CN117568289B - Protein for resisting soybean cyst nematode disease, encoding gene and application thereof - Google Patents
Protein for resisting soybean cyst nematode disease, encoding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a soybean cyst nematode disease resistant protein, and a coding gene and application thereof, and belongs to the technical field of plant disease resistance. In order to provide a soybean gene for resisting soybean cyst nematode, the technical problem of how to resist the cyst nematode in soybean is solved. The invention provides a soybean cyst nematode disease resistant protein, the sequence of which is shown as SEQ ID NO. 20. GmGH3-2 is a gene with the effect of resisting soybean cyst nematodes, and excessive expression of the gene in soybean can obviously improve the resistance of roots in recipient soybean germplasm to cyst nematodes, provide effective molecular markers and gene resources for soybean cyst nematode resistant molecular design breeding, and have important theoretical significance and practical value for realizing cyst nematode resistant strain transgenic breeding, accelerating the pest resistant breeding process and improving breeding efficiency.
Description
Technical Field
The invention belongs to the technical field of plant disease resistance, and particularly relates to a soybean cyst nematode disease resistant protein, and a coding gene and application thereof.
Background
Soybean (Glycine max) is a food or commercial crop. Soybean cyst nematode disease (SCN, heterodera Glyines Ichinohe) is the main cause of soybean yield and quality loss. 16 physiological races have been found. At present, physiological race comprises SCN1-7, 9 and 14, wherein the race 3 and the race 4 are main dominant race in northeast soybean production area; the No. 1 and No. 4 physiological race are main dominant race of Huang-Huai-Hai soybean production area, and among the identified race, the No. 4 physiological race has the strongest pathogenicity. In long breeding practice, the application of a few anti-SCN germplasm resources causes the limitation of narrow genetic foundation of disease-resistant variety resources. Many studies have shown that RNAseq-based transcriptome analysis has become one of the most effective strategies for profiling genetic basis of complex trait variations, particularly plant interactions with pests. In the research of gene excavation of soybean cyst nematode resistance, transcriptome sequencing analysis under different soybean anti-sense varieties including Harbin black beans (disease resistance), ash-skin branch black beans (disease resistance), wuzhai black beans (disease resistance), peking (disease resistance), lee (disease resistance), essex (disease resistance), soybean Williams 82 (Glycine max cv. Williams) and the like are carried out on HG type 1.2.5.7 (2 # physiological race), HG type 0 (3 # physiological race), HG type 1.2.3.5.7 (4 # physiological race) and the like under the condition of small stress, so that the expression multiple (Fold Change) of different genes between groups on the transcription level is obtained, and related genes of the pathways such as disease resistance, cell structure, signal transduction, cell wall repair, protein degradation pathway and the like are analyzed, and the gene functions are continuously verified by using methods such as over-expression, gene silencing, fluorescent signal marking and the like. Thus, the complex mechanism by which soybean interacts with SCN suggests that different soybean varieties and different physiological races can produce different gene expression changes.
Disclosure of Invention
The invention aims to provide a soybean gene for resisting soybean cyst nematodes, which solves the technical problem of how to resist soybean cyst nematodes.
The invention provides a soybean cyst nematode disease resistant protein, the sequence of which is shown as SEQ ID NO. 20.
The invention provides the encoding gene of the soybean cyst nematode disease resistant protein.
Further defined, the sequence of the coding gene is shown as SEQ ID NO. 3.
The invention provides a recombinant vector of the coding gene.
Further defined, the starting vector of the recombinant vector is pCAMBIA3300.
The present invention provides a recombinant microbial cell carrying the above gene or expressing the above protein.
Further defined, the recombinant microbial cell is a prokaryotic microbial cell or a eukaryotic microbial cell.
The invention provides a method for preparing plants resistant to soybean cyst nematode, which comprises the following specific steps:
Step 1: connecting the gene shown in SEQ ID NO.3 with a vector pCAMBIA3300 vector to obtain a recombinant vector;
step 2: transforming the recombinant vector in the step 1 into agrobacterium to obtain recombinant agrobacterium;
step 3: transferring the recombinant agrobacterium of the step 2 into soybeans to obtain transgenic soybean plants, and identifying to obtain positive transgenic soybean plants.
The invention provides a method for preparing a plant for improving the sensitivity of soybean cyst nematode, which comprises the following specific steps:
step S1: the sgRNA sequence shown in SEQ ID NO.21 is connected with a carrier PYLCRISPR/Cas9 carrier to obtain a recombinant carrier;
step S2: transforming the recombinant vector in the step 1 into agrobacterium to obtain recombinant agrobacterium;
step S3: transferring the recombinant agrobacterium of the step 2 into soybeans to obtain transgenic soybean plants, and identifying to obtain positive transgenic soybean plants.
The invention provides a method for improving soybean resistance to soybean cyst nematode, which utilizes soybean cyst nematode to infect plants which overexpress the gene shown in SEQ ID NO. 3.
The invention provides the protein for resisting the soybean cyst nematode, the gene, the recombinant vector, the recombinant microorganism cell, a soybean plant over-expressing the gene shown in SEQ ID NO.3, a soybean plant containing the gene shown in SEQ ID NO.22 or the application of knocking out the gene shown in SEQ ID NO.3 in controlling the resistance of soybean to the cyst nematode.
The beneficial effects are that: the average value of the number of the transgenic positive root females of pCAMBIA3300-GmGH3-2 is 1.83/cm, which is lower than that of the control group, the average value of the number of the mutant positive root females of GmGH3-2 is 3.79/cm, and according to the female index, the transgenic positive root and the negative control root have extremely obvious difference, so that the GmGH3-2 gene has obvious resistance to the soybean cyst nematode disease.
Drawings
FIG. 1 is a chromosome distribution of the GmGH gene of FIG. 1;
FIG. 2 shows GmGH gene structure and conserved motif composition; and (3) injection: a pedigree evolutionary tree of the GmGH3 gene; b. motif composition; c. a genetic construct;
FIG. 3 is a diagram showing the expression pattern of GmGH gene family in soybean tissue;
FIG. 4 is a GmGH gene promoter cis-acting element analysis; and (3) injection: a.GmGH3 gene promoter element distribution; b. the number of different promoter elements corresponds to GmGH; c. percentage of four types of promoter elements;
FIG. 5 is GmGH gene GO annotation and KEGG pathway information; and (3) injection: go notes GmGH protein. Blue represents molecular function (Molecular function, MF), purple represents biological process (Biological process, BP; b. Both parts of molecular function and biological process each account for a percentage; c.gmgh3 protein-related KEGG pathway;
FIG. 6 is Dongnong L-10 root RNA extraction;
FIG. 7 is a cloning of GmGH-2 genes; m: DL2000;1-2: a PCR product; 3: water control;
FIG. 8 shows transformation of E.coli with a plant expression vector; m: DL2000;1-9: a PCR product; 10 negative control;
FIG. 9 shows the PCR detection result of Agrobacterium rhizogenes bacterial liquid of pCAMBIA 3300-GmGH-2 recombinant plasmid; m: DL 2000 (+) DNA molecular weight standard; 1: a positive control; 2-10: recombining the positive transformant; 11: ddH2O negative control
FIG. 10 is a PCR identification of roots over-expressing Tonong 50; m is 2000Marker;1: a positive control; 16: a negative control; 2-15, over-expressing the Dongnong 50PCR product;
FIG. 11 is a transgenic positive root phenotype control with empty vector pCAMBIA3300 over-expressed root; and (3) injection: a. over-expressing pCAMBIA3300 empty control; pCAMBIA3300-GmGH3-2 transgenic positive roots, c.pYLCRISPR/Cas9-GmGH3-2 non-mutated positive roots; pYLCRISPR/Cas 9-GmGH-2 gene mutation positive root;
FIG. 12 is a diagram showing the result of PCR;
FIG. 13 is a graph showing mutation results;
FIG. 14 is a graph showing the results of soybean cyst nematode disease resistance.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The pharmaceutical agents used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Plant variety: dongnong L10 (disease resistance), sueinong 14 (disease susceptibility), dongnong 50 (disease susceptibility).
The anti-cyst nematode variety Dongnong L-10 and the susceptible variety Suiunong 14 are described in Wu Depeng, zhao Yue, cheng Bihan, et al, dongnong L-10's genetic model analysis of soybean cyst nematode No.3 physiological race [ J ]. Soy science, 2016,35 (3): 6.DOI: 10.11861/j.issn.1000-9841.2016.03.0367.
Disease soil type: the genetic model analysis of soybean cyst nematode HG type 1.2.3.5.7 (physiological race No. 3) is described in Wu Depeng, zhao Yue, cheng Bihan, et al, dongnong L-10 on soybean cyst nematode No. 3 physiological race [ J ]. Soy science, 2016,35 (3): 6.DOI:10.11861/J. Issn.1000-9841.2016.03.0367, from the Soy research institute of North-east agricultural university, test field.
Example 1.GmGH3-2 Gene-based bioinformatics analysis and cloning thereof
1) And Phytozome, searching homologous sequences of the GH3 protein of the arabidopsis by using Blastp, and identifying 28 soybean GH3 gene family members, named GmGH-1-GmGH-28 respectively, by analyzing a GH3 conserved domain in a pfam database. GmGH3 genes are distributed predominantly on 10 chromosomes of soybean (FIG. 1), predominantly on chr.8 and chr.12, containing 4 GmGH genes, respectively. In arabidopsis, GH3 proteins are divided into three subfamilies according to sequence similarity and specificity for adenylate phytohormones.
GmGH3 analysis of gene structure and conserved motifs. Early studies showed that structural diversity of genes is the primary driver of evolution of multiple gene families. To further understand the structural diversity of GmGH genes, the GmGH gene structure was analyzed using online software GSDS2.0, except GmGH3-28, where GmGH genes located in the same subfamily contain the same number of exons/introns, but differ in length. Protein conservation Motif prediction is an important method for protein analysis, and the GmGH protein Motif is identified through an online website MEME, and other GH3 genes contain Motif 1, motif 2 and Motif 3 except GmGH-7 and GmGH3-22 only contain Motif 1 (figure 2).
Further explored the role of GmGH gene in soybean growth and development, and analyzed the expression condition of GmGH gene in Phytozome database in 8 different development stages and different parts of soybean. The results show that the expression quantity of the same gene in different tissue positions is different, other members in subfamily I except GmGH < 3 > -16 and GmGH < 3 > -24 are mainly expressed in the stem tip, the expression quantity of other members is highest in the root, gmGH < 3 > -17 is not expressed in the root, leaf and lateral root, the same expression quantity is very low in other tissues, and the expression quantity of GmGH < 3 > gene in the nutrition organ is higher than that of the reproductive organ as a whole. The expression level of subfamily II is highest mainly in flowers and leaves, and when no flowers are opened, the expression levels of GmGH3-1, gmGH3-10, gmGH3-23, gmGH3-13, gmGH3-27, gmGH3-5 and GmGH3-19 are highest, and as the flowers are opened, the expression levels gradually decrease, and conversely, the expression levels of GmGH3-11, gmGH3-8, gmGH3-15, gmGH3-25, gmGH3-20, gmGH3-17 and GmGH3-18 show an upward trend. The expression levels of different genes at the same tissue site are also significantly different. The amount of GmGH-2 expressed in roots was much higher than that of other genes, and the amount of other members expressed in seeds except GmGH-11 was relatively small (FIG. 3).
Analysis of cis-acting elements in the region 2kb upstream of the GmGH gene promoter by using online software PLANTCARE shows that cis-elements in the GmGH gene promoter ARE mainly classified into 4 types, including abscisic Acid (ABRE), auxin (AuxRE and TCA-element), methyl jasmonate (CGTCA-motif), salicylic acid (TCA-element and TGA-Box), plant hormone response elements (Phytohormoe responsive) such as gibberellin (GARE-motif, TATC-Box and P-Box), light response elements (Light response element) such as GT1-motif, box4, TCCC-motif, I-Box, G-Box, and the like, elements involved in barrier mesophyll cell differentiation (HD-Zip 1), cell cycle regulation (MSA-like), growth-related elements (Plant growth and development) involved in meristem expression, stress response elements (Abiotic and biotic stresses) such as drought (MBS), low Temperature (LTR), anaerobic (ARE included, and the like, wherein the plant response elements respond to high frequency of the light response to the stress response of the plant elements (4 c) ARE shown in the figure. 28 GmGH genes each contain the cis-element ABRE, presumably members of the soybean GH3 family involved in the anabolic process with abscisic acid (fig. 4 b). The cis-elements are unevenly distributed over GmGH genes, and a number of cis-elements are preferentially present on individual genes (FIG. 4 a), gmGH contains a relatively large number of cis-element boxes 4, and is closely related to the photoreaction, and only GmGH-6 contains cis-element AuxRE, which is involved in the auxin metabolic pathway.
Note that: a.GmGH3 gene promoter element distribution; b. the number of different promoter elements corresponds to GmGH; c. percentage of four types of promoter elements
The biological function of GmGH proteins was further understood by GO annotation and KEGG pathway analysis. The GO annotation results indicated that 28 GmGH proteins were divided into 18 specific classes, including only the two processes of molecular function and biological process (FIG. 5 b) GmGH3 genes were mainly involved in plant stress processes, secondarily involved in signal transduction, catalytic activity, metabolic processes, etc., and furthermore had indole-3-acetic acid amino-synthase and ligase activity (FIG. 5 a). The KEGG pathway analysis results show that GmGH protein in subfamily I is mainly enriched in auxin pathway, and subfamily II GmGH protein is mainly enriched in JA pathway (figure 5 c), and is consistent with previous study, namely GH3 protein in subfamily II with IAA amino synthase activity can induce the expression of auxin, while subfamily GH3 protein with JA/SA amino synthase activity takes JA or SA as substrate, affects the biosynthesis of JA/SA, and participates in plant adversity stress reaction.
2) Taking soybean cyst nematode resistant variety Dongnong L-10 as material, sampling when the first group of three-leaf complex leaves grow out, extracting total RNA, detecting by agarose gel electrophoresis (figure 6), and detecting 18S and 28S RNA with clear bands by an ultraviolet spectrophotometer, wherein the OD260/OD280 ratio is between 1.9 and 2.0, which indicates that the RNA has good integrity and high purity.
3) Cloning target gene CDS. And adopting Phytozome v 12.1.1 database, carrying out blast comparison by using soybean Williams 82 (Glycine max Wm82.a2.v1) gene search sequence to obtain CDS sequence information of GmGH3-2, and designing a gene cloning primer (primer 1) by using Primer5.0 software. PCR reaction is carried out by taking cDNA as a template, and the reaction system is as follows: pre-denaturation at 98℃for 2min; changing the temperature to 98 ℃ for 10sec; annealing at 60 ℃ for 5sec; extension temperature was 68℃and 5sec/kb for 35 cycles and stored at 4 ℃. After the reaction, the PCR products were collected and subjected to agarose gel electrophoresis for detection, and the target fragment was recovered and purified by gel (FIG. 7). The PCR reaction system is as follows:
primer 1 overexpressing primer
GmGH3-2-S:AGAACACGGGGGACTATGGCAATTGCTAATTGTGATG(SEQ ID NO.1);GmGH3-2-A:AATCCTCTGTTTCTAGTTAGTGATGGTGATGGTGATGTAAAAAGAATAGAGATA(SEQ ID NO.2);
GmGH3 Gene sequence of 3-2 (SEQ ID NO.3)：ATGGCAATTGCTAATTGTGATGATAAGAACGCAAAAGCTTTGCAGTTCATTGAGGATATGACCCAAAACACTGACAGTGTCCAAAAGAGGGTCCTTGCTGAGATTCTGAGCCAAAACGCCAAGACCGAATACCTGAAACGGTTTGAGCTGAATGCAGCCACAGACCGTGACACTTTCAAGTCCAAGGTACCTGTCGTTTCATACGATGATTTGAAGCATGATATCCAACGCATTGCTAATGGTGACCGCTCTCCTATCCTATGCGCTCATCCAATTTCCGAGTTTCTCACCAGTTCTGGAACATCTGCTGGGGAGAGAAAATTGATGCCAACAATTCGTCAAGAGATGGACCGTCGTCAATTACTTTACAGCCTTCTGATGCCTGTGATGAACCAATACGTGCCTGATTTGGATAAGGGTAAGGCTCTACTCTTCTTGTTCATCAAAGCCGAGACAAAGACTCCGAGTGGGTTAGTGGCACGTCCAGTGCTAACTAGCCTCTACAAGAGCGACCAATTCAAGAACAGACCCTACGATCCATTCAACGTGTACACAAGCCCAGACGAAGCAATCCTCTGCCCCGATTCCTTCCAAAGCATGTACACCCAAATGCTGTGCGGCCTCATCATGCGCCACCAAGTCCTCAGAGTCGGAGCCGTTTTCGCCTCGGGCCTTCTCCGAGCCATCCGCTTCCTCCAGCTCAATTGGGCTGAACTAGCCCACGACATCTCCACCGGAACCCTAAACCCCAAAATCTCCGACCTTGCCATCAAACAACGCATGACCCAAATCCTCACACCAAATCCAGAACTCGCTGATTTCATTGTGAAAGAATGCTCGGGAGAAAACTGGGACCGCATAATCACAAGAATCTGGCCAAACACAAAGTATCTGGACGTGATTGTGACCGGTGCCATGGCGCAGTATATTCCGACCCTCGATTATTACAGCGGAGGCCTACCTAAAGCTTGCACCATGTACGCCTCATCCGAGTGTTACTTTGGGCTTAACCTAAACCCTATTTGCACCCCCTCTGACGTGTCCTACACCATCATGCCAAACATGGGTTACTTCGAGTTCCTTCCCCACGAAGAAGATTTATCTTCTAGCTCTTCAAGCTCCACCTTGTCACGTGACTCGCTCGACCTTGCAGATCTTGAACTCGGAAAATCCTACGAGCTCATCGTCACCACGTACTCCGGTCTGTGCCGTTACCGCGTAGGCGACATTCTCCAAGTCACGGGGTTCCACAACACCGCCCCACATTTCAGCTTCGTGAGGAGAAAAAACGTGCTTCTGAGCATCGACTCCGACAAGACCGACGAAGCGGAGTTACAGAACGCCGTCGAGAACGCCTCCGTGCTGCTGAGGGAGTTCAAGACGAGCGTGGCGGAGTACACGAGCTTCGCCGACACGAAGTCGATCCCGGGGCACTACGTGATTTACTGGGAACATCTATCTCTATTCTTTTTATAA;
GmGH3 amino acid sequence 3-2 (SEQ ID NO.20)：MAIANCDDKNAKALQFIEDMTQNTDSVQKRVLAEILSQNAKTEYLKRFELNAATDRDTFKSKVPVVSYDDLKHDIQRIANGDRSPILCAHPISEFLTSSGTSAGERKLMPTIRQEMDRRQLLYSLLMPVMNQYVPDLDKGKALLFLFIKAETKTPSGLVARPVLTSLYKSDQFKNRPYDPFNVYTSPDEAILCPDSFQSMYTQMLCGLIMRHQVLRVGAVFASGLLRAIRFLQLNWAELAHDISTGTLNPKISDLAIKQRMTQILTPNPELADFIVKECSGENWDRIITRIWPNTKYLDVIVTGAMAQYIPTLDYYSGGLPKACTMYASSECYFGLNLNPICTPSDVSYTIMPNMGYFEFLPHEEDLSSSSSSSTLSRDSLDLADLELGKSYELIVTTYSGLCRYRVGDILQVTGFHNTAPHFSFVRRKNVLLSIDSDKTDEAELQNAVENASVLLREFKTSVAEYTSFADTKSIPGHYVIYWEHLSLFFL.
EXAMPLE 2 construction of overexpression and mutation vectors and E.coli transformation
1) Referring to the kit instruction of TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 of Bao Ri biological company, the obtained gel recovery product is connected with a cloning vector pCAMBIA3300, and is converted into a competent cell DH5 alpha (figure 8), a monoclonal product is selected for bacterial liquid culture, a cloning primer is used as a positive cloning primer for identifying coliform liquid, 1 mu L of cultured bacterial liquid is selected as a template, and the bacterial liquid PCR amplification and the PCR reaction procedures are as follows: pre-denaturation at 94℃for 5min; denaturation at 94 ℃,30sec; annealing at 60 ℃ for 30sec; the extension temperature is 72 ℃,1min/kb, and 35 cycles are total; finally, the mixture was stored at 72℃for 5min and 4 ℃. After completion of the reaction procedure, about 500. Mu.L of the correct band was aspirated after separation by 1% agarose gel electrophoresis, and submitted to base sequencing by Beijing Kyoto major Gene technologies Co. Finally obtaining GmGH-2 gene of 1476bp of target fragment size, which shows that GmGH-2 gene is connected with an expression vector and successfully transformed into escherichia coli. The step of sequencing qualified coliform bacteria liquid is described with reference to TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0, and the extraction of coliform plasmid DNA is performed.
2) Based on GmRGH-2 gene full-length sequence, the 20 th base upstream of NGG is selected as target sequence. In order to improve mutation efficiency, benchling online websites are utilized to screen out 3 targets according to the middle target rate and the off target rate, and target primers, namely a primer 2, a primer 3 and a primer 4 are designed.
Primer 2:
GmRGH3-2-ATU3d-F：GTCACCCTCGATTATTACAGCGG(SEQ ID NO.4)；
GmRGH3-2-ATU3d-R：AAACCCGCTGTAATAATCGAGGG(SEQ ID NO.5)；
primer 3:
GmRGH3-2-ATU3b-F：GTCAGGCGACATTCTCCAAGTCA(SEQ ID NO.6)
GmRGH3-2-ATU3b-R：AAACTGACTTGGAGAATGTCGCC(SEQ ID NO.7)；
primer 4:
GmRGH3-2-ATU6-1F：ATTGCGACATTCTCCAAGTCACG(SEQ ID NO.8)；
GmRGH3-2-ATU6-1R：AAACCGTGACTTGGAGAATGTCG(SEQ ID NO.9)；
primer 5:
U-F：CTCCGTTTTACCTGTGGAATCG(SEQ ID NO.10)；
primer 6:
gRNA-R：CGGAGGAAAATTCCATCCAC(SEQ ID NO.11)；
Primers 2,3,4 were dissolved in ddH 2 O to 100. Mu.M stock solution, 1. Mu.L each was added to 98mL ddH 2 O and mixed to dilute to 1 μm, and the mixture was cooled to room temperature for 30s at 90℃to complete annealing. PYLCRISPR/Cas9-DH (N, B) strain and CRISPR/gRNA vector strain are respectively inoculated in LB solid culture medium (Kan, 25 mug/mL; amp,50 mug/mL), cultured for about 12 hours at the constant temperature of 37 ℃, single colony is selected and inoculated in 50mL LB liquid culture medium (Kan, 25 mug/mL; amp,50 mug/mL), and shake culture is carried out at 200rpm at 37 ℃ until bacterial liquid is turbid, so that plasmids are extracted. Carrying out single enzyme digestion on PYLCRISPR/Cas9-DH (N, B) plasmid by Bsa I endonuclease, wherein the reaction condition is that the temperature is 37 ℃ for 60min; 20min at 65 ℃. And (3) detecting the enzyme digestion product by 1% agarose gel electrophoresis, and then carrying out gel recovery. The gRNA expression cassette is connected by adopting Bsa I endonuclease and T4 DNA ligase and a side-cutting connection method, the reaction program is 5min at 37 ℃ and 5min at 20 ℃, and 5 times of circulation are carried out.
gRNA：SEQ ID NO.21：ACCCTCGATTATTACAGCGG；
Reagent name and amount
| Reagent(s) | Dosage of |
| Bsa I Ligase | 0.5μL(-5U) |
| Bsa I Ligase Buffer(10×) | 1 ΜL (final ATP concentration 0.5-1.0 mM) |
| PYLgRNA-AtU # plasmid | 1μL(20ng/μL) |
| T4 DNA Ligase | 0.1μL(-35U) |
| T4 DNA ligase buffer(10×) | 0.5μl |
| ddH2O | To 10μL |
PCR amplification was performed using the above-mentioned ligation products as templates, and the reverse adapter primers (reaction 1) of primers 5/primers 2, 3 and 4 and the forward adapter primers (reaction 2) of primers 2, 3 and 4, respectively, and the forward adapter primers (reaction 6). Denaturation was performed for a total of 35 cycles of extension, 3. Mu.L of PCR product was aspirated after the reaction was completed, agarose gel electrophoresis was performed, and the correct target band was subjected to gel recovery and purification.
Reaction 1 reagent name and amount
Reaction 2 reagent name and amount
| Reaction system | Volume of |
| KOD One TM PCR Master Mix(1×) | 10μL |
| Forward adapter primers | 0.8μL |
| gRNA-R | 0.8μL |
| Ligation products | 1μL |
| ddH2O | To 20μL |
Reaction 1 and reaction 2PCR reaction procedure
| Program | Temperature (temperature) | Time of |
| Pre-deformation | 98℃ | 3min |
| Deformation of | 98℃ | 10s |
| Annealing | 60℃ | 5s |
| Extension of | 68℃ | 10s |
| Final extension | 68℃ | 10min |
| Preservation of | 4℃ | Pause |
1. Mu.l of each of the recovered reaction 1,2 gel was diluted 10-fold with ddH 2 O, and 1. Mu.l of each diluted product was mixed as a template, amplified with primers 7, 8, 9, and purified.
Primer 7:
Uctcg-B1’：TTCAGAGGTCTCTCTCGACTAGTGGAATCGGCAGCAAAGG(SEQ ID NO.12)；
gRctga-B2：AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC(SEQ ID NO.13)；
primer 8:
Uctga-B2’：TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG(SEQ ID NO.14)；
gRaaga-B3：AGCGTGGGTCTGTCTTGGTCCATCCACTCCAAGCTC(SEQ ID NO.15)；
Primer 9:
Uaaga-B3’：TTCAGAGGTCTCTAAGACACTGGAATCGGCAGCAAAGG(SEQ ID NO.16)；
gRcggt-BL：AGCGTGGGTCTCGACCGACGCGTCCATCCACTCCAAGCTC(SEQ ID NO.17)；
Reference Vazyme company IIOne Step Cloning Kit, connecting the PCR amplification purification product with PYLCRISPR/Cas9-DH (N, B) subjected to Bsa I digestion by using a homologous recombination technology, transforming the connection product into E.coli DH 5 alpha competent cells to form PYLCRISPR/Cas9-mGmGH3-2 recombinant plasmids, and carrying out recombinant plasmid extraction, identification and K599 transformation.
EXAMPLE 3 recombinant vector transformation of Agrobacterium rhizogenes
1) Competent preparation and transformation of agrobacterium rhizogenes.
Agrobacterium rhizogenes (K599) shock competent preparation, reference Chen Anle (2014). The agrobacterium rhizogenes (K599) electric shock transformation method specifically comprises placing 1mm of electric transformation cup in ice bath for 30min; adding a competent K599 (100 mu L) into 1 mu LpCAMBIA3300-GmGH3-2 recombinant plasmid, lightly blowing with a pipette, moving to the bottom of a 1mm point rotating cup, and placing into an electric excitation tank; performing electric shock reaction by using a preset parameter Agrobacterium; immediately sucking 500 mu L of SOC culture medium, transferring to a shock cup, gently mixing, and sucking out liquid to a 1.5mL EP tube; placing at 28 ℃ and 150rpm, and renaturating for 1-3h; 100 mu L of the vector is evenly smeared on a screening mark plate which takes kanamycin (kan) and streptomycin (str) resistance genes Bar, after inversion and dark culture for 24 hours at 28 ℃, monoclonal is selected for bacterial liquid PCR verification, a target strip with the length of 1554bp is obtained, and pCambia 3300-GmGH-2 is proved to be successfully transferred into Agrobacterium rhizogenes K599 to obtain Agrobacterium rhizogenes K599 containing pCambia3300-GmGH3-2 vector (figure 9). The PYLCRISPR/Cas9-mGmGH3-2 recombinant plasmid was transferred into Agrobacterium rhizogenes K599 according to the procedure described above (FIG. 10)
EXAMPLE 4 Agrobacterium rhizogenes mediated genetic transformation
1) Healthy Dongnong 50 soybean seeds are selected, sterilized by chlorine (96mL NaClO,6mLHCl), germinated in a wet environment at 28 ℃ for 16 hours, sowed in sterilized stones the next day, and grown in a greenhouse under a photoperiod condition of 24 ℃ and 12/12 hours. The Agrobacterium single spot was picked and inoculated in 2mL LB (50 mg/Str) liquid medium at 28℃and 180rpm overnight for cultivation. Bacterial liquid is added with 1:100 is transferred to 50mL LB (containing 50 mg/Str) culture solution, and cultured at 28 ℃ and 200rpm until OD reaches about 1.0-1.2. The 3-4d seedling is collected by a disposable 1mL injector, the thallus is agrobacterium rhizogenes K599 containing pCambia 3300-GmGH-2 vector and agrobacterium rhizogenes K599 containing PYLCRISPR/Cas9-mGmGH3-2 recombinant plasmid, and the node or near hypocotyl at the cotyledon of the seedling is injected for 2-3 times to inoculate bacterial liquid. Dark culture at 28 ℃ after bacterial liquid injection and infecting once again for 24 hours by using thalli. And (3) placing the infected soybean seedlings into vermiculite wetted by the B & D nutrient solution, covering wounds with the vermiculite, and finally covering the culture pot with a preservative film. After 3d infection, the preservative film is lifted, the healing wound is observed, and the part about 1cm below the protrusion is cut off. Transplanting the hair roots with the length of 5-10cm into water of the B & D nutrient solution after growing for 2-3 weeks, and continuously ventilating and culturing for 3-5 days. And (3) carrying out positive detection after waiting for root growth to a certain length, then moving to 24 ℃, growing under 12/12h of illumination condition, and preparing the cyst nematode egg suspension after 2-3 days until seedlings grow normally. By using the method, gmGH-2 gene over-expression hairy roots and mutant hairy roots can be obtained rapidly.
Example 5 molecular detection of transgenic hairy root and functional verification of candidate Gene
1) Soybean genome DNA extraction. In this study, soybean genomic DNA was extracted by SDS-miniextraction to extract genomic DNA of transgenic hairy roots. For GmGH3-2 overexpressed hairy roots, PCR detection was performed using Bar protein test strips (FIG. 11); for GmGH3-2 mutant hairy roots, PCR verification is performed by using a primer 10 (FIG. 12), sequencing verification is performed on the PCR amplification product with the correct target band, and the sequencing result shows that double peaks appear at the position of the target point 1, which indicates that the target point 1 is successfully edited and mutation occurs (FIG. 13).
Mutated sequences ：(SEQ ID NO.22)ATGGCAATTGCTAATTGTGATGATAAGAACGCAAAAGCTTTGCAGTTCATTGAGGATATGACCCAAAACACTGACAGTGTCCAAAAGAGGGTCCTTGCTGAGATTCTGAGCCAAAACGCCAAGACCGAATACCTGAAACGGTTTGAGCTGAATGCAGCCACAGACCGTGACACTTTCAAGTCCAAGGTACCTGTCGTTTCATACGATGATTTGAAGCATGATATCCAACGCATTGCTAATGGTGACCGCTCTCCTATCCTATGCGCTCATCCAATTTCCGAGTTTCTCACCAGTTCTGGAACATCTGCTGGGGAGAGAAAATTGATGCCAACAATTCGTCAAGAGATGGACCGTCGTCAATTACTTTACAGCCTTCTGATGCCTGTGATGAACCAATACGTGCCTGATTTGGATAAGGGTAAGGCTCTACTCTTCTTGTTCATCAAAGCCGAGACAAAGACTCCGAGTGGGTTAGTGGCACGTCCAGTGCTAACTAGCCTCTACAAGAGCGACCAATTCAAGAACAGACCCTACGATCCATTCAACGTGTACACAAGCCCAGACGAAGCAATCCTCTGCCCCGATTCCTTCCAAAGCATGTACACCCAAATGCTGTGCGGCCTCATCATGCGCCACCAAGTCCTCAGAGTCGGAGCCGTTTTCGCCTCGGGCCTTCTCCGAGCCATCCGCTTCCTCCAGCTCAATTGGGCTGAACTAGCCCACGACATCTCCACCGGAACCCTAAACCCCAAAATCTCCGACCTTGCCATCAAACAACGCATGACCCAAATCCTCACACCAAATCCAGAACTCGCTGATTTCATTGTGAAAGAATGCTCGGGAGAAAACTGGGACCGCATAATCACAAGAATCTGGCCAAACACAAAGTATCTGGACGTGATTGTGACCGGTGCCATGGCGCAGTATATTCCGACCCTCGATTATTACAGGGAGGCCTACCTAAAGCTTGCACCATGTACGCCTCATCCGAGTGTTACTTTGGGCTTAACCTAAACCCTATTTGCACCCCCTCTGACGTGTCCTACACCATCATGCCAAACATGGGTTACTTCGAGTTCCTTCCCCACGAAGAAGATTTATCTTCTAGCTCTTCAAGCTCCACCTTGTCACGTGACTCGCTCGACCTTGCAGATCTTGAACTCGGAAAATCCTACGAGCTCATCGTCACCACGTACTCCGGTCTGTGCCGTTACCGCGTAGGCGACATTCTCCAAGTCACGGGGTTCCACAACACCGCCCCACATTTCAGCTTCGTGAGGAGAAAAAACGTGCTTCTGAGCATCGACTCCGACAAGACCGACGAAGCGGAGTTACAGAACGCCGTCGAGAACGCCTCCGTGCTGCTGAGGGAGTTCAAGACGAGCGTGGCGGAGTACACGAGCTTCGCCGACACGAAGTCGATCCCGGGGCACTACGTGATTTACTGGGAACATCTATCTCTATTCTTTTTATAA;
Primer 10 GmGH-2 gene mutation detection primer
SP-DL：GTCGTGCTCCACATGTTGACCGG(SEQ ID NO.18)；
SP-R：CCCGACATAGATGCAATAACTTC(SEQ ID NO.19)；
2) Acid fuchsin staining identifies resistance of transgenic root systems to SCN. Histological examination of SCN infestation indicated that on day 15 of inoculation, nematodes had begun feeding, and international acid fuchsin staining was used to observe the invasion and development of nematodes in roots, the method comprising 1) rinsing: washing the residual soil at the root with clear water; 2) Soaking: soaking the root with 3% NaClO water solution for 1 hr (this time can be adjusted, depending on the decolorization situation, naClO concentration is slightly higher if decolorization is fast), and ensuring complete decolorization of the root; 3) Root staining: the roots NaClO were rinsed with tap water, immersed in distilled water for 15 minutes, diluted 30 times, and then the acidic fuchsin staining solution (mother liquor: 3.5g fuchsin, 250mL glacial acetic acid, 750mL distilled water) was boiled, and the decolorized roots were placed in fuchsin and boiled for 45s-2 minutes (the time for coloring was as short as possible, and the root system was relatively compact), and the roots were wiped with absorbent paper at the time of removal, and observation was performed.
3) 15 Plants with GmGH-2 gene positive roots, gmGH-2 gene mutation positive roots and GmGH3-2 gene wild soybean roots (the wild root system is the root system of Dongnong 50) in the molecular detection result are inoculated with No. 3 physiological egg suspension for identification, 5-6 lateral roots are taken for repetition under a 20X and 100X optical microscope by an acid fuchsin staining method, the average value of each female is calculated, and paired t test is adopted. As shown in table 1, the average number of wild-type control root females was 2.60 pieces/cm, the average number of pCAMBIA3300-GmGH3-2 transgenic positive root females was 1.83 pieces/cm, lower than the control group, the results of the paired sample t-test were 2.14 and 3.00 for double tail t-values at p=0.05 and 0.01 levels, respectively, and both of the double tail P-values (Sig) were 0.007;
The average value of GmGH3-2 mutant positive root females was 3.79 pieces/cm, the double tail t values were 2.14 and 2.98 at the p=0.05 and 0.01 levels, respectively, the double tail P value (Sig) was 0.004, and according to the female index, there was a very significant difference between the transgenic positive roots and the negative control roots, and obvious resistance of the GmGH3-2 gene to soybean cyst nematode disease was primarily confirmed (fig. 14).
TABLE 1 average number of root females (individual cm -1)
Claims (3)
1. A method for preparing soybean plants resistant to soybean cyst nematode disease, characterized by the specific steps of:
Step 1: connecting the gene shown in SEQ ID NO.3 with a vector pCAMBIA3300 vector to obtain a recombinant vector;
step 2: transforming the recombinant vector in the step 1 into agrobacterium to obtain recombinant agrobacterium;
Step 3: transferring the recombinant agrobacterium of the step 2 into soybeans to obtain transgenic soybean plants, and identifying to obtain positive transgenic soybean plants; the gene shown in SEQ ID NO.3 is overexpressed in transgenic soybean plants that obtained positive.
2. A method for improving soybean resistance to soybean cyst nematode disease, characterized in that soybean plants overexpressing the gene shown in SEQ ID No.3 are infected with soybean cyst nematode.
Use of a soybean protein as set forth in seq ID No.20 for increasing resistance of soybean to cyst nematode disease.
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