阅读说明：本技术 基因g20e03、其编码的蛋白及其在提高烟草植株抗大豆孢囊线虫病中的应用 (Gene G20E03, protein coded by same and application thereof in improving soybean cyst nematode resistance of tobacco plants ) 是由 宋雯雯 史倩倩 刘兰亭 赵洪海 梁晨 段方猛 于 2020-07-29 设计创作，主要内容包括：本发明公开了一种基因G20E03、其编码的蛋白及其在提高烟草植株抗大豆孢囊线虫病中的应用,属于烟草抗病技术领域,其技术方案包括提供了一种基因G20E03,基因G20E03编码的蛋白,蛋白在提高烟草植株抗大豆孢囊线虫病中的应用及基因G20E03在提高烟草植株抗大豆孢囊线虫病中的应用,通过转基因技术获得基因G20E03的RNAi转基因烟草植株,在转基因烟草植株中表达基因G20E03的dsRNA,使大豆孢囊线虫基因G20E03沉默,缓解基因G20E03对烟草植株的免疫抑制作用,提高烟草植株的抗大豆孢囊线虫病性。本发明应用于烟草抗病方面,具有能够大量表达烟草大豆孢囊线虫效应蛋白基因的dsRNA,及显著抑制大豆孢囊线虫寄生的特点。通过本发明可获得强抗大豆孢囊线虫病的烟草植株。(The invention discloses a gene G20E03, a protein coded by the gene G20E03 and application of the protein to improvement of soybean cyst nematode resistance of a tobacco plant, and belongs to the technical field of tobacco disease resistance. The invention is applied to the aspect of tobacco disease resistance, and has the characteristics of expressing dsRNA of tobacco soybean cyst nematode effector protein genes in a large amount and obviously inhibiting the parasitism of soybean cyst nematodes. The tobacco plant with strong soybean cyst nematode resistance can be obtained by the method.)

1. The gene G20E03 is characterized in that the full-length sequence of the gene G20E03 is shown as SEQ ID NO. 1.

2. The gene G20E03 of claim 1, wherein the full-length gene sequence of the gene G20E03 includes a 5 'non-coding region, a 3' non-coding region and a cDNA region, and the gene sequence of the cDNA region is shown in SEQ ID NO. 2.

3. The gene G20E03 according to claim 2, wherein the gene G20E03 is cloned by the following method:

extracting 2-instar larva RNA of a soybean cyst nematode tobacco population by using a TRIZOL method, and performing reverse transcription to obtain cDNA;

designing a primer pair G20E03-part-F and G20E03-part-R according to a transcriptome sequencing result of SCNT, carrying out PCR amplification by taking the cDNA as a template to obtain a PCR product, purifying the PCR product and carrying out T-A connection to obtain a connection product, transferring the connection product into escherichia coli, randomly picking a single colony, carrying out PCR screening on the single colony by utilizing the primer pair G20E03-part-F and G20E03-part-R to obtain a positive single clone colony and sequencing;

according to the partial sequence of the G20E03 gene, specific primers G20E03-5 '-R and G20E 03-3' -F of 5 'end non-coding region and 3' end non-coding region genes are designed, PCR amplification is carried out, 260bp fragments are amplified at the 5 'end, and 666bp fragments are amplified at the 3' end;

and splicing the sequences, and cloning to obtain the gene full-length sequence of the gene G20E 03.

4. The protein encoded by the gene G20E03 according to any one of claims 1 to 3, wherein the amino acid sequence of the protein is shown in SEQ ID No. 3.

5. Use of the protein according to claim 4 for improving the resistance of tobacco plants against soybean cyst nematode disease.

6. Use of the gene G20E03 according to any one of claims 1 to 3 for improving the resistance of tobacco plants against soybean cyst nematode disease.

7. Use according to claim 6, characterized in that it comprises the following steps: RNAi transgenic tobacco plants of the gene G20E03 are obtained through a transgenic technology, dsRNA of the gene G20E03 is expressed in the transgenic tobacco plants, the soybean cyst nematode gene G20E03 is silenced, the immunosuppressive effect of the gene G20E03 on the tobacco plants is relieved, and the soybean cyst nematode resistance of the tobacco plants is improved.

8. The use according to claim 7, wherein the obtaining of the RNAi transgenic tobacco plant of gene G20E03 by transgenic technology comprises the following steps:

constructing a recombinant expression vector pFGC5941-RNAi-G20E 03;

transforming agrobacterium LBA4404 by a recombinant expression vector pFGC5941-RNAi-G20E03 to obtain recombinant agrobacterium;

tobacco is transformed by an agrobacterium-mediated leaf disc method, tissue culture is carried out, and transgenic tobacco plants which are positive in detection by using primers G20E03-part-F, G20E03-part-R, Bar-F and Bar-R are RNAi transgenic tobacco plants of G20E 03.

9. The use according to claim 8, wherein the construction of the recombinant expression vector pFGC5941-RNAi-G20E03 comprises the following steps:

cloning a G20E03 gene by an RACE method by taking soybean cyst nematode cDNA as a template to obtain a complete gene sequence;

designing a primer pair G20E03-ORF-R-F and G20E03-ORF-R-R, carrying out PCR amplification to obtain an ORF reverse sequence containing Xbal and BamH I enzyme cutting sites, carrying out enzyme cutting by using restriction enzymes Xbal and BamH I respectively, purifying and recovering, and connecting to a plant interference vector pFGC5941 cut by the restriction enzymes Xbal and BamH I to obtain a recombinant expression vector pFGC5941-G20E 03;

designing a primer pair G20E03-ORF-F-F and G20E03-ORF-F-R, carrying out PCR amplification to obtain an ORF forward sequence containing Nco I and Swa I enzyme cutting sites, carrying out enzyme cutting on a recombinant expression vector pFGC5941-G20E03 by using restriction enzymes Nco I and Swa I respectively, purifying and recycling, and connecting to a vector pFGC5941-G20E03 cut by the restriction enzymes Nco I and Swa I to obtain a recombinant expression vector pFGC5941-RNAi-G20E 03.

10. The use of claim 9, wherein obtaining an RNAi transgenic tobacco plant of gene G20E03 by transgenic technology further comprises:

carrying out plug seedling on T0 generation transgenic tobacco seeds by adopting seed plugs, and respectively carrying out PCR detection by utilizing specific primers G20E03-part-F, G20E03-part-R, Bar-F and Bar-R to obtain T1 generation transgenic tobacco;

and (4) carrying out generation-by-generation culture according to the method to obtain a homozygous transgenic line.

Technical Field

The invention belongs to the technical field of tobacco disease resistance, and particularly relates to a gene G20E03, a protein coded by the gene and application of the gene in improving soybean cyst nematode resistance of tobacco plants.

Background

Soybean Cyst Nematode (SCN) is a parasitic nematode in plants, mainly infests leguminous plants such as soybean (Glycine max), and tobacco (Nicotiana tabacum) is a non-host. However, in recent years, a special SCN population (SCNT) capable of infecting Tobacco has been found in the eastern Shandong. The cyst nematodes of tobacco parasitizing Shandong province are identified as soybean cyst nematodes (Heterodera glycines) by identification means such as morphology and molecular biology. The bifurcation of the root system of the tobacco infected by SCNT is increased, white or yellow spherical particles (female worms of cyst nematodes) with the sizes of millet grains can grow at the later stage, the infected root system of part of the tobacco is brown and necrotic, and finally the whole root is rotten and withered. Cyst nematodes damage root systems of tobacco, not only can cause yield loss and quality reduction of tobacco leaves, but also can aggravate other diseases (such as tobacco blight) on the tobacco, and become one of important factors which seriously restrict tobacco production in Shandong, Henan and other tobacco areas in China.

The existing methods for preventing and controlling cyst nematodes mainly comprise crop rotation, chemical prevention and breeding of disease-resistant varieties, but due to the limitation of crop rotation, huge pollution to the ecological environment caused by chemical prevention and control and shortage of nematode-resistant germplasm resources, the search for an efficient and sustainable method for preventing and controlling the cyst nematodes of soybeans is the key point in the research field of plant parasitic nematodes at present.

However, the research on SCNT in the prior art only stays in the aspect of comparing the pathogenic difference of SCN and SCNT, and the special pathogenic force of SCNT still has high technical difficulty in obtaining the tobacco plant resisting the soybean cyst nematode by screening strong pathogenic factors through the gene technology.

Disclosure of Invention

Aiming at the defects in the prior art, the technical problem to be solved by the invention is to overcome the difficulty in the prior art, obtain a strong pathogenic factor by screening by a gene technology and obtain a soybean cyst nematode resistant tobacco plant, thereby effectively solving the technical problem of tobacco diseases, and providing dsRNA capable of expressing a large amount of tobacco soybean cyst nematode effector protein genes and a method for effectively preventing and treating tobacco soybean cyst nematode.

In order to solve the technical problem, the technical scheme adopted by the invention is as follows:

the invention provides a gene G20E03, and the full-length sequence of the gene G20E03 is shown as SEQ ID NO. 1.

Preferably, the gene full-length sequence of the gene G20E03 comprises a 5 'end non-coding region, a 3' end non-coding region and a cDNA region, and the gene sequence of the cDNA region is shown as SEQ ID NO. 2.

Preferably, the gene G20E03 is cloned by the following method:

extracting 2-instar larva RNA of a soybean cyst nematode tobacco population by using a TRIZOL method, and performing reverse transcription to obtain cDNA;

designing a primer pair G20E03-part-F and G20E03-part-R according to a transcriptome sequencing result of SCNT, carrying out PCR amplification by taking the cDNA as a template to obtain a PCR product, purifying the PCR product and carrying out T-A connection to obtain a connection product, transferring the connection product into escherichia coli, randomly picking a single colony, carrying out PCR screening on the single colony by utilizing the primer pair G20E03-part-F and G20E03-part-R to obtain a positive single clone colony and sequencing;

according to the partial sequence of the G20E03 gene, specific primers G20E03-5 '-R and G20E 03-3' -F of 5 'end non-coding region and 3' end non-coding region genes are designed, PCR amplification is carried out, 260bp fragments are amplified at the 5 'end, and 666bp fragments are amplified at the 3' end;

and splicing the sequences, and cloning to obtain the gene full-length sequence of the gene G20E 03.

In another aspect, the invention provides a protein encoded by the gene G20E03 according to any one of the above technical schemes, wherein the amino acid sequence of the protein is shown as SEQ ID NO. 3.

The invention also provides application of the protein in improving soybean cyst nematode resistance of tobacco plants.

The invention also provides application of the gene G20E03 in any technical scheme in improving soybean cyst nematode resistance of tobacco plants.

Preferably, the method comprises the following steps: RNAi transgenic tobacco plants of the gene G20E03 are obtained through a transgenic technology, dsRNA of the gene G20E03 is expressed in the transgenic tobacco plants, the soybean cyst nematode gene G20E03 is silenced, the immunosuppressive effect of the gene G20E03 on the tobacco plants is relieved, and the soybean cyst nematode resistance of the tobacco plants is improved.

Preferably, the obtaining of the RNAi transgenic tobacco plant of the gene G20E03 by the transgenic technology comprises the following steps:

constructing a recombinant expression vector pFGC5941-RNAi-G20E 03;

transforming agrobacterium LBA4404 by a recombinant expression vector pFGC5941-RNAi-G20E03 to obtain recombinant agrobacterium;

tobacco is transformed by an agrobacterium-mediated leaf disc method, tissue culture is carried out, and transgenic tobacco plants which are positive in detection by using primers G20E03-part-F, G20E03-part-R, Bar-F and Bar-R are RNAi transgenic tobacco plants of G20E 03.

Preferably, the construction of the recombinant expression vector pFGC5941-RNAi-G20E03 comprises the following steps:

cloning a G20E03 gene by an RACE method by taking soybean cyst nematode cDNA as a template to obtain a complete gene sequence;

designing a primer pair G20E03-ORF-R-F and G20E03-ORF-R-R, carrying out PCR amplification to obtain an ORF reverse sequence containing Xbal and BamH I enzyme cutting sites, carrying out enzyme cutting by using restriction enzymes Xbal and BamH I respectively, purifying and recovering, and connecting to a plant interference vector pFGC5941 cut by the restriction enzymes Xbal and BamH I to obtain a recombinant expression vector pFGC5941-G20E 03;

designing a primer pair G20E03-ORF-F-F and G20E03-ORF-F-R, carrying out PCR amplification to obtain an ORF forward sequence containing Nco I and Swa I enzyme cutting sites, carrying out enzyme cutting on a recombinant expression vector pFGC5941-G20E03 by using restriction enzymes Nco I and Swa I respectively, purifying and recycling, and connecting to a vector pFGC5941-G20E03 cut by the restriction enzymes Nco I and Swa I to obtain a recombinant expression vector pFGC5941-RNAi-G20E 03.

Preferably, the RNAi transgenic tobacco plant for obtaining the gene G20E03 by transgenic technology further comprises:

carrying out plug seedling on T0 generation transgenic tobacco seeds by adopting seed plugs, and respectively carrying out PCR detection by utilizing specific primers G20E03-part-F, G20E03-part-R, Bar-F and Bar-R to obtain T1 generation transgenic tobacco;

and (4) carrying out generation-by-generation culture according to the method to obtain a homozygous transgenic line.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a gene G20E03 capable of improving soybean cyst nematode resistance of tobacco plants and protein coded by the gene, and proves that the protein coded by the gene plays an important role in soybean cyst nematode parasitizing tobacco, can inhibit tobacco immune response, realizes silent expression of the gene G20E03 by a transgenic technology, thereby improving the cyst nematode resistance of the tobacco and laying a good theoretical and practical foundation for cultivating soybean cyst nematode resistant tobacco varieties.

Drawings

FIG. 1 shows the expression levels of the G20E03 gene at different time points before and after invading the host, wherein pre-J2 represents 2-instar larvae before SCNT infection, and lower case letters represent that the difference significance P is less than 0.05;

FIG. 2 shows the result of electrophoresis for amplifying a partial sequence of the G20E03 gene, wherein M: DL2000Marker, 1: G20E03 partial fragment;

FIG. 3 shows the electrophoresis result of RACE clone G20E03 gene, wherein A: electrophoresis result of amplified G20E03 gene 5' region sequence, M: DL2000Marker, 1: G20E 035' fragment, B: electrophoresis result of amplifying the sequence of the 3' region of the G20E03 gene, M: DL2000Marker, 2: a G20E 033' fragment;

FIG. 4 shows the result of electrophoresis for amplifying the ORF region sequence of the G20E03 gene, wherein M: DL2000Marker, 1: a G20E03ORF fragment;

FIG. 5 is a graph demonstrating the immunosuppressive function of G20E 03;

FIG. 6 shows the gene silencing efficiency test of G20E03 after SCNT invades tobacco, wherein WT is wild type tobacco, and L17 and L26 are RNAi strain tobacco;

FIG. 7 shows the cyst and female numbers of wild-type and interferent lines L17 and L26 after inoculation with SCNT 35d, wherein WT, L17 and L26 represent wild-type and RNAi lines, respectively, inoculated with SCNT 35 d.

Detailed Description

The technical solutions in the embodiments of the present invention will be fully described in detail below with reference to the accompanying drawings. It is obvious that the described embodiments are only some specific embodiments, not all embodiments, of the general technical solution of the present invention. All other embodiments, which can be derived by a person skilled in the art from the general idea of the invention, fall within the scope of protection of the invention.

The invention provides a gene G20E03, and the full-length sequence of the gene G20E03 is shown as SEQ ID NO. 1.

In a preferred embodiment, the full-length gene sequence of the gene G20E03 comprises a 5 'end non-coding region, a 3' end non-coding region and a cDNA region, and the gene sequence of the cDNA region is shown as SEQ ID NO. 2.

In a preferred embodiment, the gene G20E03 is cloned by the following method:

extracting 2-instar larva RNA of a soybean cyst nematode tobacco population by using a TRIZOL method, and performing reverse transcription to obtain cDNA;

designing a primer pair G20E03-part-F and G20E03-part-R according to a transcriptome sequencing result of SCNT, carrying out PCR amplification by taking the cDNA as a template to obtain a PCR product, purifying the PCR product and carrying out T-A connection to obtain a connection product, transferring the connection product into escherichia coli, randomly picking a single colony, carrying out PCR screening on the single colony by utilizing the primer pair G20E03-part-F and G20E03-part-R to obtain a positive single clone colony and sequencing;

according to the partial sequence of the G20E03 gene, specific primers G20E03-5 '-R and G20E 03-3' -F of 5 'end non-coding region and 3' end non-coding region genes are designed, PCR amplification is carried out, 260bp fragments are amplified at the 5 'end, and 666bp fragments are amplified at the 3' end;

and splicing the sequences, and cloning to obtain the gene full-length sequence of the gene G20E 03.

In the above technical scheme, G20E03-part-F and G20E03-part-R are used as specific primer pairs to perform PCR amplification to obtain cDNA fragments, and G20E03-5 '-R and G20E 03-3' -F are used as specific primer pairs to perform PCR amplification to obtain 5 'end non-coding region fragments and 3' end non-coding region fragments, wherein the primer pairs are not primer pairs obtained by conventional selection, but are designed according to sequences obtained by sequencing the transcriptome of SCNT 2 instar larvae.

In another aspect, the invention provides a protein encoded by the gene G20E03 according to any one of the above technical schemes, wherein the amino acid sequence of the protein is shown as SEQ ID NO. 3.

The invention also provides application of the protein in improving soybean cyst nematode resistance of tobacco plants. dsRNA of a gene G20E03 in a transgenic tobacco plant enters a nematode body through the feeding of the nematode to initiate the inactivation of the gene, so that the parasitic capacity of cyst nematodes is remarkably reduced, the infection, the parasitism, the propagation and the spread of the cyst nematodes are inhibited, and the disease resistance of the tobacco plant is further improved. The effector protein plays an important role in the interaction process of the plant parasitic nematode and the host, determines the pathogenic strength of the nematode to the host, and is also a key factor for generating plant resistance. The effect protein synthesized by the nematode is injected into the host plant body through the oral needle, thereby being beneficial to the infection, the parasitism and the growth and the development of the nematode. It is to be noted that the amino acid sequence of the protein G20E03 encoded by the gene G20E03 is shown in SEQ ID NO. 3. Also, derivative proteins which are substituted and/or deleted and/or added with one or more amino acid residues and are related to soybean cyst nematode parasitism are also within the scope of the present invention.

The invention also provides application of the gene G20E03 in any technical scheme in improving soybean cyst nematode resistance of tobacco plants. It should be noted that, in the prior art, the cyst nematode parasitizing tobaccos in shandong province is identified as soybean cyst nematode (Heterodera glycines) by using identification means such as morphology and molecular biology, the population is SCNT, and research is performed on the pathogenic difference between SCN and SCNT, however, on the basis, a strong pathogenic gene of SCNT can be obtained, and still a great deal of creative labor is required, including: firstly, obtaining 13 differentially expressed genes for coding effector protein by comparing transcriptomics, and determining genes most probably causing the tobacco plants to be infected with soybean cyst nematode from the genes; then, whether the gene regulates and controls the immune response of the host plant is researched; to further clarify the effect of the effector protein G20E03 on the ability of SCNT to infect tobacco, research on RNAi transgenic plants of G20E03 gene was also conducted. Specifically, the method comprises the following steps:

firstly, 13 differentially expressed genes for coding effector protein are obtained through comparison transcriptomics, and the expression quantity of the 13 genes of SCNT 2-instar larvae at different time when the nematode infects the host is researched by utilizing fluorescent quantitative PCR. And (3) respectively inoculating the 2-instar larvae of the SCNT into the tobacco for 1, 2, 3, 4, 5 and 6 days, separating the 2-instar larvae of the root system of the tobacco, extracting RNA of the 2-instar larvae, and detecting the absolute expression quantity of 13 genes of the 2-instar larvae of the SCNT invading the root system by fluorescence quantitative PCR. The results show that compared with 2-instar larvae (pre-J2), the expression level of the G20E03 gene of the 2-instar larvae in the tobacco roots is obviously higher than that of the 2 nd, 3 rd, 4 th, 5 th and 6 th d before infection. Wherein the expression level is the highest at 3d infection, which is up to 33.15 times higher than that of pre-J2 (shown in FIG. 1). Therefore, the gene is related to early infection of the host by the nematode.

On the basis of the research, in order to confirm whether the gene regulates the immune response of the host plant, firstly, the full length of the gene is cloned by using a RACE method to obtain the full sequence of the gene (shown in figures 2, 3 and 4); secondly, a pGR107 vector containing a cDNA sequence of the G20E03 gene is constructed, the recombinant vector is transformed into the rhizoctonia solani, a negative control GFP gene and a G20E03 gene are respectively permeated at the left side and the right side of the back of the tobacco leaf of the indigenous tobacco under high pressure by using a 1mL injector, and obvious necrosis (PCD) can be observed in a GFP injection area after 7 days; whereas the leaf blade injected with G20E03 showed no or relatively little necrosis (as shown in fig. 5). The above results indicate that G20E03 can inhibit the immune response of tobacco.

In order to further confirm the influence of the effector protein G20E03 on the SCNT infection capacity of tobacco, the invention also carries out the research of RNAi transgenic plants of the G20E03 gene. The RNAi plant expression vector of the G20E03 gene is successfully constructed, after agrobacterium LBA4404 is transformed, the recombinant vector is transferred into tobacco by a tobacco leaf disc method, dsRNA of G20E03 is generated in the tobacco, the plant can obviously inhibit parasitism of soybean cyst nematode, the tobacco with strong resistance to cyst nematode is obtained, and a solid theoretical basis and practical guidance are provided for preventing and treating soybean cyst nematode diseases of the tobacco.

DNA molecules which hybridize with the above effector gene G20E03 and encode proteins associated with soybean cyst nematode parasitism, or DNA molecules which have more than 90% homology with the above soybean cyst nematode effector gene G20E03 and encode proteins associated with soybean cyst nematode parasitism are also within the scope of the present invention.

In a preferred embodiment, the method comprises the following steps: RNAi transgenic tobacco plants of the gene G20E03 are obtained through a transgenic technology, dsRNA of the gene G20E03 is expressed in the transgenic tobacco plants, the soybean cyst nematode gene G20E03 is silenced, the immunosuppressive effect of the gene G20E03 on the tobacco plants is relieved, and the soybean cyst nematode resistance of the tobacco plants is improved. The Inplanta RNA interference (RNAi) is characterized in that dsRNA with a hairpin structure is expressed in a plant host body, and a nematode feeds into the host body through a mouth needle to induce RNAi effect of a target gene, so that the target gene is silenced, the growth, development and reproduction of the nematode where the target gene is located are influenced, and even a lethal effect is generated. Because of the special parasitism of soybean cyst nematodes, RNAi is widely applied to the research of nematode gene functions as a reverse genetics technology.

In a preferred embodiment, the obtaining of the RNAi transgenic tobacco plant of the gene G20E03 by transgenic technology comprises the following steps:

constructing a recombinant expression vector pFGC5941-RNAi-G20E 03;

transforming agrobacterium LBA4404 by a recombinant expression vector pFGC5941-RNAi-G20E03 to obtain recombinant agrobacterium;

tobacco is transformed by an agrobacterium-mediated leaf disc method, tissue culture is carried out, and transgenic tobacco plants which are positive in detection by using primers G20E03-part-F, G20E03-part-R, Bar-F and Bar-R are RNAi transgenic tobacco plants of G20E 03.

The pFGC5941 is a plant RNAi vector, forward and reverse sequences of a tobacco population effect gene G20E03 containing soybean cyst nematodes are respectively cloned into a plant expression vector, the recombinant expression vector is transformed into agrobacterium and then is transformed into a tobacco body through a tobacco leaf disc method, dsRNA of the gene can be generated in the tobacco, and after the cyst nematodes infect the plant, the dsRNA enters the cyst nematodes to degrade mRNA of the gene, so that the inactivation of the gene is initiated, the parasitic capacity of the cyst nematodes is remarkably reduced, and the infection, the parasitism, the propagation and the spread of the cyst nematodes are inhibited.

In a preferred embodiment, the construction of recombinant expression vector pFGC5941-RNAi-G20E03 comprises the following steps:

cloning a G20E03 gene by an RACE method by taking soybean cyst nematode cDNA as a template to obtain a complete gene sequence;

designing a primer pair G20E03-ORF-R-F and G20E03-ORF-R-R, carrying out PCR amplification to obtain an ORF reverse sequence containing Xbal and BamH I enzyme cutting sites, carrying out enzyme cutting by using restriction enzymes Xbal and BamH I respectively, purifying and recovering, and connecting to a plant interference vector pFGC5941 cut by the restriction enzymes Xbal and BamH I to obtain a recombinant expression vector pFGC5941-G20E 03;

designing a primer pair G20E03-ORF-F-F and G20E03-ORF-F-R, carrying out PCR amplification to obtain an ORF forward sequence containing Nco I and Swa I enzyme cutting sites, carrying out enzyme cutting on a recombinant expression vector pFGC5941-G20E03 by using restriction enzymes Nco I and Swa I respectively, purifying and recycling, and connecting to a vector pFGC5941-G20E03 cut by the restriction enzymes Nco I and Swa I to obtain a recombinant expression vector pFGC5941-RNAi-G20E 03.

In a preferred embodiment, the obtaining of the RNAi transgenic tobacco plant of gene G20E03 by transgenic technology further comprises:

carrying out plug seedling on T0 generation transgenic tobacco seeds by adopting seed plugs, and respectively carrying out PCR detection by utilizing specific primers G20E03-part-F, G20E03-part-R, Bar-F and Bar-R to obtain T1 generation transgenic tobacco;

and (4) carrying out generation-by-generation culture according to the method to obtain a homozygous transgenic line.

In order to more clearly and specifically describe the gene G20E03, the protein encoded by the gene and the application of the gene in improving the soybean cyst nematode resistance of tobacco plants provided by the embodiments of the present invention, the following description will be provided with reference to specific examples.

Tobacco K326: purchased from the tobacco institute of the Chinese academy of agricultural sciences; soybean cyst nematode tobacco population: storing in a plant nematode research room of Qingdao agricultural university; vectors pFGC5941, Agrobacterium strains GV3101 and LBA4404 were all stored by the university of Qingdao plant nematode research laboratory.