阅读说明：本技术 一种可提高大豆蚜虫抗性基因的重组载体及其应用 (Recombinant vector capable of improving soybean aphid resistance gene and application thereof ) 是由 姚陆铭 王彪 马晓红 武天龙 于 2021-06-10 设计创作，主要内容包括：本发明公开了一种可提高大豆蚜虫抗性基因的重组载体及其应用,涉及植物细胞技术领域,该重组载体为pGm109-35S-GmPti5重组载体,包括植物双元表达载体pGM109和GmPti5基因；GmPti5基因核苷酸序列如SEQ ID NO.1所示。公开了该重组载体在构建抗蚜虫大豆株系中的应用。还公开了一种可提高大豆蚜虫抗性基因在构建抗蚜虫大豆株系中的应用,该基因为GmPti5基因,编码蛋白质的氨基酸序列如SEQ ID NO.2所示。本发明公开了调控大豆对蚜虫抗性的基因GmPti5,构建重组载体,该基因过量表达能够提高大豆对于蚜虫的抗性,能减少农药及人力的投入,保证大豆产量的稳定性,应用前景广阔。(The invention discloses a recombinant vector capable of improving soybean aphid resistance genes and application thereof, and relates to the technical field of plant cells, wherein the recombinant vector is pGm109-35S-GmPti5 recombinant vector and comprises plant binary expression vector pGM109 and GmPti5 genes; the nucleotide sequence of the GmPti5 gene is shown in SEQ ID NO. 1. Discloses the application of the recombinant vector in constructing aphid-resistant soybean strains. Also discloses an application of the gene capable of improving the resistance of soybean aphids in constructing an aphid-resistant soybean strain, wherein the gene is a GmPti5 gene, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 2. The invention discloses a gene GmPti5 for regulating and controlling resistance of soybean to aphids, a recombinant vector is constructed, the resistance of soybean to aphids can be improved by overexpression of the gene, the investment of pesticide and manpower can be reduced, the stability of soybean yield is ensured, and the application prospect is wide.)

1. A recombinant vector capable of improving soybean aphid resistance genes is characterized in that the recombinant vector is pGm109-35S-GmPti5, and the pGm109-35S-GmPti5 recombinant vector comprises plant binary expression vectors pGM109 and GmPti5 genes; the nucleotide sequence of the GmPti5 gene is shown in SEQ ID NO. 1.

2. The recombinant vector according to claim 1, wherein the plant binary expression vector pGM109 comprises the cauliflower mosaic virus 35S promoter, Nde I and Sal I sites; the Nde I and Sal I sites are located after the cauliflower mosaic virus 35S promoter; the GmPti5 gene was inserted in the forward direction between the Nde I and Sal I sites.

3. Use of a recombinant vector according to claim 1 or 2 for the construction of an aphid-resistant soybean line.

4. A use according to claim 3, characterized in that the method of application comprises the steps of:

step 1, synthesizing soybean first strand cDNA: extracting total RNA of the aphid-resistant soybean variety, carrying out reverse transcription to obtain first-strand cDNA, and carrying out first PCR amplification by taking the first-strand cDNA as a template to obtain an amplification product GmPti5 gene full-length cDNA;

step 2, constructing an over-expression recombinant vector pGm109-35S-GmPti5, taking the full-length cDNA of the GmPti5 gene obtained in the step 1 as a template, performing second PCR amplification by using a specific primer containing Nde I and Sal I joints to obtain a second amplification product, performing double enzyme digestion on the second amplification product through Nde I and Sal I, recovering to obtain a double enzyme digestion product, and positively inserting the double enzyme digestion product between Nde I and Sal I sites behind a cauliflower mosaic virus 35S promoter of a plant binary expression vector pGM109 to obtain a recombinant vector pGm109-35S-GmPti 5;

step 3, transforming the recombinant vector pGm109-35S-GmPti5 obtained in the step 2 into a soybean susceptible aphid material through an agrobacterium-mediated cotyledonary node method to obtain a transformed soybean susceptible aphid material; co-culturing, bud inducing, rooting culturing and planting the transformed soybean susceptible aphid material to obtain transgenic soybeans; transforming and screening the transgenic soybean, and analyzing the expression quantity to obtain a positive strain; and continuously screening the positive strains to obtain the aphid-resistant soybean material with stable inheritance.

5. The method of claim 4, wherein in step 1, the first PCR amplification primer sequence is:

an upstream primer: 5'-ATGGGAAGCAAGAAAACCTAC-3'

A downstream primer: 5'-TTAACCTGTGCCACTTGTGA-3' are provided.

6. The method according to claim 4, wherein in step 2, the specific primers containing Nde I and Sal I linkers are:

an upstream primer: 5'-CGCCATATGATGGGAAGCAAGAAAACCTAC-3'

A downstream primer: 5'-CGCGTCGACTTAACCTGTGCCACTTGTGA-3' are provided.

7. The method according to claim 4, wherein said positive lines are subjected to said continuous screening for 3 generations in said step 3, obtaining said stably inherited aphid-resistant soybean material.

8. The method of claim 4, wherein step 3 further comprises detecting aphid resistance in said stably inherited aphid-resistant soybean material.

9. The use of claim 4, wherein said co-cultivation time in step 3 is 5 days and said shoot induction cultivation time is 3 weeks.

10. The application of the gene capable of improving the soybean aphid resistance in constructing an aphid-resistant soybean strain is characterized in that the gene capable of improving the soybean aphid resistance is a GmPti5 gene, the nucleotide sequence of the GmPti5 gene is shown as SEQ ID No.1, and the amino acid sequence of the protein coded by the GmPti5 gene is shown as SEQ ID No. 2.

Technical Field

The invention relates to the technical field of plant cells, in particular to a recombinant vector of a gene capable of improving soybean aphid resistance and application thereof.

Background

Aphids are one of the major pests encountered in soybean production and are also important causes affecting soybean yield. The natural resistance of the soybeans to aphids is improved, so that the normal growth of the soybeans can be guaranteed, the investment of pesticides and manpower can be reduced, and the stability of the yield of the soybeans is guaranteed. At present, no soybean variety resisting aphids appears in production, and the prevention and the treatment of the soybean aphids are still mainly carried out through chemical prevention and treatment. Therefore, the improvement of the resistance of the soybean to aphids is an important problem to be solved urgently in soybean production.

Different soybean varieties have great difference in resistance to aphids, and the insect-resistant varieties have higher aphid resistance. The discovery and utilization of the key gene of aphid resistance in soybean have important significance for breeding aphid-resistant soybean varieties.

Accordingly, those skilled in the art are devoted to the discovery and cloning of genes that increase soybean aphid resistance and to the development of a method for increasing soybean aphid resistance by overexpressing an ethylene response factor and the product encoded thereby.

Disclosure of Invention

In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to develop a method for improving aphid resistance of soybean by over-expressing an ethylene response factor and a product coded by the ethylene response factor.

In order to realize the purpose, the invention provides a recombinant vector capable of improving soybean aphid resistance genes and an application method thereof in constructing aphid-resistant soybean strains; provides a gene capable of improving soybean aphid resistance and an application method thereof in constructing an aphid-resistant soybean strain.

The invention provides a recombinant vector capable of improving soybean aphid resistance genes, which is pGm109-35S-GmPti5 recombinant vector, pGm109-35S-GmPti5 recombinant vector comprises plant binary expression vector pGM109 and GmPti5 genes; the nucleotide sequence of the GmPti5 gene is shown in SEQ ID NO. 1.

Further, the plant binary expression vector pGM109 includes a cauliflower mosaic virus 35S promoter, Nde I and Sal I sites; nde I and Sal I sites are located after the cauliflower mosaic virus 35S promoter; the GmPti5 gene was inserted forward between Nde I and Sal I sites.

The invention also provides application of the recombinant vector capable of improving the soybean aphid resistance gene in constructing an aphid-resistant soybean strain.

The method for applying the recombinant vector in constructing the aphid-resistant soybean strain comprises the following steps:

step 1, synthesizing soybean first strand cDNA: extracting total RNA of the aphid-resistant soybean variety, carrying out reverse transcription to obtain first-strand cDNA, and carrying out first PCR amplification by taking the first-strand cDNA as a template to obtain an amplification product GmPti5 gene full-length cDNA;

step 2, constructing an over-expression recombinant vector pGm109-35S-GmPti5, taking the full-length cDNA of the GmPti5 gene obtained in the step 1 as a template, performing second PCR amplification by using a specific primer containing Nde I and Sal I joints to obtain a second amplification product, performing double enzyme digestion on the second amplification product through Nde I and Sal I, recovering to obtain a double enzyme digestion product, and positively inserting the double enzyme digestion product into a position between Nde I and Sal I sites behind a cauliflower mosaic virus 35S promoter of a plant binary expression vector pGM109 to obtain a recombinant vector pGm109-35S-GmPti 5;

step 3, transforming the recombinant vector pGm109-35S-GmPti5 obtained in the step 2 into a soybean susceptible aphid material through an agrobacterium-mediated cotyledonary node method to obtain a transformed soybean susceptible aphid material; co-culturing, bud inducing, rooting culturing and planting the transformed soybean susceptible aphid material to obtain transgenic soybean; transforming and screening the transgenic soybean, and analyzing the expression quantity to obtain a positive strain; and continuously screening the positive strains to obtain the aphid-resistant soybean material with stable inheritance.

Further, the first PCR amplification primer sequence is:

an upstream primer: 5'-ATGGGAAGCAAGAAAACCTAC-3'

A downstream primer: 5'-TTAACCTGTGCCACTTGTGA-3' are provided.

Further, specific primers containing Nde I and Sal I linkers were:

an upstream primer: 5'-CGCCATATGATGGGAAGCAAGAAAACCTAC-3'

A downstream primer: 5'-CGCGTCGACTTAACCTGTGCCACTTGTGA-3' are provided.

Further, the positive strains are continuously screened for 3 generations in the step 3, and the aphid-resistant soybean material with stable inheritance is obtained.

Further, step 3 also comprises detecting aphid resistance of the stably inherited aphid-resistant soybean material.

Further, the co-culture time in step 3 was 5 days, and the shoot induction culture time was 3 weeks.

The invention also provides application of the gene capable of improving the resistance of soybean aphids in constructing an aphid-resistant soybean strain, wherein the gene capable of improving the resistance of the soybean aphids is a GmPti5 gene, the nucleotide sequence of the GmPti5 gene is shown in SEQ ID No.1, and the amino acid sequence of the protein coded by the GmPti5 gene is shown in SEQ ID No. 2.

In the preferred embodiment of the invention, the discovery and cloning method of the GmPti5 gene which can improve the resistance of soybean to aphids by over-expression is detailed;

in another preferred embodiment of the present invention, a method for constructing an aphid-resistant soybean line using the GmPti5 gene is described in detail.

The technical effect of the invention is shown in that the gene GmPti5 for regulating and controlling the resistance of soybean to aphids is disclosed, a recombinant vector capable of improving the resistance gene of soybean aphids is constructed, and the resistance of soybean to aphids can be improved by the overexpression of the gene. The natural resistance of the soybeans to aphids is improved, the normal growth of the soybeans can be guaranteed, the input of pesticides and manpower can be reduced, and the stability of the yield of the soybeans is guaranteed. The GmPti5 has wide application prospect in breeding aphid-resistant soybean varieties.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 is a schematic diagram showing the construction of the GmPti5 gene expression vector according to a preferred embodiment of the present invention;

FIG. 2 is a histogram of expression levels of soybean lines overexpressing GmPti5 of a preferred embodiment 2 of the present invention;

FIG. 3 is a histogram of aphid resistance of soybean lines overexpressing GmPti5 of a preferred embodiment 2 of the present invention.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

Example 1 discovery and cloning of GmPti5 Gene

(1) Discovery of GmPti5 gene: a recombinant inbred line is constructed by an aphid-resistant soybean material P746 and an aphid-susceptible variety Dongnong 47, a high-density genetic linkage map and map-based cloning are constructed, differential genes related to aphid resistance are found, the genes are compared with a public database, and the genes are found to belong to an ethylene dependent factor family, which is named as Glycine max genetic-related genes transgenic activator 5, GmPti 5.

(2) Acquisition of GmPti5 gene: extracting RNA of aphid-resistant soybean material P746, obtaining first strand cDNA through reverse transcription, and carrying out PCR amplification of GmPti5 gene by using the first strand cDNA as a template, wherein the amplification primers are as follows:

an upstream primer: 5'-ATGGGAAGCAAGAAAACCTAC-3'

A downstream primer: 5'-TTAACCTGTGCCACTTGTGA-3' are provided.

Example 2 construction of aphid-resistant Soybean lines Using the GmPti5 Gene

(1) Construction of Soybean transformation vector

The full-length cDNA of the GmPti5 gene obtained in example 1 was used as a template, PCR amplification was performed using a specific primer containing Nde I and Sal I linkers, and the amplification product was digested with Nde I and Sal I, recovered, and inserted forward between the Nde I and Sal I sites behind the cauliflower mosaic virus 35S promoter of the plant binary expression vector pGM109, to obtain a recombinant vector pGm109-35S-GmPti5, as shown in FIG. 1.

The specific primers are as follows:

an upstream primer: 5'-CGCCATATGATGGGAAGCAAGAAAACCTAC-3'

A downstream primer: 5'-CGCGTCGACTTAACCTGTGCCACTTGTGA-3'

(2) Agrobacterium-mediated cotyledonary node method for transforming soybean susceptible aphid material

Transforming the recombinant vector constructed in the step (1) into soybean susceptible aphid materials by an agrobacterium-mediated cotyledonary node method, and then carrying out co-culture, bud induction, rooting culture, planting, positive clone detection and continuous screening for 3 generations to obtain the aphid-resistant soybean materials with stable inheritance.

The specific operations of agrobacterium activation are as follows: single colonies containing the target gene were picked from the plate, inoculated into 5mL of YEP broth, supplemented with rifampicin 100. mu.g and kanamycin 50. mu.g per mL of YEP broth, and shake-cultured at 28 ℃ and 200rpm for 24h until OD600 reached 1.0.

The switching process specifically comprises the following steps: the activated bacterial liquid is prepared according to the following steps of 1: 100, transferring the mixture into LB liquid culture medium containing corresponding antibiotics at 28 ℃, shaking the shaking table at 200rpm for 6h, and using the mixture for transformation when OD600 reaches 0.6.

The infection process is as follows: infecting the growing point of the tender stem tip, after the seeds germinate for 1 day, cutting off embryo and radicle, soaking the rest part in the bacterial liquid for 30 minutes, taking out and placing on sterile filter paper to absorb the attached bacterial liquid.

The co-culture process comprises the steps of laying the infected explants on a culture dish containing the co-culture medium, sealing the culture dish by a sealing film, and performing black culture at 28 ℃ for 5 days.

Co-cultivation medium was supplemented with 0.25mg GA3 and 1.67mg BAP per liter MSB medium.

The bud induction culture process comprises the following steps: and transferring the explants after co-culture to a plate containing a selective differentiation solid culture medium, and culturing under the conditions that the temperature is 26 ℃, the illumination intensity is 5000lux, and the light is 16h/8h in dark.

The bud induction medium is prepared by adding 100mg Cef and 1.67mg BAP into each liter MSB medium.

Rooting culture, selecting bud, inducing and culturing for 3 weeks, cutting the adventitious bud when the adventitious bud grows to 2cm, and transferring to a triangular flask containing rooting culture medium for rooting culture.

The rooting medium is added with 100 mug IBA per liter of MSB medium.

The planting process specifically comprises the following steps: transplanting into a flowerpot when the root grows to 2cm and the seedling is 7cm high, and culturing in a greenhouse.

(3) Screening and expression quantity analysis of transformation positive strain

Extracting genome DNA of the transgenic soybean seedlings, and further detecting and screening positive plants after PCR amplification.

The sequence of the amplification primer is as follows:

an upstream primer: 5'-GCCACAAATTCATAACACAACAAG-3'

A downstream primer: 5'-TACATCACAATCACACAAAACTAAC-3'

And extracting the total RNA of the obtained positive plants, performing qPCR analysis, detecting the gene expression conditions of different transgenic lines, and obtaining the transgenic line with stably inherited GmPti5 gene over-expression.

The qPCRR specific primer sequence is as follows:

an upstream primer: 5'-AAAGAAGCACTACAGAGGAGTC-3'

A downstream primer: 5'-GCAACAATTTCAGCTGGGAAAT-3'

The specific primer sequence of the soybean internal reference gene Fbox used for qPCR is as follows:

an upstream primer: 5'-TTGCTTATTCAATCCGCTTGTC-3'

A downstream primer: 5'-TAAACAAATTCTTGAGCACCCG-3' are provided.

The screening was continued to observe segregation at T1, and this was repeated until T3 generation obtained genetically stable transgenic lines, and the genetically stable transgenic lines and the control line were tested for aphid resistance.

The aphid resistance detection method comprises the following steps:

and (3) planting the soybean transgenic line and the control strain in greenhouse pot plants, wherein each line is planted with 10 pots, 1 plant is planted in each pot plant, aphids which are hatched for 1 day are inoculated on the front surfaces of the first pair of three compound leaves, and the number of the aphids on the plants is counted after 7 days.

As shown in fig. 2, in the 4T 0 generation strains overexpressing the GmPti5 gene obtained in example 2, the expression level of the GmPti5 gene in L1 and L6 was significantly higher than that of the control variety, compared to the control variety. As shown in fig. 3, the resistance of these 4 stably inherited GmPti5 overexpressing transgenic material to aphids was increased by 16.5% -60.1% compared to the control variety.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Sequence listing

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