CN110205325B - Application of soybean VQ motif coding gene GmVQ58 - Google Patents
Application of soybean VQ motif coding gene GmVQ58 Download PDFInfo
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- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
Abstract
The invention discloses application of a soybean VQ motif coding gene GmVQ 58. Application of soybean VQ motif coding gene GmVQ58 shown in SEQ ID No.1 in genetic engineering modification of resistance of soybean to prodenia litura. Overexpression of GmVQ58 restored the sensitivity of jav1 Arabidopsis mutants to Spodoptera litura. The expression of the gene is inhibited, and the resistance of the soybean to prodenia litura can be obviously improved. The soybean VQ motif coding gene GmVQ58 can be transformed into soybean hairy roots through genetic engineering, and finally, the resistance of soybeans to prodenia litura can be negatively regulated and controlled.
Description
Technical Field
The invention relates to application of a soybean VQ motif coding gene GmVQ58, belonging to the field of genetic engineering.
Background
Soybean, dicotyledonous herbaceous plant, originated in our country, is rich in vegetable oil and vegetable protein, is one of five crops in the world. Soybeans are often subjected to various biotic stresses, of which prodenia litura has become the major binge eating pest in south China. Prodenia litura poses great threat to the yield and quality of soybean, thus bringing serious economic loss to farmers. Although chemical pesticides can control pests, long-term use of them not only pollutes the environment but also causes resistance to pests. The use of insect-resistant varieties is the most economical and effective pest control measure. The traditional breeding method has long year limit for breeding new varieties, is difficult to obtain varieties with excellent resistance, wastes time and labor and may be futile. With the gradual application of genetic engineering means and the gradual maturity of transgenic technology, the insect-resistant gene can be introduced into the body of the existing variety. The transgenic breeding method can avoid the side effect caused by chemical pest killing method, and has the advantages of short breeding period, fast effect, etc. thus opening up a new path for preventing and controlling pests.
GmVQ58 encodes a soybean VQ protein that belongs to the VQ family with a single and conserved amino acid sequence fxxxhvqxhtg. In Arabidopsis thaliana isotype plants, the VQ gene regulates various growth processes including response to biotic and abiotic stresses, seed development and photomorphogenesis, etc. In arabidopsis, AtVQ22(JAV1) negatively regulated the resistance response of arabidopsis to phytophagous pests (spodoptera exigua, aphids and mosquito larvae) and saprophytic fungi (botrytis cinerea) by the JA pathway without affecting growth and development. AtVQ12 responds strongly to gray mold infestation and is functionally redundant with AtVQ29 in regulating the basal resistance of plants to gray mold. In rice, the OsVQs gene family members are found to have different performances in the aspects of responding to rice bacterial blight, ABA and drought stress. In bananas, MaVQ5 responds to low temperature stress and may act as a repressor of the cold-responsive transcription factor MaWRKY26 to regulate MeJA-mediated low temperature stress responses. Of soybean, only GmVQ35 and GmVQ47 were reported to negatively regulate plant resistance to botrytis cinerea in arabidopsis thaliana. However, there is no report in soybean that VQ gene is involved in insect-resistant function.
Disclosure of Invention
The invention aims to disclose the insect-resistant gene engineering application of soybean VQ gene GmVQ58, wherein the gene is mainly expressed in soybean leaves and roots and is quickly up-regulated and expressed in leaves after the spodoptera litura is induced by stress. Furthermore, subcellular localization analysis revealed that GmVQ58 is a nuclear localization protein. GmVQ58 can be introduced into arabidopsis thaliana and soybean hairy roots as a target gene, the resistance of a plant to prodenia litura is negatively regulated, meanwhile, GmVQ58 can respectively influence the expressions of arabidopsis thaliana AtPR4 and AtVSP1 and soybean GmPR1 and GmVSP beta, and a negative correlation exists on the gene expression level.
The purpose of the invention can be realized by the following technical scheme:
the application of the soybean VQ motif coding gene GmVQ58 in genetic engineering modification of resistance of soybean to prodenia litura is disclosed, wherein the soybean VQ motif coding gene GmVQ58 has a coding region sequence shown as SEQ ID No. 1.
In arabidopsis, overexpression of GmVQ58 restored the sensitivity of the jav1 mutant to prodenia litura. In soybeans, the expression of the gene is inhibited, and the insect resistance of the soybeans can be obviously improved.
Advantageous effects
GmVQ58 is a soybean VQ gene, and the soybean GmVQ58 gene negatively regulates the resistance of transgenic arabidopsis thaliana and soybean hairy roots to prodenia litura. Through expression analysis, GmVQ58 is mainly expressed in soybean leaves and roots, and is up-regulated in leaves after insect induction. By subcellular localization analysis, GmVQ58 was demonstrated to be a nuclear localization protein. Meanwhile, the resistance of transgenic arabidopsis thaliana and soybean hairy roots to prodenia litura is found to be negatively regulated by functional verification, the expressions of arabidopsis thaliana AtPR4 and AtVSP1 and soybean GmPR1 and GmVSP beta are respectively influenced, and a negative correlation relationship exists. Therefore, GmVQ58 can be used as a target point for regulating the resistance of soybeans to prodenia litura and is used for insect resistance modification of the soybeans.
Drawings
FIG. 1 agarose gel electrophoresis picture after PCR cloning of GmVQ 58. The size of the target fragment is 486 bp. Marker: DL2000
FIG. 2 GmVQ58 tissue expression pattern. N is 3.
FIG. 3 GmVQ58 was able to respond to prodenia litura stress induction. Change in expression level of GmVQ58 in soybean leaves 1, 6, 12, 24, 48, 72h after insect induction (N ═ 3).
FIG. 4 subcellular localization analysis of GmVQ58 protein. GFP, GFP fluorescence; light, brightfield; merge, fusion protein; 35S-GFP, no-load control; 35S GmVQ58-GFP and GmVQ58 protein with GFP labels. Scale bar: 50 μm.
FIG. 5 expression levels of JAV1, GmVQ58 and two stress-related genes AtPR4 and AtVSP1 in control and transgenic Arabidopsis thaliana. (A)28 days of wild type Col-0, jav1 mutant and 3 GmVQ58-OE jav1 transgenic lines. Scale bar 1 cm. (B) Relative expression levels of JAV1 in Col-0 and JAV1 mutants. (C) Relative expression levels of GmVQ58 in Col-0 and 3 GmVQ58-OE jav1 transgenic lines. Relative expression levels of AtPR4(D) and AtVSP1(E) in Col-0, jav1 mutant and 3 GmVQ58-OE jav1 transgenic lines. 1#, 2#, 3# GmVQ58-OE jav1 represent 3 independent transgenic Arabidopsis lines over-expressing GmVQ58 in the background of Arabidopsis jav1 mutant, respectively. Two-tailed test, N ═ 3,: p < 0.05; **: p < 0.01; ***: p <0.001, error bars represent ± SEM.
Fig. 6, overexpression of GmVQ58 in the jav1 mutant attenuated the resistance of plants to prodenia litura. (A) Typical rosette leaves in Col-0, jav1 mutant and 3 GmVQ58-OE jav1 transgenic lines 24 hours after feeding Control (CK) or spodoptera litura. Scale bar 4 mm. (B) The average insect weight was weighed on 0, 2, 3 and 4 days after feeding prodenia litura. Two-tailed test, N ═ 3. **: p <0.01, error bars represent ± SEM. (C) Col-0, jav1 mutant and 3 GmVQ58-OE jav1 transgenic lines were fed to Prodenia litura with larvae sizes at 0 and 4 days. Scale bar 1 cm. 1#, 2#, 3# GmVQ58-OE jav1 represent 3 independent transgenic Arabidopsis lines over-expressing GmVQ58 in the background of Arabidopsis jav1 mutant, respectively.
FIG. 7 relative expression levels of GmVQ58 and two stress-related genes in transgenic soybean hairy roots. (A) Soybean over-expressing hairy root GmVQ58-OE, over-expressing no-load hairy root Control-OE, RNA interference hairy root GmVQ58-RNAi and RNA interference no-load hairy root Control-RNAi in 25 days. Scale bar 2 cm. (B) Relative expression levels of GmVQ58 in 4 genotype soybean hairy roots. Relative expression levels of GmPR1(C) and GmVSP beta (D) in hairy roots of 4 genotypes of transgenic soybean. Two-tailed test, N ═ 3. ns: there was no significant difference; *: p < 0.05; **: p < 0.01. Error bars represent ± SEM.
FIG. 8 identification of resistance of transgenic hairy roots to prodenia litura. (A) Soybean over-expressing hairy root GmVQ58-OE and over-expressing no-load hairy root Control-OE feed Spodoptera litura with larvae sizes of 0, 4 and 6 days. Scale bar 1 cm. (B) Soybean over-expressed hairy root GmVQ58-OE and over-expressed no-load hairy root Control-OE were fed to Spodoptera litura with average weights measured on days 0, 2, 3, 4, 5 and 6. (C) Soybean interfering hairy root GmVQ58-RNAi and RNA interfering No-load hairy root Control-RNAi raised Prodenia litura 0, 4 and 6 days old larva size. Scale bar 1 cm. (D) Soybean interfering hairy root GmVQ58-RNAi and RNA interfering No-load hairy root Control-RNAi fed Spodoptera litura with average weight measured on days 0, 2, 3, 4, 5 and 6. Two-tailed test, N ═ 3. *: p < 0.05; **: p < 0.01. Error bars represent ± SEM.
FIG. 9 PCR assay of transgenic Arabidopsis. (A) PCR detection of Arabidopsis jav1 mutant (T-DNA insertion). Within a certain time, the Arabidopsis jav1 mutant could not amplify a 1329bp sized fragment, while the Col-0 plant could amplify a 1329bp sized fragment. (B) PCR detection of GmVQ58 transgenic Arabidopsis thaliana, and the size of the target fragment is 855 bp. M: marker DL2000, P: positive plasmid, Col-0: arabidopsis wild type Col-0, jav 1: arabidopsis jav1 mutant, 1-6: different GmVQ58-OE jav1 transgenic plants, H2O: blank control.
FIG. 10 PCR detection of transgenic hairy roots. (A) PCR detection of over-expressed hairy root GmVQ58-OE and over-expressed empty hairy root Control-OE. The size of the target fragment is 855 bp. M: marker DL2000, P: positive plasmid (pMDC83-GmVQ58), H2O: blank control, ck: negative hairy root, 1-6: different positive hairy roots carrying pMDC83-GmVQ58 plasmid. (B) Interfering with the PCR detection of hairy root GmVQ58-RNAi and interfering with the PCR detection of empty hairy root Control-RNAi. The size of the target fragment is 625 bp. M: marker DL2000, P: positive plasmid [ pB7GWIWG2(II) -GmVQ58]， H2O: blank control, ck: negative hairy root, 1-6: different positive hairy roots carrying pB7GWIWG2(II) -GmVQ58 plasmid.
Detailed Description
The invention is further explained below with reference to the drawings and the embodiments.
The methods used in the following examples are conventional methods unless otherwise specified.
Example 1
1) Cloning of Soybean VQ Gene GmVQ58
Taking soybean variety Williams 82 as a material-taking object, taking leaves of the soybean variety Williams 82, grinding the leaves by using a mortar, adding the leaves into a 1.5mL EP tube containing a lysate, fully oscillating the obtained product, transferring the product into the 1.5mL EP tube, extracting Total RNA (Total RNA Kit (Tiangen, Beijing, China), identifying the quality of the Total RNA by formaldehyde denaturing gel electrophoresis, measuring the RNA content by using a spectrophotometer, taking the obtained Total RNA as a template, and carrying out reverse transcription according to a reverse transcription Kit provided by TaKaRa company of Japan(TaKaRa Primer ScriptTMRT reagent kit, japan) to obtain the first strand of cDNA, and PCR amplification was performed as follows: pre-denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 15 sec, annealing at 60 ℃ for 15 sec, extension at 72 ℃ for 90 sec for 35 cycles, final incubation at 72 ℃ for 5 min, followed by incubation at 12 ℃ to obtain cDNA of Williams 82.
Finding out a gene corresponding to GmVQ58 from an NCBI database and a Phytozome v12 soybean database (Glyma.14g002800, GeneID:102665271), designing specific primer primers according to nucleotide sequences provided by the database, wherein the primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, amplifying the gene from a gene coding region (CDS) sequence of a soybean variety Williams 82, carrying out PCR product tapping purification, connection and transformation after PCR cloning, picking out positive monoclonal clones for sequencing, and obtaining the CDS sequence of the soybean GmVQ58 gene with the length of 486bp and a complete coding region, wherein the coding region sequence is shown in SEQ ID NO.1, the size is 486bp (figure 1), and the nucleotide sequence and the amino acid sequence (SEQ ID NO.1 and SEQ ID NO.2) are obtained after sequencing.
2) Tissue expression analysis of GmVQ58
To identify the expression levels of GmVQ58 in different tissues, roots, stems, leaves, flowers, pods and seeds of soybean variety Williams 82 at different developmental stages were collected: roots, stems and leaves are at stage V4; mature flower is in stage R2; seeds and pods were collected 15 days after flowering. Samples were snap frozen in liquid nitrogen and stored at-80 ℃. The total RNA extraction was performed in the same manner as in step 1). The total RNA obtained by sampling each tissue was used as a template and inverted to cDNA. The fluorescent quantitative primer sequence of GmVQ58 is shown in SEQ ID NO.5 and SEQ ID NO.6, the change of the expression quantity of the GmVQ58 gene in each tissue is detected, the soybean composition reference gene Tubulin is used as an internal reference, the primer sequence is shown in SEQ ID NO.7 and SEQ ID NO.8, and Real-time fluorescent quantitative PCR reaction (Real-time RT-PCR) is carried out.
GmVQ58 was mainly expressed in soybean leaf and root tissues with lower expression levels in stems, flowers, pods, and seeds (fig. 2), suggesting that GmVQ58 may play a role in leaves and roots.
3) Expression analysis of GmVQ58 under stress induction of prodenia litura
Spodoptera litura is respectively placed on the true leaves and the second compound leaves of the V4 stage soybean variety Williams 82, and two ends of each plant are arranged. And after 48 hours, taking out the insects, and finishing the induction treatment, wherein the loss area of the soybean leaves reaches about 20 percent. Fresh leaves are respectively taken from the induction groups 1, 6, 12, 24, 48 and 72h after induction treatment and the control group (non-induction group), and are stored at-80 ℃ after being quickly frozen by liquid nitrogen. The total RNA extraction was performed in the same manner as in step 1). The total RNA obtained by sampling the induction group and the control group is used as a template, the template is inverted into cDNA, and Real-time fluorescent quantitative PCR reaction (Real-time RT-PCR) is carried out. The fluorescent quantitative primer sequence of the GmVQ58 and the primer sequence of the internal reference gene Tubulin are the same as the sequence in the step 2).
After soybean leaves are treated by prodenia litura, the expression level of GmVQ58 is remarkably increased, reaches a peak value at 48h and is sharply reduced at 72h (figure 3), which shows that GmVQ58 is induced to express by the stress of prodenia litura.
Example 2
1) Cloning of Soybean VQ Gene GmVQ58
Taking the total RNA of the leaves of a soybean variety Williams 82 as a template, synthesizing a first cDNA chain by reverse transcription, and carrying out PCR amplification, wherein the primer sequences are shown in SEQ ID NO.9 and SEQ ID NO.10, and the PCR program is as follows: pre-denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 15 sec, annealing at 60 ℃ for 15 sec, extension at 72 ℃ for 90 sec, 35 cycles in total, final incubation at 72 ℃ for 5 min, subsequent incubation at 4 ℃ for 5 min, and sequencing to obtain the CDS sequence of soybean GmVQ58 gene without stop codon.
2) Construction of subcellular localization vectors
When constructing a subcellular localization vector, the CDS sequence of the soybean GmVQ58 gene without a stop codon was inserted into the pFGC5941 expression vector containing a GFP tag. The vector has a 35S promoter, and can strongly induce the expression of a target gene GmVQ58 in a receptor. The vector was then transferred into Agrobacterium tumefaciens strain EHA105 by freeze-thaw methods. Also, unloaded pFGC5941 was also transformed into EHA105 as an unloaded control.
3) Subcellular localization of GmVQ58
The bacterial suspension containing 35S: GmVQ58-GFP and 35S: GFP was injected into the leaf blades of Nicotiana benthamiana of 7-8 weeks size, cultured for 36-48 hours, and then observed by a Leica TCS SP2 laser confocal microscope to determine the reporter gene GFP. The GFP signal showed that the GmVQ58-GFP fusion protein localized only to the nucleus, and no-load to the whole tobacco cell (FIG. 4).
Example 3 genetic engineering application of Gene GmVQ58
1) Cloning of Soybean VQ Gene GmVQ58
Taking the total RNA of the leaves of a soybean variety Williams 82 as a template, synthesizing a first cDNA chain by reverse transcription, and carrying out PCR amplification, wherein the primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, and the PCR program is as follows: pre-denaturation at 95 ℃ for 3 minutes, denaturation at 95 ℃ for 15 seconds, annealing at 60 ℃ for 15 seconds, extension at 72 ℃ for 90 seconds, 35 cycles, finally heat preservation at 72 ℃ for 5 minutes, constant temperature at 4 ℃ and sequencing to obtain the CDS sequence of the soybean GmVQ58 gene with the complete coding region and the length of 486bp, wherein the sequence is shown in SEQ ID NO. 1.
2) Construction of plant expression vectors
When constructing an overexpression vector, the GmVQ58 gene sequence was compared with that of Invitrogen
Technology with ClonaseTMCarrying out BP reaction on a pDONR221 vector in the II kit, carrying out bacteria liquid PCR sequencing verification, wherein primer sequences are shown in SEQ ID NO.11 and SEQ ID NO.12, and the specific PCR process is the same as that in the step 1), so as to obtain entry clone. And performing recombination exchange on the obtained entry clone and a target expression vector pMDC83 developed by Invitrogen company to obtain a pMDC83-GmVQ58 plant over-expression vector, wherein the plant transformation vector pMDC83 contains a 2x 35S strong promoter and can strongly induce the expression of a target gene GmVQ58 in a receptor. The vectors were then transferred into Agrobacterium tumefaciens strains EHA105 and K599 by freeze-thaw methods. Also, unloaded pMDC83 was also transformed into K599 as an unloaded control.
When an RNA interference vector is constructed, the total length of GmVQ58CDS is less than 500bp, and the length of an interference fragment is met, so that the full length of the interference vector is amplified and connected, the sequence of a primer and the following steps are the same as those of the construction of an over-expression vector, and only the pMDC83 vector is replaced by a pB7GWIWG2(II) vector. The constructed vectors pBI-GmVQ58 and empty pB7GWIWG2(II) were transformed into K599.
3) Obtaining transgenic Arabidopsis plants
Transforming Arabidopsis by using a flower soaking method, soaking jav1 Arabidopsis mutant buds in the bacterial solution of the Agrobacterium tumefaciens strain EHA105 containing pMDC83-GmVQ58 in the step 2), and infecting for 30 s-1 min. Wrapping the plants with a preservative film or a preservative bag to keep humidity, and culturing in dark for 24 h. Then, according to the growth condition of the plants, the new batch of inflorescences are transformed again after growing. The arabidopsis thaliana was grown continuously under normal conditions and seeds were harvested. Selection of three homozygous T3Phenotypic analysis and identification of resistance to prodenia litura were performed on the GmVQ58-OE jav1 transgenic Arabidopsis lines (FIG. 5A). In order to detect whether the mutant is jav1 Arabidopsis thaliana and whether the mutant is transgenic Arabidopsis thaliana with overexpression GmVQ58, PCR detection (SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16) is respectively carried out on the extracted DNA fragments by using specific primers, and a PCR detection gel diagram is shown in FIG. 9. Real-time fluorescent quantitative qPCR found that expression levels of JAV1 were significantly reduced in the JAV1 arabidopsis mutant compared to the wild-type control (fig. 5B). Jav1 and Arabidopsis thaliana internal reference Tubulin fluorescent quantitative primer sequences are shown in SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO. 20. The expression level of the GmVQ58 gene in three GmVQ58-OE jav1 transgenic Arabidopsis thaliana is significantly higher than that of the wild-type control (FIG. 5C). The fluorescent quantitative primer sequences of the GmVQ58 and the soybean internal reference Tubulin are the same as above.
3-year-old prodenia litura is fed indoors by using a culture dish, and the wild type control Col-0 and jav1 arabidopsis mutants and three GmVQ58-OE jav1 arabidopsis transgenic lines of about 28 days are picked to feed the prodenia litura. Each dish had 5 beetles, 3 replicates. Fresh leaves were replaced daily and weighed on days 0, 2, 3 and 4 of feeding, respectively. And calculating the average worm weight of each dish, and taking the average worm weight of the prodenia litura as a resistance identification index. When fed for 4 days, the prodenia litura larvae fed with the jav1 mutant swallowed a small number of leaves and grew slowly, while Col-0 or GmVQ58-OE jav1 transgenic arabidopsis promoted the growth of the larvae (fig. 6A and C). Meanwhile, the average insect weight of spodoptera litura larvae fed with Col-0 or GmVQ58-OE jav1 arabidopsis was significantly higher than that of larvae fed with the jav1 mutant (fig. 6B). In addition, qRT-PCR also detected the expression levels of two insect-resistance related genes. The expression level of AtPR4(At3g04720) in the jav1 mutant was significantly higher than that in GmVQ58-OE jav1 Arabidopsis thaliana or Col-0 (FIG. 5D). Also, expression of AtVSP1 (nutritional storage protein 1, At5g24780) in the jav1 mutant was significantly accumulated compared to GmVQ58-OE jav1 Arabidopsis or Col-0 (FIG. 5E). The sequences of the AtPR4 and AtVSP1 fluorescent quantitative primers are shown in SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO. 24.
4) Obtaining transgenic soybean hairy root
Wound treatment is carried out on the backs of cotyledons of soybean seedlings growing for 5 days, agrobacterium tumefaciens strain K599 bacterial liquids respectively containing pMDC83-GmVQ58 and pBI-GmVQ58 vectors and corresponding no-load control obtained in the step 2) are inoculated to the wound on the backs of the soybean cotyledons, and the inoculated cotyledons are placed in White culture medium containing 500 mu g/mL carbethoxyl and 50 mu g/mL cephalosporin. After 3-4 weeks of dark culture at 25 ℃, hairy roots can grow out from the wounds on the backs of cotyledons, the over-expression hairy roots are called GmVQ58-OE, and the no-load contrast is Control-OE; the interfering hairy root is called GmVQ58-RNAi, and the no-load Control is Control-RNAi. In order to detect whether the hairy root is positive, PCR detection is carried out on the extracted DNA fragment by using a specific primer. The over-expression hairy root detection primer sequence is consistent with the over-expression Arabidopsis thaliana detection sequence in the 3), and a PCR positive hairy root detection gel diagram is shown in a figure 10A. The sequences of the primers for detecting RNA interference hairy roots are shown in SEQ ID NO.25 and SEQ ID NO.26, and the detection of PCR positive hairy roots is shown in FIG. 10B. The real-time fluorescent quantitative qPCR found that the expression level of GmVQ58 gene in the over-expressed hairy root (GmVQ58-OE) is significantly higher than that in Control-OE, while the expression level of GmVQ58 gene in the RNA-interfered hairy root (GmVQ58-RNAi) is significantly lower than that in Control-RNAi (FIG. 7B). The fluorescent quantitative primer sequences of the GmVQ58 and the soybean internal reference Tubulin are the same as above.
3-year-old prodenia litura is fed indoors by using a culture dish, and soybean hairy roots (GmVQ58-OE, Control-OE, GmVQ58-RNAi and Control-RNAi) with 4 genotypes are picked for about 25 days and fed with the prodenia litura. Each dish had 5 beetles, 3 replicates. Fresh hairy roots were replaced daily and weighed at 0, 2, 3, 4, 5 and 6 days of feeding, respectively. And calculating the average worm weight of each dish, and taking the average worm weight of the prodenia litura as a resistance identification index. The average worm weight of spodoptera litura fed with GmVQ58-OE transgenic hairy roots was not significantly different from that fed with its controls, whereas the average worm weight of spodoptera litura fed with GmVQ58-RNAi transgenic hairy roots was significantly lower than that fed with its controls. Meanwhile, relative expression amounts of insect-resistant related genes GmPR1(Glyma.15g062500) and GmVSP beta (Glyma.08g200100) in the hairy roots are measured, and the expression amounts of the GmPR1 and the GmVSP beta genes are found to have no obvious difference between the GmVQ58-OE hairy roots and Control-OE hairy roots. However, the transcription levels of GmPR1 and GmVSP β were significantly increased in GmVQ58-RNAi hairy roots compared to Control-RNAi hairy roots. The sequences of the GmPR1 and GmVSP beta fluorescence quantitative primer are shown in SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29 and SEQ ID NO. 30.
Sequence listing
<110> Nanjing university of agriculture
Application of <120> soybean VQ motif coding gene GmVQ58
<160> 30
<170> SIPOSequenceListing 1.0
<210> 1
<211> 486
<212> DNA
<213> Soybean (Glycine max)
<400> 1
atgactgcta ccatgtctgg ccctattcct aatgatcact ggctccattt ctatcaacaa 60
aaccaattcc cctcttccgt ttccgacgcc acaaccgtga caaccaccgc cgcgccggcc 120
aagctaagcc cagaaggcgg agtctcaaag cccattagac ggcgttcacg ggcctcaagg 180
agaactccca ccactctgct gaacacggac accagcaatt tccgggccat ggtgcaacag 240
ttcactggag ctccttctgc gcctgatatg tttgggagcc cagtccctgt caacccaaat 300
ggcctcatgt tggccccttc tcagtccctt tatcaccaac aaaaccactt agcatacaga 360
gatggtggag acaagggttt ttttcagaga ctgagcaacc caacagcaac agctccaaat 420
aatgatcgag ctgatgaggt tttggtggac catggcgggc gtttcttccc aacaacttct 480
tcttga 486
<210> 2
<211> 161
<212> PRT
<213> Soybean (Glycine max)
<400> 2
Met Thr Ala Thr Met Ser Gly Pro Ile Pro Asn Asp His Trp Leu His
1 5 10 15
Phe Tyr Gln Gln Asn Gln Phe Pro Ser Ser Val Ser Asp Ala Thr Thr
20 25 30
Val Thr Thr Thr Ala Ala Pro Ala Lys Leu Ser Pro Glu Gly Gly Val
35 40 45
Ser Lys Pro Ile Arg Arg Arg Ser Arg Ala Ser Arg Arg Thr Pro Thr
50 55 60
Thr Leu Leu Asn Thr Asp Thr Ser Asn Phe Arg Ala Met Val Gln Gln
65 70 75 80
Phe Thr Gly Ala Pro Ser Ala Pro Asp Met Phe Gly Ser Pro Val Pro
85 90 95
Val Asn Pro Asn Gly Leu Met Leu Ala Pro Ser Gln Ser Leu Tyr His
100 105 110
Gln Gln Asn His Leu Ala Tyr Arg Asp Gly Gly Asp Lys Gly Phe Phe
115 120 125
Gln Arg Leu Ser Asn Pro Thr Ala Thr Ala Pro Asn Asn Asp Arg Ala
130 135 140
Asp Glu Val Leu Val Asp His Gly Gly Arg Phe Phe Pro Thr Thr Ser
145 150 155 160
Ser
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgactgcta ccatgtctgg c 21
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tcaagaagaa gttgttggga ag 22
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
<210> 7
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
cctcgttcga attcgctttt tg 22
<210> 8
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
caactgtctt gtcacttggc at 22
<210> 9
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gctctagaat gactgctacc atgtctggc 29
<210> 10
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ccgctcgaga gaagaagttg ttgggaagaa ac 32
<210> 11
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ggggacaagt ttgtacaaaa aagcaggctc catgactgct accatgtctg gc 52
<210> 12
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ggggaccact ttgtacaaga aagctgggtt caagaagaag ttgttgggaa g 51
<210> 13
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ttttctctac cacaaaaccg c 21
<210> 14
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ccaacaagtt gacgaaattg g 21
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
tgaagaagat ggtcctctcc tg 22
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
<210> 19
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ctcaagaggt tctcagcagt a 21
<210> 20
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
tcaccttctt catccgcagt t 21
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
<210> 22
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
atacttgctc cgccatgc 18
<210> 23
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gtttggatct ttgacctaga cga 23
<210> 24
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ctctaaccac gaccagtacg c 21
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
<210> 26
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
aagaaggaag caagtcaaca cac 23
<210> 27
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
aactatgctc cccctggcaa ctatattg 28
<210> 28
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
tctgaagtgg tagcttctac atcgaaacaa 30
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
<210> 30
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Claims (1)
1. Soybean VQ motif coding geneGmVQ58Application of genetically engineered soybean in resistance to prodenia litura, wherein soybean VQ motif coding geneGmVQ58，The sequence of the coding region is shown as SEQ ID number 1, and the inhibition isGmVQ58The expression of the gene can obviously improve the resistance of the soybean to prodenia litura.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105177028A (en) * | 2015-09-22 | 2015-12-23 | 南京农业大学 | Soybean gmsams1 gene and application thereof |
| CN108504664A (en) * | 2018-06-06 | 2018-09-07 | 南京农业大学 | The application of Protein G mCDF1 encoding genes is discharged in soybean cation |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105177028A (en) * | 2015-09-22 | 2015-12-23 | 南京农业大学 | Soybean gmsams1 gene and application thereof |
| CN108504664A (en) * | 2018-06-06 | 2018-09-07 | 南京农业大学 | The application of Protein G mCDF1 encoding genes is discharged in soybean cation |
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