybean production. Although the use of highly toxic insecticides such as DDT, carbofuran, and aldicarb can kill nematodes, they will cause serious environmental pollution, so they have been banned or restricted. The problems encountered in cultivating soybean resistant varieties using traditional breeding programs are cumbersome backcrossing procedures and complex resistance identification. It takes at least ten years to breed a variety by backcross breeding method, which greatly limits the breeding of new varieties resistant to soybean cyst nematodes.

The emergence of RNA interference technology (RNAi) has made it possible to obtain high-yielding soybean varieties and other crop varieties with broad-spectrum resistance to cyst nematodes through transgenic technology. The phenomenon of RNAi was first discovered in plants and revealed in the non-parasitic nematode Caenorhabditis elegans, and then further confirmed in plants, fruit flies and vertebrates. After Caenorhabditis elegans ingests dsRNA-containing Escherichia coli, it is digested in its gut, and the dsRNA in it can be absorbed and passed between cells, reaching most tissues and cells, causing the specific degradation of the mRNA encoded by the target gene, thereby affect the function of the target gene.

Contents of the invention

The object of the present invention is to provide a soybean cyst nematode RNA helicase CGH-1 and its coding gene and application.

The protein provided by the present invention, obtained from soybean cyst nematode (Heterodera glycines, Soybean cystnematode), is as follows (a) or (b):

(a) a protein consisting of the amino acid sequence shown in Sequence 1 in the Sequence Listing;

(b) A protein derived from Sequence 1 in which the amino acid sequence of Sequence 1 has undergone substitution and/or deletion and/or addition of one or several amino acid residues and is related to the development of soybean cyst nematode.

In order to facilitate the purification of the protein in (a), the amino-terminal or carboxy-terminal of the protein consisting of the amino acid sequence shown in Sequence 1 in the Sequence Listing can be linked with the tags shown in Table 1.

Table 1 Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The protein in (b) above can be synthesized artificially, or its coding gene can be synthesized first, and then obtained by biological expression. The protein-encoding gene in (b) above can be deleted by deleting one or several amino acid residue codons in the DNA sequence shown in Sequence 2 in the sequence listing, and/or carrying out one or several base pairs of missense mutation, and/or link the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end.

The gene encoding the protein also belongs to the protection scope of the present invention.

The gene can be the DNA molecule described in any of the following 1) to 4):

1) The DNA molecule shown in the 499th to 1839th nucleotides from the 5' end of Sequence 2 in the sequence listing;

2) The DNA molecule shown in sequence 2 in the sequence listing;

3) a DNA molecule that hybridizes to the DNA sequence defined in 1) or 2) under stringent conditions and encodes a protein related to soybean cyst nematode development;

4) A DNA molecule that has more than 90% homology with the DNA sequence defined in 1) or 2) and encodes a protein related to soybean cyst nematode development.

The above-mentioned stringent conditions can be hybridization and membrane washing in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS at 65° C.

Recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the genes all belong to the protection scope of the present invention.

The present invention also protects the recombinant plasmid (interfering plasmid) containing double-stranded DNA fragment A and double-stranded DNA fragment B; the double-stranded DNA fragment B and the double-stranded DNA fragment A are reverse complementary sequences; the double-stranded DNA Fragment A is shown in sequence 2 of sequence 2 from 523 to 1090 nucleotides at the 5' end. The recombinant plasmid can 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 polyA signal directs the addition of polyA to the 3' end of the pre-mRNA. When constructing the recombinant plasmid, any enhanced promoter or constitutive promoter can be added before its transcription start nucleotide, and they can be used alone or in combination with other promoters; constructing the recombinant plasmid In addition, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be in the same reading frame as the coding sequence to ensure that the entire correct translation of the 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 plants, the recombinant plasmids used can be processed, such as adding genes that encode enzymes that can produce color changes or light-emitting compounds that can be expressed in plants, antibiotic markers with resistance, or chemical resistance. Reagent marker genes, etc. It is also possible to directly select transformed plants by stress without adding any selectable marker gene. The recombinant plasmid can be a recombinant plasmid obtained by respectively inserting the double-stranded DNA fragment A and the DNA fragment B into different multiple cloning sites of the vector pZH01. The recombinant plasmid can specifically be inserted between the Xba I and Sal I restriction sites of the backbone vector pZH01, inserting the double-stranded DNA fragment A, and inserting the double-stranded DNA fragment between the Kpn I and Sac I restriction sites B obtained recombinant plasmid.

The invention also protects a method for cultivating transgenic plants, which is to introduce the recombinant plasmid into a target plant to obtain a transgenic plant with higher soybean cyst nematode resistance than the target plant. The recombinant plasmid can transform plant cells by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium-mediated (such as Agrobacterium rhizogenes K599), gene gun, etc. or tissue, and growing the transformed plant tissue into a plant. The target plant can be either a monocotyledon or a dicotyledon. Specifically, the dicotyledonous plant can be soybean, such as soybean Kefeng 1. The soybean cyst nematode may specifically be race 4 of soybean cyst nematode.

The protein, or the gene, or the recombinant expression vector, expression cassette, transgenic cell line or recombinant bacteria, or the recombinant plasmid can be used for plant breeding, such as cultivating soybean cyst nematode-resistant plants. The target plant can be either a monocotyledon or a dicotyledon. Specifically, the dicotyledonous plant can be soybean, such as soybean Kefeng 1. The soybean cyst nematode may specifically be race 4 of soybean cyst nematode.

The present invention provides soybean cyst nematode RNA helicase CGH-1 and its encoding gene, and based on the encoding gene, a RNAi expression vector is designed, and the RNAi expression vector is introduced into plants, and the dsRNA of the gene can be expressed in plants , when the pests ingest transgenic plants, dsRNA enters the intestinal tract, and the siRNA produced can further enter other tissues and cells, triggering the inactivation of the gene, causing serious obstacles to the reproduction or development of the pests, thereby inhibiting Infestation and spread of pests. Through the method provided by the invention, transgenic plants with strong insect-resistant activity can be obtained, which lays a foundation for further breeding new soybean varieties with broad-spectrum insect-resistant properties.

Description of drawings

Figure 1 is the electrophoresis result of the SCN cgh-1 gene fragment amplified with KOD Plus DNA polymerase; 2 is the SCN cgh-1 gene fragment; M is the molecular weight marker; other swimming lanes are samples not related to this patent.

Fig. 2 is the electrophoresis result of the fragments obtained by gel recovery; 3 is the SCN cgh-1 gene fragment; M is the molecular weight marker; other swimming lanes are samples irrelevant to this patent.

Figure 3 is the electrophoresis results of fragments amplified by 5'-RACE and 3'-RACE; 2: SCN cgh-15'-RACE; 7, 8: SCN cgh-13'-RACE; M is the molecular weight marker; other lanes All are samples irrelevant to this patent.

Figure 4 is a schematic structural view of the SCN cgh-1 RNAi expression vector.

Figure 5 is the PCR identification electrophoresis of the recombinant Agrobacterium liquid; M: 2000bp Marker; Swimming lane 3 is the PCR amplification product (cgh-1 gene fragment) of the recombinant Agrobacterium liquid.

Figure 6 is a photograph of GUS histochemical staining.

7 is the electrophoresis graph of total RNA of soybeans in each group; 1-5: total RNA of soybean roots in experimental groups; CK: total RNA of soybean roots in control group A.

Figure 8 is the RT-PCR identification electrophoresis of soybeans in each group; M: 2000bp Marker; 1-5: PCR amplification products of soybeans in the experimental group; CK: PCR amplification products of soybeans in the control group.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

The solvent of X-Gluc solution (pH 7.0) is water; the solute and its concentration are as follows: 50mM Na ₃ PO ₄ , 10mM Na ₂ EDTA, 0.1% (volume ratio) Triton X-100, 0.1M K ₃ [Fe(CN) ₆ ] , 0.1M K ₄ [Fe(CN) ₆ ], 0.5 g/L X-Gluc and 20% (by volume) methanol.

Soybean cyst nematode (race No. 4): The public can obtain it from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences; references: Lu Weiguo, Gai Junyi, Li Weidong Sampling survey and analysis of soybean cyst nematode physiological races in the Huanghuai area Research. Chinese Agricultural Sciences, 2006, 39(2): 306-312.

Soybean Kefeng No. 1: Publicly available from Institute of Genetics and Developmental Biology, Chinese Academy of Sciences; References: Qi-Yun Zhou, Ai-Guo Tian, Hong-Feng Zou, Zong-Ming Xie, Gang Lei, Jian Huang, Chun -Mei Wang, Hui-Wen Wang, Jin-Song Zhang, and Shou-Yi Chen. SoybeanWRKY-type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54, conferdifferential tolerance to abiotic stresses in transgenic Arabidopsis no. 6, 486-503.

Soybean susceptible variety Lee: The public can obtain it from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences; references: Lu Weiguo, Yuan Daohua, Li Jinying, Li Haichao, Wen Zixiang, Zhang Hui. Changes in heritability of soybean cyst nematode resistance genes in different generations , Henan Agricultural Sciences, 2010, (2): 24-27.

Vector pZH01: Publicly available from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences; References: Junhuang Zou, Shuying Zhang, Weiping Zhang, Gang Li, Zongxiang Chen, WenxueZhai, Xianfeng Zhao, Xuebiao Pan, Qi Xie and Lihuang Zhu.The rice HIGH-TILLERING DWARF1 encoding an ortholog of Arabidopsis MAX3 is required for negative regulation of the outgrowth of axillary buds. The Plant Journal, 2006, 48, 687-696.

Agrobacterium rhizogenes K599: The public can obtain it from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences; References: Attila Kereszt, Dongxue Li, Arief Indrasumunar, Cuc DT Nguyen, SureepornNontachaiyapoom, Mark Kinkema & Peter M Gresshoff.Agrobacterium rhizogenes-mediated transformation of soybean to study root biology, NATURE PROTOCOLS, 2007, 2(4): 948-952.

Embodiment 1, the discovery of soybean cyst nematode SCN cgh-1 protein and its coding gene

1. Selection of soybean cyst nematode SCN cgh-1 protein and its coding gene

In the NCBI database, as of May 2009, there were 108,045 related sequences of soybean cyst nematode, including 81,873 nucleotide sequences, 26,172 EST sequences, 183 mRNA and 252 protein sequences. Alkharouf et al processed 24,438 EST sequences and 231 genome sequences of soybean cyst nematode, and compared them with the gene sequences of Caenorhabditis elegans, and found that there were 8,334 gene sequences similar between the two, and among them, 1,508 Genes with lethal phenotype in C. elegans were grouped according to sequence similarity, and these genes were divided into 6 groups, among which there were 150 genes with the most similar (E-values between 0 and 1E-100). The inventor compared the genetic relationship between 150 genes and humans, and selected 13 candidate lethal genes for cloning considering factors such as the length of the amino acid sequence, one of which was the gene discovered and protected by the present invention.

2. Discovery of soybean cyst nematode SCN cgh-1 protein and its coding gene

1. TRIZOL method was used to extract total RNA from the cyst of soybean cyst nematode (physiological race No. 4).

2. Reverse transcribe the total RNA into cDNA (using Clontech's RACE kit: SMART RACE cDNA Amplification Kit, Cat No. 634923).

3. Acquisition of gene fragments

(1) By comparing the soybean cyst nematode EST sequence homologous to Caenorhabditis elegans cgh-1 in the NCBI database, and designing the following primer pair (target sequence 628bp) according to the EST sequence:

cgh-1HG Nhe I NS: 5'-CCGCTAGCGTTTCTTCTTCGCGTCCGAT-3';

cgh-1HG Xho I CAS: 5'-GGCTCGAGTAGAAACTGAACAAGACGGTC-3'.

(2) Use the cDNA in step 2 as a template, and perform PCR amplification with the primer pair designed in step 3.

PCR amplification system: cDNA 2ul, two primers each 2ul, MgSO ₄ solution 2ul, KOD Plus DNA polymerase 1ul, KOD Plus DNA polymerase buffer 5ul, dNTP 5ul, DMSO 1ul, ddH ₂ O 30ul.

PCR amplification program (BIO-RAD iCycler): 94°C for 2min, 35× (94°C for 15s, 57°C for 30s, 68°C for 2min), 68°C for 5min, and store at 12°C.

The results of agarose gel electrophoresis of PCR amplification products are shown in Figure 1.

(3) Use the OMEGA Gel Extraction Kit (D2500-01) to cut the gel and recover the bands. The agarose gel electrophoresis of the recovered bands is shown in Figure 2.

(4) Digest the recovered PCR fragment with NEB restriction endonuclease.

Enzyme digestion system: gel recovery product 43.5ul, Nhe I 1ul, Xho I 1ul, Buffer 25ul, BSA 0.5ul. Digest overnight at 37°C, then use OMEGA Gel Extraction Kit (D2500-01) to cut the gel and recover.

(5) Digest vector pPD129.36 with NEB restriction endonuclease.

Enzyme digestion system: pPD129.365ul, Hind III 1ul, Xho I 1ul, Buffer 25ul, BSA 0.5ul. Digest overnight at 37°C, then use OMEGA Gel Extraction Kit (D2500-01) to cut the gel and recover.

(6) The fragments recovered in step (4) and step (5) are connected with NEB T4 Ligase.

Ligation system: pPD129.36 (after enzyme digestion) 1ul, gene fragment (after enzyme digestion) 12ul, T4 Ligase Buffer 1.5ul, T4 Ligase 1ul.

(7) Transform DH5α competent cells by the heat shock method of the ligated product, spread on LB plate (Amp+), and culture overnight at 37°C. After the clones grow out, single clones are identified by Cloning PCR method, and positive clones are obtained (8) (11) , sent to Yingwei Jieji (Shanghai) Trading Co., Ltd. for sequencing. See sequence 3 in the sequence listing for the sequencing results.

4. Obtaining 5'-RACE fragments and 3'-RACE fragments

(1) Prepare the PCR Master Mix listed in Table 2, and vortex gently to avoid air bubbles during the process.

Table 2 Composition of PCR Master Mix

Element Each response (ul) 9 responses (ul) PCR-Grade Water 34.5 310.5 10X Advantage 2PCR Buffer 5.0 45.0 dNTP Mix(10mM) 1.0 9.0 50X Advantage 2 Polymerase Mix 1.0 9.0 Total Volume 41.5 373.5

(2) On the basis of step (1), see Table 3 and Table 4 for the reaction components of 5'-RACE PCR and 3'-RACE PCR.

Table 3 Reaction components of 5’-RACE PCR

Element Each response (ul) 5'-RACE-Ready cDNA 2.5 UPM(10X) 5 GSP1 1

Master Mix 41.5 Total Volume 50.0

Table 4 Reaction components of 3’-RACE PCR

Element Each response (ul) 5'-RACE-Ready cDNA 2.5 UPM(10X) 5 GSP2 1 Master Mix 41.5 Total Volume 50.0

(3) Use the BioRad iCycler PCR instrument to run the program (Touchdown PCR), the conditions are: 5×(94°C for 30s, 72°C for 3min), 5×(94°C for 30s, 70°C for 30s, 72°C for 3min), 27×(94 ℃ 30s, 68℃ 30s, 72℃ 3min), 4℃ forever. The amplification results are shown in Figure 3.

(4) After agarose gel electrophoresis, the gel was cut and recovered with OMEGA Gel Extraction Kit (D2500-01).

(5) Construct clones with the pEASY-Blunt Simple Cloning Kit of Quanshijin Company, obtain positive clones, and send them to INVITROGEN for sequencing. For the sequencing results of 3'-RACE, see sequence 4 and 5' in the sequence list - See sequence 5 in the sequence listing for the sequencing results of RACE.

The protein shown in Sequence 1 of the sequence listing is named as SCN cgh-1 protein (composed of 446 amino acid residues), which is an ATP-dependent RNA helicase. The coding gene of SCN cgh-1 albumen is called SCN cgh-1 gene, its cDNA is as shown in sequence 2 (2128bp) of sequence listing, 5'UTP length 498bp, coding region length 1341bp (sequence 2 of sequence listing is from 5' 499-1839 nucleotides at the end), and the 3'UTR is 289bp long.

Example 2, Functional verification of SCN cgh-1 protein

1. Construction of SCN cgh-1 RNAi expression vector

1. Extract total RNA from the cyst of soybean cyst nematode (physiological race No. 4), and reverse transcribe it into cDNA.

2. Using cDNA as a template, carry out PCR with a primer pair consisting of Primer-F and Primer-R (the target sequence is sequence 2 from the 5' end 523-1090 nucleotides in the sequence listing, i.e. the SCN cgh-1 gene fragment) Amplify to obtain PCR amplification products.

Primer-F: 5'-CGC TCT AGA GAG CTC CGT CCG ATG GAA GAT TG-3' (Xba I restriction recognition site and Sac I restriction recognition site are underlined);

Primer-R: 5'-ACG C GT CGA CGG TAC C GG AAG TCC TGG GAA AGG-3' (Sal I enzyme digestion recognition site and Kpn I enzyme digestion recognition site are underlined);

PCR amplification system (50 μl): cDNA 1 μl, 10×buffer 5 μl, dNTP (10 mM) 1 μl, Primer-F 1 μl, Primer-R 1 μl, Pfu DNA Polymerase (TaKaRa) 1 μl, ddH ₂ O 40 μl.

PCR amplification conditions: 94°C for 3min; 94°C for 30s, 50°C for 30s, 72°C for 30s, 30 cycles; 72°C for 10m.

The PCR amplified product was subjected to 1% agarose gel electrophoresis, and the target fragment of about 500 bp was recovered with a gel recovery kit.

3. Construction of SCN cgh-1 RNAi expression vector

① Digest the target fragment in step 2 with restriction enzymes Sac I and Kpn I, and recover the digested product.

② Digest the vector pZH01 with restriction enzymes Sac I and Kpn I to recover the vector backbone (about 11.5Kp).

③ Ligate the digested product of step ① with the vector backbone of step ② to obtain recombinant plasmid A.

④ Digest the target fragment in step 2 with restriction endonucleases XbaI and SalI, and recover the digested product.

5. Digest the recombinant plasmid A of step 3. with restriction endonucleases Xba I and Sal I to reclaim the vector backbone (about 12Kp).

⑥ Ligate the digested product of step ④ with the vector backbone of step ⑤ to obtain recombinant plasmid B, which is the SCNcgh-1 RNAi expression vector. According to the sequencing results, the structure of the SCN cgh-1 RNAi expression vector is described as follows: the backbone vector is pZH01, and a double-stranded DNA fragment A is inserted between the Xba I and Sal I restriction sites of the backbone vector (such as sequence 2 from 5' 523-1090 nucleotides at the end), double-stranded DNA fragment B is inserted between the KpnI and Sac I restriction sites of the backbone vector; double-stranded DNA fragment B and double-stranded DNA fragment A are reverse complementary sequences . The schematic diagram of the structure of the SCN cgh-1RNAi expression vector is shown in Figure 4.

2. Transformation and expression identification of soybean root system

1. Transform the SCN cgh-1 RNAi expression vector into Agrobacterium rhizogenes K599 to obtain recombinant Agrobacterium. Figure 5 shows the electrophoresis of the PCR identification of the recombinant Agrobacterium liquid (primer pair consisting of Primer-F and Primer-R). The recombinant Agrobacterium liquid was amplified to a band with the same size as the target fragment, indicating that the SCN cgh-1RNAi expression vector had been integrated into Agrobacterium rhizogenes K599.

The vector pZH01 was used to transform Agrobacterium rhizogenes K599 to obtain the control Agrobacterium.

2. Transformation of soybean roots

(1) Transformation of experimental group soybean

The seeds of Soybean Kefeng No. 1 were sterilized and sowed in the sterilized calcite; 5 days later, the seedlings germinated; the recombinant Agrobacterium solution was inoculated into the cotyledon nodes of the seedlings, and then the seedlings were transplanted to keep moisture and grow; 2-3 weeks later, the roots Occur; when hair root 5-10 centimetre, cut off primary root, transplant in the soil (the plastic cup of d6cm * h12cm) grow, each plant a plastic cup.

(2) transformation of control group A soybean

The operation of step (1) is carried out by replacing the recombinant Agrobacterium with the control Agrobacterium.

(3) transformation of control group B soybean

Carry out the operation of step (1), but do not inoculate the recombinant Agrobacterium bacteria liquid.

3. Identification of GUS gene expression in hairy roots

In step 2, after 4 weeks of Agrobacterium infection, the roots of soybeans in the experimental group and control group B were collected for histochemical staining to identify the expression of the GUS gene. The specific steps are as follows: put the root system into X-Gluc solution and culture overnight at 37°C in the dark; then wash it with 70% (volume ratio) ethanol water solution at room temperature for 30 minutes; then put the root system into water, the blue color under the white background is GUS expression site.

The results are shown in Figure 6. The roots of soybeans in the control group B did not turn blue, and the roots of soybeans in the experimental group all turned blue, and the transformation efficiency was about 100%. The results of GUS staining showed that the technique of transforming Soybean Kefeng No. 1 by introducing recombinant plasmids into Agrobacterium rhizogenes was effective.

4. RT-PCR analysis of the expression of SCN cgh-1 fragment in soybean roots

In step 2, after 4 weeks of Agrobacterium infection, take the root system of 1 control group A soybean and 5 experimental group soybeans, extract total RNA (see Figure 7), reverse transcribe into cDNA, and dilute the cDNA template by 5 times , carry out RT-PCR amplification of cgh-1 gene (using the primer pair consisting of Primer-F and Primer-R). Adjust the amount of template with the tubulin gene, and when the brightness of the amplified products is basically the same, it can be determined that the template concentration is consistent.

Trizol Reagent was purchased from Invitrogen, and the reverse transcription kit was purchased from Treasure Bioengineering (Dalian) Co., Ltd., TaKaRa Code: D6210A. PCR reaction system (25 μl): cDNA 2 μl, Primer-F 1 μl, Primer-R 1 μl, 2×TagPCR Master Mix 12.5 μl, ddH ₂ O 8.5 μl. PCR amplification program: 94°C 4min; 94°C 40s; 52°C 40s; 72°C 50s (30cycles); 72°C 5min; 10°C 1h.

In Fig. 7, the band patterns of the extracted RNA 28S ribosomal RNA and 18S ribosomal RNA are relatively clear without tailing phenomenon, indicating that the extracted RNA has good integrity.

The amplification results are shown in Figure 8. The control group A soybean did not amplify the target band, while the roots of the five experimental group soybeans all amplified the 568bp target band. It indicated that the cyst nematode cgh-1 gene fragment was correctly transcribed in soybean root genome.

5. Identification of resistance to soybean cyst nematode

In step 2, 4 weeks after Agrobacterium infection, 5 ml of pathogenic solution (each ml containing about 400 eggs of soybean cyst nematode race 4) was added to each plastic cup. After inoculating the pathogenic solution, keep the soil temperature at 24-30°C, and count the number of cysts after 30 days. The experiment was repeated three times, and the results were averaged. The degree of resistance of soybean lines to soybean cyst nematodes is generally expressed by the parasitism index. A parasitic index between 0-9 is a highly resistant variety, between 10-30 is a moderately resistant variety, between 31-60 is a moderately susceptible variety, and greater than 60 is a susceptible variety.

Parasitic index = (the number of cysts per gram of root of the test plant/the number of cysts per gram of root of susceptible soybean variety Lee)×100.

The statistical results of the parasitic index of each group are shown in Table 5.

Table 5 Statistical results of parasitic index in each group

Experimental group soybean Control group A soybean Control group B soybean Soybean susceptible variety Lee

Number of inoculated plants 32 37 5 twenty two Average number of cysts/g root 65 111 109 276 P value 0.0002 parasitic index twenty three 40 39 100 IP identification result neutral resistance middle sense middle sense Number of eggs/g root 8798 18811 P value ＜0.0001 The number of eggs contained in a single cystworm 128 194 P value 0.122

The results in Table 5 show that the number of cysts per gram of root of the plants introduced into the SCN cgh-1RNAi expression vector is significantly reduced, the number of eggs per gram of root is significantly reduced, and the number of eggs contained in a single cyst is also greatly reduced. That is, the SCNcgh-1RNAi expression vector inhibits the growth and reproduction ability of cyst nematodes. According to the criteria for judging the parasitic index, it can be seen from Table 5 that the parasitic index of the soybean containing the experimental group is 23, and the identification standard of the parasitic index is moderately resistant. The perennial identification results of soybean Kefeng 1 proved to be a moderately susceptible soybean cyst nematode No. 4. Small species (parasitic index about 40), so the transfer of SCN cgh-1 RNAi expression vector significantly improved the resistance of soybean roots to cyst nematodes.

Claims (11)

1. protein, the protein that is formed by the aminoacid sequence shown in sequence in sequence table 1.

2. the gene of coding claim 1 described albumen.

3. gene as claimed in claim 2, it is characterized in that: described gene is following 1) or 2) described DNA molecular:

1) sequence 2 of sequence table is from the DNA molecular shown in 5 ' end the 499th to 1839 Nucleotide;

2) DNA molecular shown in sequence 2 in sequence table;

4. the recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium that contain claim 2 or 3 described genes.

5. the recombinant plasmid that contains double chain DNA fragment A and double chain DNA fragment B; Described double chain DNA fragment B and described double chain DNA fragment A are reverse complementary sequence; Described double chain DNA fragment A as the sequence 2 of sequence 2 from as shown in 523 to 1090 Nucleotide of 5 ' end.

6. recombinant plasmid as claimed in claim 5 is characterized in that: described recombinant plasmid is to insert respectively in the different multiple clone site of carrier pZH01 the recombinant plasmid that described double chain DNA fragment A and described DNA fragmentation B obtain.

7. recombinant plasmid as claimed in claim 6 is characterized in that: described recombinant plasmid is for inserting the recombinant plasmid that described double chain DNA fragment B obtains inserting between the Xba of skeleton carrier pZH01 I and Sa1 I restriction enzyme site between described double chain DNA fragment A, Kpn I and Sac I restriction enzyme site.

8. a method of cultivating transgenic plant, be that arbitrary described recombinant plasmid in claim 5 to 7 is imported in the purpose plant, obtains the soybean cyst nematode Heterodera glycines resistance higher than the transgenic plant of described purpose plant.

9. method as claimed in claim 8, it is characterized in that: described purpose plant is monocotyledons or dicotyledons.

10. method as claimed in claim 9, it is characterized in that: described dicotyledons is soybean.

11. the described albumen of claim 1, or the described gene of claim 2 or 3, or the described recombinant expression vector of claim 4, expression cassette, transgenic cell line or recombinant bacterium, or the application of arbitrary described recombinant plasmid in cultivating Chinese People's Anti-Japanese Military and Political College beans Cyst nematode plant in claim 5 to 7.

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