CN117925701B - Application of GhTPS gene in regulation and control of verticillium resistance of cotton - Google Patents
Application of GhTPS gene in regulation and control of verticillium resistance of cotton Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering, in particular to application of GhTPS gene in regulating and controlling verticillium wilt resistance of cotton. According to the invention, through a VIGS experiment, the function of GhTPS gene in resisting verticillium wilt is verified, and silencing GhTPS gene in cotton can lead to improvement of plant susceptibility, increase of fungus content and more serious browning of vascular bundles, which shows that GhTPS gene has positive regulation and control effect in resisting verticillium wilt in cotton. Provides gene resources for the development of effective gene resources for improving the verticillium wilt resistance of cotton in cotton breeding in future.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to application of GhTPS gene in regulating and controlling verticillium wilt resistance of cotton.
Background
TPS genes are widely distributed in plants and their function has been studied extensively, especially in terms of disease resistance. OsTPS19 was identified in rice as (S) -limonene synthase, which effectively inhibited the germination of rice blast (Magnaporthe oryzae) spores. Over-expression and RNAi experiments show that OsTPS19 plays an important role in resisting rice blast bacteria 【Chen, X., Chen, H., Yuan, J.S., Köllner, T.G., Chen, Y., Guo, Y., Zhuang, X., Chen, X., Zhang, Y.J., Fu, J., Nebenführ, A., Guo, Z., Chen, F., 2018. The rice terpene synthase gene OsTPS19 functions as an (S)-limonene synthase in planta, and its overexpression leads to enhanced resistance to the blast fungus Magnaporthe oryzae. Plant Biotechnol. J. 16, 1778-1787.】.. In alfalfa tribulus, mtTPS codes for a sesquiterpene synthase, and mutant Mttps plants are more susceptible to pathogenic bacteria Aphanomyces euteiches than wild type plants 【Yadav, H., Dreher, D., Athmer, B., Porzel, A., Gavrin, A., Baldermann, S., Tissier, A., Hause, B., 2019. Medicago TERPENE SYNTHASE 10 is involved in defense against an oomycete root pathogen. Plant Physiol. 180, 1598-1613.】.
Cotton is a world leading renewable textile fiber resource and is also an important source of edible oil. Cotton is characterized by having small and dark colored pigment glands containing sesquiterpene gossypol and its derivatives as plant defenses, providing basic and inducible protection against pests and diseases. GhTPS1, ghTPS and GhTPS3 have been identified in cotton as having mechanical injury, fungal inducers and methyl jasmonate (MeJA) induced sesquiterpene production, while transgenic tobacco plants that also induced expression 【Yang, C.Q., Wu, X.M., Ruan, J.X., Hu, W.L., Mao, Y.B., Chen, X.Y., Wang, L.J., 2013. Isolation and characterization of terpene synthases in cotton (Gossypium hirsutum). Phytochemistry 96, 46-56.】. of the corresponding synthase gene over-expressed GhTPS12 produced relatively more (3S) -linalool, and showed stronger resistance to herbivorous insects than wild-type plants as demonstrated by 【Huang, X.Z., Xiao, Y.T., Köllner, T.G., Jing, W.X., Kou, J.F., Chen, J.Y., Liu, D.F., Gu, S.H., Wu, J.X., Zhang, Y.J., Guo, Y.Y., 2018. The terpene synthase gene family in Gossypium hirsutum harbors a linalool synthase GhTPS12 implicated in direct defence responses against herbivores. Plant Cell Environ. 41, 261-274.】. virus-induced gene silencing (VIGS) experiments in which three GhTPS genes were associated with glandular activity 【Zhang, C.P., Zhang, J.L., Sun, Z.R., Liu, X.Y., Shu, L.Z., Wu, H., Song, Y., He, D.H., 2022. Genome-wide identification and characterization of terpene synthase genes in Gossypium hirsutum. Gene 828, 146462.】. however, TPS genes were rarely reported to be effective against the most severe soil transmission of cotton and verticillium wilt of the phytopathogenic fungus that causes vascular wilt.
Disclosure of Invention
In order to solve the problems, the invention provides an application of GhTPS gene in regulating and controlling verticillium wilt resistance of cotton. The invention discovers that GhTPS gene can regulate and control verticillium wilt resistance of cotton, and silencing GhTPS gene in cotton can lead to plant susceptibility improvement, fungus content increase and vascular bundle browning more serious.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides an application of GhTPS gene in regulating and controlling verticillium wilt resistance of cotton; the amino acid sequence of GhTPS gene code is shown in SEQ ID NO. 12.
Preferably, the modulation comprises reducing the amount of GhTPS gene expression to reduce verticillium resistance in cotton.
Preferably, the nucleotide sequence of GhTPS gene is shown as SEQ ID NO. 13.
The invention provides a recombinant vector for reducing verticillium wilt resistance of cotton, which comprises a part sequence of GhTPS gene and pTRV2 vector; the amino acid sequence of GhTPS gene code is shown in SEQ ID NO. 12.
Preferably, the nucleotide sequence of the part sequence of GhTPS gene is shown as SEQ ID NO. 5.
The invention provides an engineering bacterium for reducing verticillium wilt resistance of cotton, which comprises a recombinant vector and an original bacterium.
Preferably, the primordial bacteria comprise Agrobacterium (Agrobacterium).
Preferably, the agrobacterium comprises agrobacterium tumefaciens (Agrobacterium tumefaciens).
The invention provides an application of the recombinant vector or the engineering bacteria in the technical scheme in reducing the verticillium wilt resistance of cotton.
The beneficial effects are that:
The invention provides an application of GhTPS gene in regulating and controlling verticillium wilt resistance of cotton; the amino acid sequence of GhTPS gene code is shown in SEQ ID NO. 12. According to the invention, through a VIGS experiment, the function of GhTPS gene in resisting verticillium wilt is verified, and silencing GhTPS gene in cotton can lead to improvement of plant susceptibility, increase of fungus content and more serious browning of vascular bundles, which shows that GhTPS gene has positive regulation and control effect in resisting verticillium wilt in cotton. Provides gene resources for the development of effective gene resources for improving the verticillium wilt resistance of cotton in cotton breeding in future.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows subcellular localization of GhTPS-GFP fusion protein in tobacco leaves;
FIG. 2 shows the albino phenotype and the silencing efficiency of qRT-PCR detection GhTPS gene 16 days after pTRV2-GhCLA1 inoculation of plants;
FIG. 3 shows the results of post-silencing plant resistance to verticillium wilt of GhTPS genes in cotton.
Detailed Description
The invention provides an application of GhTPS gene in regulating and controlling verticillium wilt resistance of cotton; the GhTPS gene codes an amino acid sequence shown in SEQ ID NO.12, and the amino acid sequence is specifically as follows ： MASQASPVLASPHPAISSENRPKADFHPGIWGDMFLNCPDTDIDAATELQYEEFKAQVRKMIMEPVDDSNQKLPFIDAVQRLGVSYHFEKEIEDELENIYRDTNNNDAENDLYTTALRFRLLREHGFDISCDAFNKFKDEAGNFKPSLTSDVQGLLELYEASYMRVHGEEILDEAISFTTAQLTLALPTLDHPLSEQVGHALKQSIRRGLPRVEARNFISIYQDLESHNKSLLQFAKIDFNLLQLLHRKELSEICRWWKDLDFTRKLPFARDRVVEGYFWIMGVYFEPQYSLGRKMLTKVIAMASIVDDTYDSYATYDELIPYTNAIQRWDIKCMNQLPDYMKISYKALLDVYEEMEQLLANQGRQYRVEYAKKAMIRLAQAYLLEAKWTHQNYKPTFEEFRDNALPTSGYAMLAITAFVGMGEVITPETFKWAANDPKIIKASTIICRFMDDIAEHKFNHRREDDCSAIECYMEQYGVTAQEAYNEFNKHIESSWKDVNEEFLKPTEMPTPVLCRSLNLARVMDVLYREGDGYTHVGKAAKGGITSLLIDPIQI;
In the present invention, the nucleotide sequence of GhTPS gene is preferably shown in SEQ ID NO.13, specifically as follows ：5'-ATGGCTTCACAAGCTTCTCCAGTTCTCGCTTCACCCCATCCCGCCATTTCATCCGAAAATCGACCCAAGGCTGATTTTCATCCCGGTATTTGGGGTGATATGTTCCTCAACTGTCCTGATACGGATATCGATGCTGCAACTGAATTACAATATGAAGAATTTAAAGCTCAAGTGAGGAAGATGATTATGGAACCTGTTGATGATTCAAACCAAAAGTTGCCCTTCATTGATGCTGTTCAAAGATTAGGTGTGAGTTATCATTTTGAGAAAGAGATTGAAGATGAACTAGAGAATATTTACCGTGACACCAACAACAATGATGCTGAGAACGATCTCTACACTACAGCTCTTCGATTCCGGTTACTTAGAGAGCACGGCTTCGATATTTCATGTGATGCATTCAACAAGTTCAAAGATGAGGCAGGGAACTTCAAGCCATCATTGACGAGTGATGTGCAAGGGTTGTTGGAACTTTATGAAGCTTCCTATATGAGGGTCCATGGGGAAGAAATACTTGATGAAGCCATTTCTTTCACCACTGCTCAACTTACACTTGCTCTACCAACTTTAGACCATCCTTTATCGGAACAGGTCGGCCATGCCTTAAAGCAGTCTATCCGAAGGGGCTTGCCAAGGGTTGAGGCCCGGAATTTCATTTCGATATACCAAGATTTAGAATCCCACAACAAATCGTTGCTTCAATTTGCAAAGATTGATTTCAACTTGTTGCAGCTTTTGCATAGGAAAGAGCTAAGTGAGATCTGCAGGTGGTGGAAAGATTTAGACTTCACAAGAAAACTACCATTTGCAAGAGATAGAGTGGTTGAAGGCTATTTTTGGATAATGGGAGTTTACTTTGAACCCCAATACTCCCTTGGTAGAAAGATGTTGACAAAAGTCATAGCGATGGCTTCCATTGTTGATGATACTTATGATTCATATGCAACATATGATGAACTCATTCCTTATACAAATGCAATTCAAAGGTGGGATATTAAATGCATGAACCAACTTCCAGATTACATGAAAATAAGTTACAAGGCACTATTAGATGTTTATGAAGAAATGGAACAGCTGTTGGCAAATCAAGGGAGACAGTATCGAGTTGAGTATGCGAAAAAGGCGATGATACGCCTTGCTCAAGCTTACCTTTTGGAGGCCAAATGGACTCATCAAAACTATAAACCAACCTTCGAGGAATTTAGAGATAATGCATTACCAACCTCTGGCTATGCCATGCTTGCTATAACGGCGTTTGTCGGCATGGGCGAAGTTATAACCCCTGAAACCTTCAAATGGGCCGCCAATGACCCCAAGATCATTAAGGCTTCCACCATTATTTGCAGGTTCATGGATGATATTGCGGAACATAAGTTCAACCATAGGAGAGAAGACGATTGCTCAGCGATCGAATGTTACATGGAACAATATGGGGTGACAGCGCAAGAAGCATACAATGAATTCAACAAACACATTGAGAGTTCATGGAAAGATGTAAATGAAGAGTTCTTGAAACCGACAGAAATGCCGACACCCGTTCTTTGTCGTAGCCTCAACCTTGCTAGGGTTATGGATGTACTTTACAGAGAAGGTGACGGTTATACACATGTTGGGAAAGCTGCTAAAGGTGGGATCACTTCATTATTGATTGATCCAATCCAAATTTGA-3'.
In the present invention, the regulation preferably includes reducing the amount of GhTPS gene expression to reduce verticillium resistance in cotton. According to the invention, through a VIGS experiment, the function of GhTPS gene in resisting verticillium wilt is verified, and silencing GhTPS gene in cotton can lead to improvement of plant susceptibility, increase of fungus content and more serious browning of vascular bundles, which shows that GhTPS gene has positive regulation and control effect in resisting verticillium wilt in cotton.
The invention provides a recombinant vector for reducing verticillium wilt resistance of cotton, which comprises a part sequence of GhTPS gene and pTRV2 vector; the amino acid sequence of GhTPS gene code is shown in SEQ ID NO. 12.
In the present invention, the nucleotide sequence of the part sequence of GhTPS gene is preferably shown as SEQ ID NO. 5.
The invention provides an engineering bacterium for reducing verticillium wilt resistance of cotton, which comprises a recombinant vector and an original bacterium. In the present invention, the original bacteria preferably include agrobacterium; the agrobacterium preferably includes agrobacterium tumefaciens, more preferably agrobacterium tumefaciens GV3101.
The invention provides an application of the recombinant vector or the engineering bacteria in the technical scheme in reducing the verticillium wilt resistance of cotton. The recombinant vector or engineering bacteria provided by the invention can reduce the expression quantity of GhTPS gene, thereby reducing the verticillium resistance of cotton.
For further explanation of the present invention, the use of GhTPS gene provided in the present invention for controlling verticillium wilt resistance of cotton is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The CDS of GhTPS47 gene with the stop codon removed is subjected to homologous recombination with GFP in pCAMBIA2300-GFP vector by ClonExpress ® Ultra One Step Cloning Kit to obtain a recombinant plasmid 35S, ghTPS47-GFP, which is prepared by the following specific method:
1. Obtaining the target fragment: the cDNA of TM-1 cotton is used as a template, and a high-fidelity enzyme PRIMESTAR ® GXL Premix amplification sequence of TAKARA is utilized, and a PCR reaction system is as follows: PRIMESTAR GXL PREMIX 25 [ mu ] L, primer 2300-GhTPS-F1.5 [ mu ] L, primer 2300-GhTPS-R1.5 [ mu ] L, template 2 [ mu ] L, ddH 2 O20 [ mu ] L; the nucleotide sequence of the primer 2300-GhTPS-F is shown as SEQ ID NO.1, and the specific steps are as follows: 5'-ATACTAGTGGATCCGGTACCATGGCTTCACAAGCTTCTCCAGTT-3'; the nucleotide sequence of the primer 2300-GhTPS47-R is shown as SEQ ID NO.2, and the specific steps are as follows: 5'-CCCTTGCTCACCATGGTACCAATTTGGATTGGATCAATCAATAA-3';
The PCR conditions were as follows: 98. pre-denaturation at 5 min ℃; 98. denaturation at 10 s, annealing at 58℃for 15 s, extension at 68℃for 1 min/kb for a total of 35 cycles; 68. the final extension is 10 min deg.C.
2. Expression vector linearization: the expression vector is incubated at 37 ℃ for 3 h double enzyme digestion to obtain a linearization vector, and the reaction system is as follows: pCAMBIA2300-GFP 1 μg, bamHI 1 μL, sacI 1 μL, cutSmart μL, ddH 2 O complements to 50 μL.
3. And (3) recovering and purifying the PCR product and the linearization expression vector through Beijing full gold EasyPure ® Quick Gel Extraction Kit.
4. Carrying out homologous recombination on the linearization vector and the target fragment by utilizing ClonExpress ® Ultra One Step Cloning Kit of Nanjinopran to construct the vector, and preparing the following system on ice: linearization carrier 1 mu L, target fragment 4 mu L, 2X ClonExpress Mix mu L. 50. The reaction was allowed to react at 10 min c and immediately cooled on ice. The recombinant product was then transformed into E.coli competence. And (3) after the sequencing comparison is successful, extracting the escherichia coli plasmid by using Beijing full gold EasyPure ® PLASMID MINIPREP KIT.
The recombinant plasmid 35S GhTPS-GFP and empty pCAMBIA2300-GFP vector were transformed into Agrobacterium tumefaciens strain GV3101 and transiently expressed by injecting 5-week-old tobacco leaves until 80% of the leaves were wetted. GFP fluorescence signal was then detected by laser scanning confocal microscopy. The results are shown in fig. 1, scale bar = 20 μm.
As can be seen from FIG. 1, GFP fluorescence of GhTPS-GFP fusion protein was observed in both the nucleus and cytoplasm.
Example 2
Plant material and virus-induced gene silencing (VIGS)
1. Obtaining the target fragment: the cDNA of TM-1 cotton is used as a template, and a high-fidelity enzyme PRIMESTAR ® GXL Premix amplification sequence of TAKARA is utilized, and a PCR reaction system is as follows: PRIMESTAR GXL PREMIX 25 [ mu ] L, a primer VIGS-GhTPS 47-F1.5 [ mu ] L, a primer VIGS-GhTPS 47-R1.5 [ mu ] L, a template 2[ mu ] L and a ddH 2 O20 [ mu ] L; the nucleotide sequence of the primer VIGS-GhTPS-F is shown as SEQ ID NO.3, and is specifically as follows: 5'-gtgagtaaggttaccgaattcTCTCGCTTCACCCCATCCC-3'; the nucleotide sequence of the primer VIGS-GhTPS-R is shown as SEQ ID NO.4, and is specifically as follows: 5'-cgtgagctcggtaccggatccGCATCATTGTTGTTGGTGTCACG-3';
The PCR conditions were as follows: 98. pre-denaturation at 5 min ℃; 98. denaturation at 10 s, annealing at 58℃for 15 s, extension at 68℃for 1 min/kb for a total of 35 cycles; 68. the final extension is 10 min deg.C.
2. Expression vector linearization: the expression vector is incubated at 37 ℃ for 3 h double enzyme digestion to obtain a linearization vector, and the reaction system is as follows: pTRV 21 μg, bamhi 1 μl, ecori 1 μl, cutSmart μl, ddH 2 O complements to 50 μl.
3. Recovering and purifying the PCR product and the linearization expression vector through Beijing full gold EasyPure ® Quick Gel Extraction Kit; the nucleotide sequence of the PCR product is shown as SEQ ID NO.5, and is specifically as follows ：5'-TCTCGCTTCACCCCATCCCGCCATTTCATCCGAAAATCGACCCAAGGCTGATTTTCATCCCGGTATTTGGGGTGATATGTTCCTCAACTGTCCTGATACGGATATCGATGCTGCAACTGAATTACAATATGAAGAATTTAAAGCTCAAGTGAGGAAGATGATTATGGAACCTGTTGATGATTCAAACCAAAAGTTGCCCTTCATTGATGCTGTTCAAAGATTAGGTGTGAGTTATCATTTTGAGAAAGAGATTGAAGATGAACTAGAGAATATTTACCGTGACACCAACAACAATGATGC-3'.
4. Carrying out homologous recombination on the linearization vector and the target fragment by utilizing ClonExpress cube Ultra One Step Cloning Kit of Nanjinouzan, constructing the vector, and preparing the following system on ice: linearization carrier 1 mu L, target fragment 4 mu L, 2X ClonExpress Mix mu L.
50. The reaction was allowed to react at 10 min c and immediately cooled on ice. The recombinant product was then transformed into E.coli competence. And after the sequencing comparison is successful, extracting the escherichia coli plasmid by using Beijing full gold EasyPure ® PLASMID MINIPREP KIT to obtain the TRV GhTPS47 recombinant vector.
5. Agrobacterium transformation
1) 1 Mu L of plasmid is added into the freshly melted agrobacterium competent GV3101, and the mixture is gently mixed.
2) The tube was allowed to stand on ice for 10 min a and then placed in liquid nitrogen for 5a min a.
3) The centrifuge tube is rapidly placed in a 37 ℃ water bath 5 min without shaking the water surface.
4) And then placed on ice for standing for 5 min.
5) And adding 800 mu L of LB liquid medium without antibiotics, and placing the LB liquid medium in a shaking table at 28 ℃ to shake for 2-3 hours.
6) Centrifuging at 5000 r/min for 2min, discarding 700 mu L of supernatant in an ultra-clean workbench, leaving 100 mu L of bacterial liquid to blow and evenly coat on an LB solid culture medium containing K + antibiotics, drying, and then culturing in a 28 ℃ incubator for 2-3 d in an inverted mode.
6. VIGS treatment of cotton seedlings
In a greenhouse at 26 ℃,16 hours of light/8 hours of dark cycle, the relative humidity is about 70%, seeds of upland cotton variety 9053 with resistance to verticillium are cultivated, when cotton seedlings grow to the cotyledon flat period, seedlings with more consistent growth are selected, and VIGS treatment is carried out on seedling cotyledons by an injection method.
1) 2 Days before VIGS treatment of cotton seedlings, helper vector pTRV1, empty vector pTRV2 (negative control), positive control pTRV2-GhCLA1 (see [ Wang Xinyu, lv Kun, cai Caiping, xu Jun, guo Wangzhen, 2014. TRV virus-mediated gene silencing System was established and used in cotton; crop theory, 40, 8.) and the desired gene TRV were each stored at-80℃in a sterile centrifuge tube of 2 mL in 50. Mu.L of GhTPS recombinant vector solution, and 500. Mu.L of sterile LB (containing kanamycin (K +) 50. Mu.g/mL and rifampicin (Rif +) 50. Mu.g/mL) medium was added to the tube, and the culture was activated at 28℃in shaker 200 r/min.
2) The activated strain was inoculated into a sterilized flask containing 50 mL LB of the culture broth (containing K +& Rif+) and placed at 28℃and shaking at 200 r/min for 16 h.
3) Culturing the agrobacterium liquid by shaking table until the OD 600 is about 0.8, collecting the liquid into a sterilized 50mL centrifuge tube, and centrifuging at 8000 r/min for 10 min. After removing supernatant in the centrifuge tube, re-adjusting the bacterial liquid OD 600 to 0.8-1.0 by using a VIGS heavy suspension, uniformly mixing heavy suspension bacterial liquids of a positive control pTRV2-GhCLA1, a target gene TRV (GhTPS: recombinant vector) and a pTRV2 empty vector with the heavy suspension bacterial liquid of an auxiliary vector pTRV1 according to the equal volume of 1:1, and standing at room temperature for 3 h.
The formula of the VIGS bacterial liquid heavy suspension is as follows: magnesium chloride, mgCl:2.03 g/L, morpholinoethanesulfonic acid MES:2.135 g/L and acetosyringone AS:0.03 g/L;
5) When the VIGS treatment is carried out, a 1mL gun head is firstly used for carrying out light-point 3-4 small holes on the back of cotyledons of cotton seedlings. Then the treated bacterial liquid is injected into cotton cotyledons along the small holes by a 1mL injector until the whole cotyledons show water stain shape. After the seedlings are subjected to the VIGS treatment, uniformly watering, covering the treated cotton seedlings with a black plastic film, and culturing the cotton seedlings in a dark place for 24 h.
6) The next day of VIGS treatment of seedling cotyledons, the black plastic film was uncovered, and the culture conditions were maintained at 25 ℃,16 h light/8 h darkness.
Verticillium bacteria culture and inoculation
The verticillium wilt strain V592 deposited in the laboratory of the national institute of agricultural sciences, three-China (disclosed in 【Sun, M., Zhang, Z., Ren, Z., Wang, X., Sun, W., Feng, H., Zhao, J., Zhang, F., Li, W., Ma, X., Yang, D., 2021. The GhSWEET42 Glucose transporter participates in Verticillium dahliae infection in cotton. Front Plant Sci 12, 690754.】）, which was dark cultured on Potato Dextrose Agar (PDA) medium at 25 ℃ for 4-5 days, then transferred to Czapek medium, and shaken in a shaker at 25 ℃ for 3 days at 120 rpm days when the plant pTRV2-GhCLA1 developed a leaf whitening phenotype, spore concentration of the bacterial solution was adjusted to 10 7/mL with deionized water and verticillium wilt was inoculated by root irrigation.
Disease assessment after verticillium inoculation
At 16 days post inoculation, cotton roots were harvested for evaluation of seedling fungal biomass and the relative expression of GhTPS gene. Real-time fluorescence quantitative PCR (qRT-PCR) is carried out by adopting fungus specific primers ITS1-F and verticillium dahliae specific reverse primer STVe-R, the relative expression quantity of GhTPS genes is detected by qRT-PCR-GhTPS-F and qRT-PCR-GhTPS-R, ghHis3 is used as an internal reference, and the primer sequence is as follows:
ITS1-F：5’-AAAGTTTTAATGGTTCGCTAAGA-3’，SEQ ID NO.6；
STVe1-R：5’-CTTGGTCATTTAGAGGAAGTAA-3’，SEQ ID NO.7；
GhHis3-F：5’-GACACCAACCTTTGCGCGAT-3’，SEQ ID NO.8；
GhHis3-R：5’-AGCGACTGATCCACACTTCTG-3’，SEQ ID NO.9；
qRT-PCR-GhTPS47-F：5’-GAGATCTGCAGGTGGTGGAA-3’，SEQ ID NO.10；
qRT-PCR-GhTPS47-R：5’-CCAAGGGAGTATTGGGGTTCAA-3’，SEQ ID NO.11；
the qRT-PCR reaction system is as follows: 10. mu L2X ChamQ Universal SYBR QPCR MASTER Mix, primers F/R0.5 mu L each, template cDNA 1 mu L, ddH 2 O to 20 mu L;
The reaction program of the qRT-PCR is as follows: pre-denaturation at 95 ℃,30 s; cycling reaction at 95 ℃,10 s,60 ℃,30 s and 40 cycles; the melting curve uses an instrument default program; the results are shown in FIG. 2, FIG. 3, A and FIG. 3, wherein A in FIG. 2 is the albino phenotype 16 days after pTRV2-GhCLA1 inoculation of the plants; b in FIG. 2 is the silencing efficiency result of qRT-PCR detection GhTPS gene; FIG. 3A is the disease symptoms of GhTPS gene-silenced plants and negative control plants (TRV:: 00) 16 days after inoculation; b in FIG. 3 is the results of qRT-PCR assay TRV::00 and TRV:: ghTPS fungal biomass in plants; * P < 0.01.
Fresh stems on cotyledonary nodes at the same position of each plant are collected and used for a verticillium dahliae recovery test. The stem is soaked in 75% alcohol for 1 minute, then soaked in 30% hydrogen peroxide for 30 minutes, and washed with sterile water for 4-5 times. The stem sections cut into 1 cm pieces were placed on PDA medium, cultured at 25℃for 3 days, and the growth of Verticillium dahliae was observed, the observation method was seen from 【 Sun, M., Zhang, Z., Ren, Z., Wang, X., Sun, W., Feng, H., Zhao, J., Zhang, F., Li, W., Ma, X., Yang, D., 2021. The GhSWEET42 glucose transporter participates in Verticillium dahliae infection in cotton. Front Plant Sci. 12, 690754.】. Disease Index (DI) score ranging from 0 (none) to 4 (severe) for evaluation of Disease severity, the DI calculation method was seen from 【Gong, Q., Yang, Z., Chen, E., Sun, G., He, S., Butt, H.I., Zhang, C., Zhang, X., Yang, Z., Du, X., Li, F., 2018. A phi-class glutathione S-transferase gene for Verticillium wilt resistance in Gossypium arboreum identified in a genome-wide association study. Plant Cell Physiol. 59, 275-289.】. results to C-E in FIG. 3, where C in FIG. 3 is TRV: 00 and TRV: ghTPS plant Disease index, D in FIG. 3 is fungus recovery experimental results, and E in FIG. 3 is vascular tissue inoculated with verticillium wilt TRV: 00 and TRV: ghTPS plants.
As can be seen from FIG. 2, when the plants of pTRV2-GhCLA had a albino leaf phenotype, qRT-PCR analysis showed that the expression level of each target gene (GhTPS) in the plants inoculated with the TRV: ghTPS47 recombinant vector was significantly reduced compared with the control plants, demonstrating the effectiveness of gene silencing.
As can be seen from FIG. 3, ghTPS.sup.47-silenced plants showed more wilting and chlorosis (A in FIG. 3) than negative control plants after 16 days of cotton seedling inoculation with the verticillium V592 strain. qRT-PCR analysis of fungal biomass showed that GhTPS had a higher fungal content in the silenced plants (B in FIG. 3). The disease index of GhTPS 47-silenced plants was significantly higher than that of control plants (C in FIG. 3). Fungus recovery experiments performed on stem segments of inoculated cotton showed that the hyphal growth of the TRV:: ghTPS plants was evident, whereas the control plants showed little hyphal growth (D in FIG. 3). Consistent with this finding, the vascular bundle browning of the TRV:: ghTPS47 plant was more severe than that of the TRV::00 plant (E in FIG. 3). In summary, cotton plants that have silenced the GhTPS47 gene are more susceptible to disease, and the GhTPS47 gene plays a key role in regulating resistance of cotton to verticillium wilt.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (2)
- Application of GhTPS47 gene in regulating cotton verticillium wilt resistance, wherein the amino acid sequence coded by GhTPS gene is shown as SEQ ID NO. 12; the regulation is to reduce the expression level of GhTPS gene to reduce verticillium wilt resistance of cotton, and the pathogenic bacteria of verticillium wilt of cotton is verticillium dahliae (Verticillium dahliae).
- 2. The use according to claim 1, wherein the nucleotide sequence of GhTPS gene is shown as SEQ ID No. 13.
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| CN102321162A (en) * | 2011-10-08 | 2012-01-18 | 左开井 | GbRPS1 gene for resisting plant fusarium wilt and verticillium wilt and application thereof |
| CN109097350A (en) * | 2018-08-31 | 2018-12-28 | 中国林业科学研究院热带林业研究所 | A kind of oil nanmu sesquiterpene synthase SgSTPS2 and its encoding gene and application |
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