Application of cotton GbCCD gene in resistance to Verticillium wilt

Abstract

This application belongs to the technical field of cotton gene resistance breeding, specifically involving two cotton GbCCD Patent application for the application of genes in cotton resistance to Verticillium wilt. In view of the important role of SLs as a new type of hormone in plant biotic and abiotic stress, in this application, the inventors used reverse genetics, genetic engineering, molecular biology and bioinformatics to synthesize SLs genes. CCD7 / 8 Expression and V. dahliae Response relationship research, and through CCD7 / 8 Gene silencing and related regulatory pathway studies have initially clarified CCD7 / 8 The regulatory mechanism and effect of genes in SLs cotton Verticillium wilt resistance can lay a good technical foundation for the prevention and control of cotton Verticillium wilt and the breeding of new plant varieties resistant to Verticillium wilt. It can also provide new reference and reference for the screening of new disease-resistant genes and the breeding of resistant plants.

Description

Technical Field

[0001] This application belongs to the technical field of cotton gene resistance breeding, specifically involving two cotton GbC Patent application for the use of genes in cotton resistance to Verticillium wilt. Background Art

[0002] Cotton is the main source of natural fiber and an important oil crop, and is also one of the important economic crops. In agricultural cultivation, cotton wilt is one of the main diseases in the cotton planting process. Cotton wilt is a soil-borne fungus Verticillium dahliae ( Verticillum dahliae ) mainly harms the stems, branches and leaves of cotton. Since Verticillium dahliae mainly exists in the form of microsclerotia, cotton Verticillium wilt is a disease that is difficult to eradicate and extremely difficult to prevent and control. Therefore, in-depth research on the mechanism of action between cotton and Verticillium wilt and analysis of the biological functions of cotton Verticillium wilt resistance-related genes are of great scientific significance for the prevention and control of cotton Verticillium wilt.

[0003] Existing research on plant defense mechanisms against pathogens shows that there are three main modes of plant immune recognition of pathogens, namely: PTI (PAMP-triggered Immunity) immune response triggered by pathogen-associated molecular patterns (PAMPs), ETI (Effector-triggered Immunity) immune response triggered by effectors, and DTI (DAMP-triggered Immunity) immune response triggered by damage-associated molecular patterns (DAMPs). Among them, plant hormones are important regulators in the PTI, ETI and SAR immune processes, and the main hormones of plant immune responses include SA, JA and ET. In the specific response mechanism, the basic immune response triggered by the recognition of PAMPs molecules by pattern recognition receptors (PRRs), including Ca 2+ Influx, ROS burst, hypersensitive response (HR), activation of MAPK (Mitogen-activated protein kinase) cascade pathway, NO production, callose deposition and defense gene expression, thereby regulating the plant's adaptability to adversity. Summary of the invention

[0004] Based on the previous screening of cotton wilt resistance genes, the main purpose of this invention is to provide two genes with application prospects for wilt resistance. GbCGene( GbCCD7 , GbC ), thus laying a certain application foundation for the prevention and control of Verticillium wilt and the cultivation of new cotton varieties resistant to Verticillium wilt.

[0005] The detailed technical solution adopted in this application is as follows.

[0006] cotton GbC Application of gene in cotton resistance to Verticillium wilt, the cotton GbC The genes include two: GbCCD7 , GbC ;

[0007] The cotton GbC Gene, which is the carotenoid cleavage dioxygenase gene in the SLs synthase; in specific application, cotton GbC The expression of the gene was upregulated in response to induction by Verticillium dahliae;

[0008] cotton GbC Gene expression is positively correlated with resistance to Verticillium wilt; Silent cotton GbC Gene expression can reduce cotton resistance to Verticillium wilt; Overexpression of cotton GbC After the gene is introduced, the resistance of cotton to Verticillium wilt can be enhanced;

[0009] cotton GbC The gene exerts resistance to Verticillium wilt through the accumulation of reactive oxygen species (ROS);

[0010] cotton GbC Gene expression is regulated by the plant hormone JA, and the transcription factor MYC2 in the JA signaling pathway is negatively regulated CCD7 / 8 expression.

[0011] A method for enhancing cotton resistance to Verticillium wilt, which can improve cotton resistance to Verticillium wilt by applying SLs or its analogs (such as rac-GR24) externally; the application mechanism is: applying SLs or its analogs (such as rac-GR24) externally can enhance defense-related genes in the JA signaling pathway MYC2 , PDF1.2 and VSP2 expression to improve resistance to Verticillium wilt; at the same time, the transcription factor MYC2 of the JA signaling pathway negatively regulates CCD7 / 8 expression.

[0012] A method for breeding new cotton varieties resistant to Verticillium wilt by overexpression GbCCD7 and GbC Genetic approaches to improve Verticillium wilt resistance.

[0013] Strigolactones (SLs) are a class of terpenoid lactones derived from carotenoids. As the cleavage product of carotenoids, they can be found in the root secretions of various plants. Since 2008, researchers have identified them as a new type of plant hormone. Studies have shown that the synthesis process of SLs is mainly composed of D27 (Dwarf 27), CCD7 / 8 (Carotenoid-cleavage dioxy-genases7 / 8, carotenoid cleavage dioxygenase) and MAX1 (Moreaxillary growth 1) is accomplished by four genes encoding proteins or enzymes. Among them, two key enzymes catalyze the cleavage of carotenoids to form strigolactones CCD7 / 8 Belongs to the carotenoid cleavage dioxygenase CCDs family. The CCDs family is a Fe 2+ It is a large family of CCD enzymes, which can be divided into five subfamilies, namely CCD1, CCD2, CCD4, CCD7 and CCD8, according to their cleavage activity and substrate properties. Among them, CCD1, CCD4 and CCD7 have broad substrate specificity, while CCD8 is believed to be specific for the synthesis of SLs.

[0014] In view of the important role of SLs as a new type of hormone in plant biotic and abiotic stress, in this application, the inventors used reverse genetics, genetic engineering, molecular biology and bioinformatics to synthesize SLs genes. CCD7 / 8 Expression and V. dahliae Response relationship research, and through CCD7 / 8 Gene silencing and related regulatory pathway studies have initially clarified CCD7 / 8 The regulatory mechanism and effect of genes in SLs cotton Verticillium wilt resistance can lay a good technical foundation for the prevention and control of cotton Verticillium wilt and the breeding of new plant varieties resistant to Verticillium wilt. It can also provide new reference and reference for the screening of new disease-resistant genes and the breeding of resistant plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The evolutionary tree and gene structure analysis of CCD7 / 8 in allotetraploid cotton sea island cotton and upland cotton, including: A, the evolutionary tree of CCD7 / 8; B, the structure of CCD7 / 8 gene;

[0016] Figure 2 The cloning and tissue expression patterns of GbCCD7 / 8a / b genes, where: A, agarose gel electrophoresis of GbCCD7 / 8a / b gene cloning; B, tissue expression pattern of GbCCD7 / 8a / b genes; C, expression pattern of GhCCD7 / 8a / b genes;

[0017] Figure 3 The expression pattern of cotton GbCCD7 / 8a / b gene after fungal infection and the germination experiment of O. aegyptiaca seeds; A, transcription level of GbCCD7 / 8a / b gene of sea island cotton after fungal infection; B, transcription level of GhCCD7 / 8a / b gene of upland cotton after fungal infection; C, representative picture of O. aegyptiaca seed germination; D, germination rate of O. aegyptiaca seeds; scale bar = 5 mm;

[0018] Figure 4 The results show that the application of rac-GR24 can enhance the resistance of the susceptible cotton variety Jimian11 to Verticillium wilt. A, symptoms of V. dahliae infection 21 days after cotton was treated with rac-GR24; B, cotton disease index; C, anatomical diagram of cotton stalk; scale bar = 2 mm; D, statistics of browning area of ​​cotton stalk; E, recovery culture of V. dahliae on cotton stalk;

[0019] Figure 5 Effects of exogenous application of rac-GR24 on the transcription level of GbCCD7 / 8b in cotton; A, transcription level of GbCCD7 / 8b in sea island cotton treated with rac-GR24; B, transcription level of GbCCD7 / 8b in upland cotton treated with rac-GR24;

[0020] Figure 6 The results showed that the application of TIS108 reduced the disease resistance of cotton to Verticillium wilt; A, the transcription levels of GbCCD7 and GbCCD8b after TIS108 treatment of cotton Hai7124; B, the picture of Orobanche seed germination; scale bar = 5 mm; C, the germination rate of Orobanche seeds; D, the symptoms of V. dahliae infection in cotton for 21 days; E, the disease index of cotton; F, the anatomical diagram of cotton stem; scale bar = 2 mm;

[0021] G, Statistics of browning area of ​​cotton stalks; H, Recovery culture of V. dahliae; Scale bar = 2 cm;

[0022] Figure 7 There was no significant difference in the resistance of cotton to Verticillium wilt after interfering with GbCCD8a gene; A, transcription level of GbCCD8a gene in interfering plants; B, germination diagram of Orobanche seeds; scale bar = 5 mm; C, germination rate of Orobanche seeds; D, albino phenotype of cotton after interfering with GbCLA gene; E, symptoms of cotton infected with V. dahliae for 21 days; F, disease index of cotton;

[0023] Figure 8The interference of GbCCD7 / 8b gene reduced the disease resistance of cotton to Verticillium wilt, where: A and B, transcription levels of GbCCD7 and GbCCD8b genes in the interference plants; C and D, germination and germination rate of Orobanche seeds treated with root extracts of GbCCD7 and GbCCD8b interference plants; scale bar = 5 mm; E, albino phenotype of GbCLA gene interference plants; F, symptoms of cotton infected with V. dahliae for 21 days after the interference of GbCCD7 and GbCCD8b respectively; G, disease index of cotton at 21 and 23 days after the onset of disease; HJ, anatomical diagram of cotton stalks, statistics of browning area and recovery culture of V. dahliae; scale bar = 2 mm (stalk dissection), scale bar = 2 cm (recovery culture);

[0024] Fig. 9 This is a T0 generation OE-GbCCD8b transgenic cotton; A, schematic diagram of the overexpression vector; B, PCR identification of the target gene in the transgenic plant; C, T0 transgenic seedling;

[0025] Fig.10 The resistance of transgenic cotton to Verticillium wilt by overexpressing OE-GbCCD8b; A, PCR verification of the target gene in T1 transgenic plants; B, immunoblotting detection of GbCCD8b protein levels in transgenic plants; C, quantitative detection of GbCCD8b gene transcription levels in transgenic plants; D, disease resistance phenotype of transgenic plants to Verticillium wilt; E, disease index of transgenic plants; F, anatomical diagram of cotton stalks; G, statistics of browning area of ​​cotton stalks;

[0026] Fig.11 Analysis of cis-acting elements in the promoter of GbCCD7 / 8b gene; A, fan-shaped graph showing the proportion of each cis-acting element in the promoter; B, bar graph showing the number of cis-acting elements that respond to hormone signals; C, distribution of cis-acting elements in promoters;

[0027] Fig.12 The three-dimensional structure and subcellular localization of GbCCD7 and GbCCD8b; A, the three-dimensional structure of GbCCD7 and GbCCD8b proteins, including monomers and homodimers; B, the expression model of GbCCD7-GFP and GbCCD8b-GFP reporter genes; C, the subcellular localization of GbCCD7-GFP and GbCCD8b-GFP in Nicotiana benthamiana; Scale bar = 50µm;

[0028] Fig.13The transcription levels of GbCCD7 / 8b in cotton treated with different hormones; A, the transcription levels of GbCCD7 and GbCCD8b in cotton treated with methyl jasmonate MeJA; B, the transcription levels of GbCCD7 and GbCCD8b in cotton treated with salicylic acid SA; C, the transcription levels of GbCCD7 and GbCCD8b in cotton treated with ABA;

[0029] Fig.14 Self-activation assay of pAbAi-proGbCCD7 and pAbAi-proGbCCD8b; AbA bait reporter Y1HGold was inhibited on SD / -Ura +700 ngml-1 AbA medium; Bar = 2 cm;

[0030] Fig.15 The interaction between GbMYC2 and GbCCD7 / 8 promoters was verified by yeast one-hybrid experiment (Y1H); A and B, GbMYC2 interacts with the promoters of GbCCD7 and GbCCD8b genes, respectively; Y1HGold reporter strain transformed with pGADT7-GbMYC2 and pAbAi-proGbCCD7 plasmids can grow in SD / -Ura+700 ng ml-1 AbA medium; Bar = 5mm;

[0031] Fig.16 Luciferase LUC imaging experiment verifies the regulatory relationship between MYC2 and GbCCD7 / 8b promoter; A, schematic diagram of LUC reporter gene expression model; B, LUC imaging analysis of the interaction between GbMYC2 and GbCCD7 and GbCCD8b promoters; EV represents empty vector as control;

[0032] Fig.17 The transcription levels of defense-related genes in cotton after rac-GR24 treatment; AB, the transcription levels of defense genes GbMYC2 and GbPDF1.2 in the hormone JA signaling pathway; CD, the transcription levels of defense genes GbNPR1 and GbPR1 in the SA signaling pathway;

[0033] Fig.18 The transcription levels of cotton defense-related genes in the presence of the SLs synthesis inhibitor TIS108 were shown in Figure 2. Among them: AC, the transcription levels of defense genes GbMYC2, GbPDF1.2 and GbVSP2 in the hormone JA signaling pathway; DE, the transcription levels of defense genes GbNPR1 and GbPR1 in the SA signaling pathway;

[0034] Fig.19To interfere with the transcription level of cotton defense-related genes of GbCCD7 / 8b gene; among them: AC, the transcription level of defense genes GbMYC2, GbPDF1.2 and GbVSP2 of hormone JA signaling pathway; DE, the transcription level of defense genes GbNPR1 and GbPR1 of SA signaling pathway;

[0035] Fig. 20 To interfere with the accumulation of ROS in leaves of GbCCD7 / 8b gene cotton plants; A, ROS staining of active oxygen in cotton leaves; B, detection of H2O2 content in cotton leaves; C, Trypan blue staining of cotton leaves;

[0036] In the relevant figures, the relevant data were statistically analyzed, and the significance analysis was t-test: **P < 0.01, ***, P < 0.001. DETAILED DESCRIPTION

[0037] The present application is further explained below in conjunction with the embodiments. Before introducing the specific embodiments, some experimental backgrounds in the following embodiments are briefly introduced as follows.

[0038] Biomaterials:

[0039] Sea Island cotton Gossypium barbadense ) Hai 7124, Upland cotton ( Gossypium hirsutum )Jilin 11, Arabidopsis wild type Col-0 ( Arabidopsis thaliana ), are commonly used experimental materials and can be obtained from public channels;

[0040] Arabidopsis mutants max3-11 and max4-6 were purchased from a public Arabidopsis mutant seed resource bank;

[0041] Escherichia coli DH5α (E. coli), Agrobacterium tumefaciens GV3101 and LBA4404 ( Agrobacterium tumefaciens ), Verticillium dahliae V991, are commonly used experimental materials and can be obtained from public channels; V991-GFP (V. dahliae) strain was provided by Professor Jiang Jing's research group at Henan University;

[0042] The cloning vector pClone007 was purchased from Qingke Biotechnology; the VIGS experiment-related vectors pTRV1, pTRV2 and pTRV2-CLA are all commonly used experimental materials and can be obtained from public channels; the gene editing vector pYL-CRISPR / Cas9 is a commonly used vector in public use and can be obtained by referring to the conventional construction of the prior art. The vectors used in the following examples were provided by Gao Wei's research group at Henan University; the overexpression vectors pCAMBIA1300 and pCAMBIA2300 are common plasmid vectors in existing biological research and can be obtained from public channels;

[0043] Experimental reagents:

[0044] RNAprep Pure Plant Total RNA Extraction Kit, a product of TIANGEN; HiScript II Q SelectRT SuperMix for qPCR (for reverse transcription and cDNA synthesis), FastPure Plant DNA Isolation Mini kit (for plant DNA extraction), ClonExpress Ultra One Step Cloning Kit (for VIGS vector construction), ChamQ TM SYBR Color qPCR Master Mix (for q-PCR gene quantification), Rapid Taq Master Mix (for PCR amplification), products of Vazyme; The EZNAGel Extraction Kit (for agarose gel recovery), TheE.ZNAplasmid mini Kit (for plasmid extraction), products of OMEGA;

[0045] PrimerSTAR Max DNA Polymerase (high-fidelity PCR amplification enzyme), a product of TaKaRa; KOD OneTM PCR Master Mix (for PCR amplification), a product of TOYOBO; Gateway® BP enzyme (for Gateway vector construction), Gateway® LR enzyme (for Gateway vector construction), a product of Invitrogen; restriction endonucleases Kpn I, Bam HI, Bsa I, a product of NEB; Anti-GFP Mouse Monoclonal Antibody (for Western blot detection), a product of Transgen; HRP-conjugated AffiniPure Goat Anti- Mouse IgG (for Western blot detection), a product of Proteintech;

[0046] GR24( rac -GR24, powder form), a product of YEASEN; TIS108 (inhibitor), a product of ChemeGen;

[0047] Main experimental equipment:

[0048] Real-time fluorescence quantitative PCR instrument (LightCycler 480Ⅱ), a product of Roche, Switzerland; PCR instrument (2990224882), a product of Applied biosystems; laser confocal microscope Nikon / A1+N-SIM, a product of Nikon; inverted fluorescence microscope LX73, a product of OLYMPUS; chemiluminescence imager Fusion-FX, a product of Vilber.

[0049] Example 1

[0050] Based on the inventor's previous research, the inventor found that after cotton was infected by Verticillium wilt, GbCCD7 and GbC The expression level is responsively upregulated. In this regard, the inventors took the sea island cotton Hai7124 as an example to analyze the evolutionary tree and expression pattern of the relevant genes, and the specific situation is briefly introduced as follows.

[0051] 1. Cotton GbCCD7 and GbC Gene evolution tree analysis

[0052] In the long-term biological evolution of cotton, interspecific hybridization of diploid wild cotton has led to the formation of allotetraploid cotton. Therefore, understanding the evolutionary relationship between genes in allotetraploid cotton is of great scientific significance for the analysis of gene function. Based on the previous research work and in view of the application prospects of plant hormone strigolactones (SLs) in plant stress resistance, the inventors selected carotenoid cleavage dioxygenase ( CCD7 / 8 ) were taken as the research object, and the cultivated species of upland cotton and sea island cotton were used as research materials, and a preliminary analysis of their phylogenetic evolutionary relationships was conducted.

[0053] Specific evolutionary tree analysis and gene structure comparison Figure 1 As shown. Analysis shows that: in sea island cotton and upland cotton, CCD7 There are two copies of the gene, one on chromosome 12 in the A genome and one on the D genome. CCD8 The gene has four copies, i.e. two pairs of alleles. CCD8a and CCD8b , they are located on chromosomes 06 and 07 of the A genome and D genome respectively; at the same time, the homologous genes between different species are closely related and are distributed on the same subgenomes and corresponding chromosomes; and the comparison of gene structures shows that they are highly conserved in gene structure, but individual genes will also undergo obvious changes in gene structure due to mutations in the long-term evolutionary process.

[0054] 2. Cloning and tissue expression pattern of GbCCD7 / 8b gene in sea island cotton

[0055] Since tissue expression patterns have an important influence on gene function, the inventors used sea island cotton Hai7124 as research material and cloned GbCCD7 , GbCCD8a and GbC The coding sequence (CDS) of the gene was analyzed and its tissue expression pattern was preliminarily analyzed. The details are briefly described as follows.

[0056] (1) Gene cloning

[0057] With reference to the prior art and the instructions of the relevant kit, total RNA was extracted from young leaves of cotton Hai7124, and reverse transcribed to prepare cDNA for later use;

[0058] Subsequently, the primer sequences for PCR amplification were designed as follows:

[0059] GbCCD7-F:5'-ATGCAAGCAAAAATATTCCATATTG-3',

[0060] GbCCD7-R: 5'-ATCATTCTTGGCCCAAAAACC-3';

[0061] GbCCD8a-F:5'-ATGGATTCCCTATGGTTTTCAGAT-3',

[0062] GbCCD8a –R: 5′-TTATTTCTTGGGTACCCAACATCC-3′;

[0063] GbCCD8b-F: 5'-ATGGGTTCCCTATCACTTTCAGG-3',

[0064] GbCCD8b –R: 5′-TTCTTTGGGTACCCAACATCCA-3′;

[0065] Then, PCR amplification was performed and the amplification primers were tested by electrophoresis (the electrophoresis results were as follows Figure 2 A) and extraction;

[0066] Finally, the extraction results were sequenced and analyzed to obtain the cloned gene sequence.

[0067] The specific sequence results are as follows:

[0068] GbCCD7 (shown in SEQ ID No.1, 1842 bp):

[0069]

[0070] GbCCD8a :

[0071]

[0072] GbC (shown in SEQ ID No.2, 1674 bp):

[0073]

[0074] (2) Analysis of tissue expression patterns

[0075] Based on the above cloning results, the inventors further analyzed the expression patterns of related genes using RT-qPCR technology (UBQ7 was an internal reference gene). It should be noted that during RT-qPCR detection, the relevant primer sequences were designed as follows:

[0076] GbCCD7-F: 5'-CCGATACTTATTACATACTGTTCG-3',

[0077] GbCCD7-R: 5'-ACTTATCAGATGACCGAGGA-3';

[0078] GbCCD8a-F: 5'-CGAAACCGTAGACCGTCGTGAT,

[0079] GbCCD8a- R: 5'-GACCTGGGCCATTTCGTAA-3';

[0080] GbCCD8b-F:5'-AGAACCCGTCGACCCGGATCA-3',

[0081] GbCCD8b- R: 5'-GACCTGGACCGTTTCTGAG-3';

[0082] GbUBQ7-F: 5'-GAAGGCATCCACCTGACCAAC-3',

[0083] GbUBQ7-R: 5'-CTTGACCTTCTTCTTCTTGTGCTTG-3'.

[0084] Tissue expression pattern results Figure 2 B. It can be seen that: in Hai7124, GbCCD7 and GbC The expression was highest in roots, followed by stems, and lowest in leaves; GbCCD8a The expression level is highest in the stem, followed by the root, and lowest in the leaf. As a control and reference, the inventors also tested the expression of related genes in the "Upland Cotton Jimian11" plant. The specific results are as follows Figure 2 C. It can be seen that the expression pattern of related tissues is similar to that of sea island cotton, except that GhCCD8b The expression in leaves was slightly higher than that in stems.

[0085] Based on the above results, we can preliminarily determine that: CCD7 / 8a / bThe gene is mainly expressed in the roots and stems of cotton, and the synthesis of SLs is mainly achieved in the roots and stems of cotton, which has certain guiding significance for studying and clarifying the function of SLs in cotton resistance to Verticillium wilt.

[0086] 3. Cotton GBCCD7 / 8 Gene response to Verticillium wilt (i.e. gene expression pattern in response to fungal invasion)

[0087] In order to clarify whether the biosynthesis of cotton SLs can respond to the induction of Verticillium dahliae, the inventors used the fungal expression mode to express the SLs synthesis gene in cotton infected with Verticillium dahliae. GBCCD7 / 8 The changes in transcription levels were studied and analyzed. The specific experimental results are briefly described as follows.

[0088] The sea island cotton (Hai7124) plants were removed from the culture pots, the soil on the roots was washed off, the moisture on the roots was absorbed with absorbent paper, the root tips were cut off, and the plants were placed in spore solution (spore solution of Verticillium dahliae V991, 1×10 7 conidia / mL) was allowed to infect for 1 min, and then the seedlings with spores on the roots were replanted in the nutrient pots and cultured normally. Samples were taken at 0, 1, 3, 5, 7 and 9 days, and the results at each time point were tested. GbCCD7 , GbCCD8a and GbC Changes in transcription levels. When specifically testing (for detailed instructions, refer to the instructions of the relevant kit, the above instructions or the existing technology):

[0089] First, total RNA was extracted from root tissues and reverse transcribed to prepare cDNA; Ubiquitin7 ( UBQ7 ) was used as the internal reference gene, and real-time quantitative PCR was performed using a real-time fluorescence quantitative PCR instrument (Roche LightCycler480). The reaction program was: 95°C, 1 min; then 95°C, 5s, 60°C, 40s, 45 cycles; after the reaction, the specificity of qRT-PCR amplification was determined by melting curves, at least three biological replicates were used, and 2 −△△Ct The expression level was calculated by

[0090] The experimental results are as follows Figure 3 As shown in A. It can be seen that after 3 days of bacterial invasion, the expression of SLs synthesis genes began to increase and showed a linear increase trend, especially GbCCD7 and GbC The up-regulation trend of genes was the most significant.

[0091] Similarly, as a reference and control, the inventors used upland cotton plants (Jimian11) as experimental materials and obtained similar results, where GhCCD7The transcription level of Figure 3 B).

[0092] To further determine V. dahliae Whether the infection of O. spp. affects the changes in SLs content in the roots of cotton plants was investigated, with the parasitic weed Orobanche deserticola ( Orobanche aegyptiaca ) was used as the experimental material, and a seed germination experiment was used to evaluate the changes in SLs content in cotton roots. The specific experimental introduction is as follows.

[0093] The SLs extract from the roots of Hai7124 cotton infected with Verticillium wilt for 9 days was used to treat the seeds of Orobanche, and the seed germination rate was observed and counted. The specific operation is as follows: First, the seeds of Orobanche were sterilized with 1% sodium hypochlorite for 3 minutes, and then rinsed with sterile water 5 times as sterile seeds; then, the seeds were spotted with water on 8 mm glass fiber filter paper and placed in a culture dish; at the same time, the cotton root samples (about 100 mg per sample) were ground into powder and ultrasonically treated in 1.5 ml of water for 30 minutes; finally, after the cotton root water extract was centrifuged, 15 µL of the extract was immediately added to the filter membrane (glass fiber filter paper) containing the seeds of Orobanche, and cultured in the dark at 25°C, and the germination rate was detected after 8 days.

[0094] The experimental results are as follows Figure 3 C. Figure 3 D. It can be seen that compared with the control group (Mock), the infection V. dahliae The germination rate of Orobancha seeds treated with cotton root extract was higher ( Figure 3 C and D), which indicates V. dahliae The infection caused the accumulation of SLs in cotton roots.

[0095] Based on the above results, we can preliminarily infer that SLs synthesis gene GbCCD7 / 8b The transcription of V. dahliae The induction of SLs is upregulated, thereby increasing the SLs hormone content in the roots of cotton plants, which may affect the resistance of cotton to Verticillium wilt.

[0096] Example 2

[0097] Based on the results of Example 1, in order to further accurately determine whether the change in SLs content has a direct impact on cotton resistance to Verticillium wilt, the inventors applied SLs analogs rac -GR24 and the exogenous application of SLs synthesis inhibitor TIS108 were used to evaluate the changes in cotton resistance to Verticillium wilt. The specific experimental results are briefly described as follows.

[0098] 1. External administration of SLs analogs rac -GR24's Effect on Cotton's Resistance to Verticillium Wilt

[0099] In the outside rac -GR24 (3 µM), the cotton varieties were infected with Verticillium wilt, and the incidence of Verticillium wilt in cotton plants was observed. The specific experiment is as follows.

[0100] First, the cotton seedlings grown to two leaves and one heart were divided into two groups. One group was irrigated with 5 mL of 3 µM GR24 solution from the surface of vermiculite to the roots (it should be explained that in the preliminary experiment, under a certain concentration range, the results of different concentrations of GR24 solution were consistent. Therefore, for the convenience of operation, this embodiment only used this concentration for the experiment), and the other group was irrigated with an equal volume of aqueous solution containing the same solvent; after the irrigating was completed, the cultivation was continued for 24 hours; then, the seedlings were taken out of the pot, the vermiculite was washed off, the end of the lateral root was cut off by 2-3 mm with scissors, and then immersed in a solution containing 1×10 7 Pieces / mL V. dahliae The seedlings were placed in the spore suspension for 1 min; then, the seedlings were replanted in the vermiculite and continued to be cultured and grown in the incubator for 3 days before sampling; in the experiment, the experimental group treated with GR24 and the control group treated with water were treated with bacteria respectively; simultaneously, cotton treated with sterile water after root injury was used as the blank control group.

[0101] The results of the test on susceptible cotton variety Jimian11 infected with Verticillium wilt are as follows Figure 4 As shown in the figure (the difference in the effect of susceptible varieties is relatively more significant). It can be seen that after 21 days of infection, the plants in the control group (Mock) showed obvious symptoms of disease, while rac -GR24 treated cotton plants showed reduced Verticillium wilt symptoms, and rac -GR24 treated cotton plants had a disease index that was about 20% lower than that of the control ( Figure 4 A and B).

[0102] Further analysis of cotton stalks showed that rac The browning degree and browning area of ​​cotton stems treated with -GR24 were significantly lower than those of Mock. Further recovery culture experiments on Verticillium dahliae in stems also showed that rac -GR24 treated cotton plants had less Verticillium dahliae content ( Figure 4 C. Figure 4 D and Figure 4 E).

[0103] In general, the above phenotypic and physiological experiments preliminarily proved that: rac -GR24 can enhance the disease resistance of susceptible variety Jimian11 to cotton Verticillium wilt to a certain extent.

[0104] Although the above experiments prove that rac -GR24 can enhance the cotton V. dahliae However, in this case, whether the content of SLs in cotton plants is affected by exogenous SL analogs still needs to be further clarified. rac -GR24 treatment, the inventors detected SLs synthesis genes at different time points GbCCD7 / 8b The changes in the expression level of (the relevant experimental operations can be referred to above and will not be repeated here).

[0105] The results are as follows Figure 5 As shown. It can be seen that: rac -GR24 makes SLs synthase gene GbCCD7 and GbC The transcription level showed a trend of first downregulation and then upregulation ( Figure 5 A), while in upland cotton GbCCD7 and GbC The transcription level first decreased and then returned to normal level ( Figure 5 B).

[0106] Based on the above experimental results, it can be preliminarily determined that: rac -GR24 method, although it can inhibit the synthesis of SLs in cotton to a certain extent in the initial stage of application, the gene synthesis in the plant will then return to normal levels or even up-regulated, especially in sea island cotton. This mode of negative feedback regulation followed by positive feedback regulation may also be one of the reasons why sea island cotton is more resistant to Verticillium wilt than upland cotton.

[0107] 2. Effect of external application of inhibitor TIS108 on cotton resistance to Verticillium wilt

[0108] After applying 3 µM TIS108 to cotton plants, further infection V. dahliae The relevant operations refer to the above operations (the experimental group was only adjusted to water the roots with 5 mL, 3 µM TIS108 solution), which will not be repeated here.

[0109] During the experiment, RT-qPCR technology was used to detect the SLs synthesis gene under the condition of exogenous TIS108. GbCCD7 and GbC The results are as follows: Figure 6 As shown in A. It can be seen that the transcription of the two genes showed a trend of first down-regulation and then up-regulation, especially GbCCD7 The transcription level of GbC The transcription level tends to return to normal transcription level.

[0110] Further experiments on the germination of Orobanche seeds showed that the application of TIS108 reduced the effect of cotton root extract on the germination of Orobanche seeds (refer to the above experimental operations for related operations and will not be repeated here). Figure 6 B), the germination rate of Orobanche seeds decreased ( Figure 6 C), this result indicates that TIS108 inhibits the synthesis of SLs in cotton roots.

[0111] The results of the final phenotypic experiment showed that the application of TIS108 could reduce the resistance of Hai7124 to cotton Verticillium wilt to a certain extent ( Figure 6 D), among which the disease index of plants treated with TIS108 was significantly increased ( Figure 6 E) Increased lesions and browning of the stems ( Figure 6 F and G), stems V. dahliae The content of Figure 6 H).

[0112] To sum up the above results, it can be seen that the SLs content is positively correlated with the resistance of cotton to Verticillium wilt, that is, when the SLs content is increased, the resistance of cotton to Verticillium wilt can be enhanced, and when the SLs content is reduced (inhibited), the resistance of cotton to Verticillium wilt is also significantly reduced.

[0113] Example 3

[0114] Based on Examples 1 and 2, GbCCD7 and GbC The inventors used VIGS gene silencing technology and overexpression technology to investigate the important role of SLs in the synthesis of SLs. GbCCD7 and GbC The genes were silenced or overexpressed, and the resistance of cotton to Verticillium wilt under the conditions of silencing or overexpression of the relevant genes was evaluated. The specific experimental conditions are briefly described as follows.

[0115] (one) GbCCD7 and GbC Changes in Verticillium wilt resistance after gene silencing

[0116] It should be noted that due to GbCCD8 There are two pairs of alleles in the allotetraploid cotton Gossypium barbadense GbCCD8a and GbC To clarify whether there is functional redundancy between the two in cotton Verticillium wilt resistance, the inventors constructed GbCCD8a and GbC Gene silencing VIGS vector to interfere with GbCCD8a and GbC The expression of genes, and thus the function of genes, can be studied by reverse genetics. The specific experimental details are briefly described as follows.

[0117] First, according to the target gene sequence information, a 300-500 bp specific gene fragment was selected for the construction of the VIGS vector, and the primer sequences containing BamHI and KpnI restriction sites were designed as follows:

[0118] VIGS-CCD7-F: 5'-agaaggcctccatggggatccACGTCTGAGCCGATCACACTCC-3',

[0119] VIGS-CCD7-R: 5'-gagacgcgtgagctcggtaccTCGCCACGAGCCGGTCAC;

[0120] VIGS-CCD8a-F: 5'-agaaggcctccatggggatccATGTCGGGGATCTATCAAAGCTC,

[0121] VIGS-CCD8a-R: 5'-gagacgcgtgagctcggtaccGGATGAGCCGAGTGTATCAATCC;

[0122] VIGS-CCD8b-F: 5'-agaaggcctccatggggatccCCGAACCACGTTGCATGG,

[0123] VIGS-CCD8b-R: 5'-gagacgcgtgagctcggtaccGAAGCACCGGAGAAAAGCTTT;

[0124] Subsequently, PCR amplification was performed using the above primer sequences, and the amplified product was extracted for later use; then, the pTRV2 plasmid was digested, and the linearized plasmid was recovered and connected with the above amplified product to recombinantly integrate the amplified fragment into the pTRV2 plasmid genome; then, the above connection product was transformed into Escherichia coli. Screening and identification were performed to obtain the correct recombinant VIGS plasmid. Then, TRV1, TRV:00, TRV:CLA (CLA, albino indicator gene) and the above recombinant plasmids were transformed into Agrobacterium, and the correctly transformed Agrobacterium strain was inoculated into LB liquid culture medium containing Rif and Kan, and cultured at 28°C and 200 rpm until OD600 = 0.8. Subsequently, the culture was centrifuged at 8000 rpm and room temperature for 15 min to collect the bacteria, and the bacteria were resuspended in VIGS infection solution (containing 10 mM MES, 10 mM MgCl2, 0.1 mM AS), and OD600 was adjusted to about 0.8. TRV:00 bacterial solution was mixed with other resuspended bacterial solutions at a ratio of 1: 1 (volume ratio), and activated at 28°C and 200 rpm for 2 h as the infection bacterial solution.

[0125] Finally, a small hole was gently pricked on the back of the cotyledon of the cotton seedling with the needle of a syringe, and then the resuspended bacterial solution was filled into the cotton cotyledon. After culturing in the dark for 24 h, normal culture was carried out under light conditions at 23°C.

[0126] After about two weeks of cultivation, it was observed that the true leaves of the seedlings injected with TRV:CLA plasmid Agrobacterium appeared bleached. This result showed that the VIGS system worked and the relevant genes were indeed silenced.

[0127] Related experimental results such as Figure 7 As shown in the analysis, the positive control plants TRV:CLA The true leaves showed albino phenotype ( Figure 7 D), indicating that the VIGS system has successfully worked. GbCCD8a In the case of gene expression, its transcription level decreased by about 60% ( Figure 7 A), at the same time, the germination experiment of Orobanche seeds also showed that the germination rate of Orobanche seeds was reduced by interfering plant treatment ( Figure 7 B and C).

[0128] Subsequently, the results of the disease resistance phenotype experiment showed that interfering GbCCD8a Cannot significantly reduce cotton Verticillium wilt resistance ( Figure 7 E), and its disease index did not increase significantly ( Figure 7 F), which indicates GbCCD8a The role of the gene in cotton Verticillium wilt resistance is not decisive; this is consistent with the aforementioned expression pattern in fungus invasion. GbCCD8a The induction of Verticillium dahliae was not significantly responsive, which was consistent with the preliminary conclusion that GbCCD8a is not the key gene that determines cotton Verticillium wilt resistance. In other words, based on the VIGS results, GbCCD8a The phenotypic differences after gene silencing were not obvious, and the response to Verticillium wilt was weak. Therefore, unless necessary, the relevant test results no longer involve too much information about GbCCD8a Analysis of genetic associations.

[0129] Interference GbCCD7 and GbC The results of the gene experiments are as follows Figure 8 As shown in the analysis, the positive control plants TRV:CLA The true leaves showed albino phenotype ( Figure 8 E), indicating that the VIGS system played a normal role. The statistical results show that GbCCD7 and GbC The transcription level in the gene interference plants was significantly reduced, by about 60% ( Figure 8 A and B). Further germination experiments of Orobanche seeds showed that the germination rates of Orobanche seeds were reduced by about 5% and 7% respectively after the treatment of Orobanche seeds with plant root extracts. Figure 8 C and D).

[0130] The results of disease resistance phenotype experiments showed that GbCCD7 and GbC The interference plants all reduced the resistance to Verticillium wilt in cotton, and GbC Intervention plants had more severe disease and higher disease index ( Figure 8 F and G). Stalk dissection experiment, statistics of browning area and V. dahliae The recovery culture experiments showed that GbCCD7 and GbC Interference with plants V. dahliae The resistance is weakened ( Figure 8 H, I and J).

[0131] Based on the above results, it can be preliminarily determined that the SLs synthase gene GbCCD7 and GbC Positive regulation of cotton V. dahliae disease resistance, especially GbC The role of genes is even more significant.

[0132] (two) GbC Changes in Verticillium wilt resistance after gene overexpression

[0133] Combined with the above experimental results, the inventors further constructed relevant overexpression vectors (recombinant vector structure as shown in Fig. 9 The plant physiological and phenotypic conditions after gene overexpression were further measured and evaluated. The specific experimental process is briefly described as follows.

[0134] First, the relevant PCR amplification primer sequences were designed as follows:

[0135] OE-CCD7-F:ggggacaagtttgtacaaaaaagcaggctccATGCAAGCAAAAATATTCC,

[0136] OE-CCD7-R:ggggacccactttgtacaagaaagctgggtcTCAATCATTCTTGGCCCAAAAACC;

[0137] OE-CCD8-F:ggggacaagtttgtacaaaaaagcaggctggATGGGTTCCCTATCACTTTCAGGTA,

[0138] OE-CCD8-R:ggggacccactttgtacaagaaagctgggtcTTATTCTTTGGGTACCCAACATCCAT;

[0139] Subsequently, PCR amplification was performed using the above primers, and the amplified product was recovered; then, the above PCR amplified product was used to construct an overexpression vector using the Gateway cloning method, and the vector construction process included entry cloning (BP reaction) and expression cloning (LR reaction), wherein the BP cloning reaction system was: PCR product, 0.5 µL; pDONR221, 0.5 µL; TE, 1 µL; BP enzyme, 0.5 µL; reaction conditions: incubation at 25°C for 1 h; after the reaction, transformation to Escherichia coli was performed, and screening and sequencing were performed for identification; the LR cloning reaction system was: the above identified correctly entry cloning plasmid, 0.5 µL; expression vector, 0.5 µL; TE, 1 µL; LR enzyme, 0.5 µL; reaction conditions: incubation at 25°C for 1 h; after the reaction, transformation to Escherichia coli was performed for screening and identification;

[0140] Then, the successfully constructed plasmid vector was transformed into Agrobacterium LBA4404, and the correctly transformed strain was screened and identified; then, a single colony with the correct transformation was picked and inoculated into YEP liquid medium (containing 50 mg / L Kan), and cultured at 28°C and 200 rpm to amplify to OD 600= about 0.8, centrifuge at 8000rpm for 1 min to collect the bacteria, and resuspend the bacteria in an equal volume of liquid culture medium (for cotton tissue culture) to use this as the transfection solution; in advance, disinfect the kernels of cotton seeds and culture them at 30℃ for 2~3d until the hypocotyls extend to 2-3cm; cut the cotton hypocotyls into small sections of about 1 cm, and infect them with the above-mentioned transfection solution at 28℃ and 100rpm for 30 min; after the infection is completed, transfer to culture medium, culture at 28℃ in the dark for 2d, transfer to screening (kanamycin resistance) culture medium, culture at 28℃ in light for 20d, and further perform callus induction and subculture until seedlings are formed.

[0141] Combined with the above results, given GbC The effect of genes is more obvious, so only GbC The results related to gene overexpression are briefly described below.

[0142] OE- GbC Overexpression transgenic lines such as Fig. 9 B and Fig. 9 C Fig. 9 B and Fig. 9 C shows the obtained T0 generation transgenic positive seedlings and PCR identification results). The T0 generation positive transgenic plants were further propagated to the T1 generation, and then the T1 generation transgenic plants were used to identify the resistance to cotton Verticillium wilt. The results are as follows.

[0143] First, the four transgenic lines (L1, L3, L14 and L15) of the T1 generation were identified as positive plants and their protein expression levels were tested by PCR and Western blotting (WB) technology (the relevant operations can be referred to the conventional operations of the existing technology and will not be repeated here). The results showed that the four positive plant lines all expressed protein levels ( Fig.10 A and B).

[0144] Then, RT-qPCR was used to GbC The transcription level of the gene was detected, and the quantitative results of the gene showed that the expression levels of strains L1, L14 and L15 were 100-500 times higher, while that of L3 was relatively low. Therefore, L1, L14 and L15 were selected for the next step of the disease resistance identification experiment against Verticillium wilt ( Fig.10 C).

[0145] Infection of T1 positive transgenic plants V. dahliae Spore suspension (10 7The disease began to occur around 15 days after infection, and the difference in the disease symptoms of cotton reached the maximum around 23 days. The disease index and disease symptoms of transgenic lines L1, L14 and L15 were significantly milder than those of the wild type (WT), with the disease index reduced by 15-20% ( Fig.10 D and E). Further observation of the plant stems showed that the browning degree of the overexpression plant stems was lighter, and the browning area was significantly lower than that of the WT ( Fig.10 F and G).

[0146] In summary, overexpression in cotton GbC Genes can improve cotton's resistance to Verticillium wilt.

[0147] Example 4

[0148] In view of the complexity of the regulatory relationship between hormones, in order to clarify the relevant regulatory mechanism, the inventors GbCCD7 and GbC The cis-acting elements of the promoter were preliminarily analyzed, and the regulatory relationship between hormones was studied through yeast one-hybrid and LUC experiments. The specific process is briefly described as follows.

[0149] one) GbCCD7 and GbC Gene promoter sequence analysis

[0150] Combined with the relevant analysis website, the inventors selected 2000 bp upstream of the gene CDS as the promoter. GbCCD7 / 8b The cis-acting elements of the promoter were preliminarily analyzed. Fig.11 The analysis results show that:

[0151] GbCCD7 / 8b In addition to the most abundant core promoter elements (TATA-box) and enhancer elements (CAAT-box), the promoter sequence also contains MYB elements that can respond to abiotic stresses and MYC elements that respond to biotic stresses, ABRE elements that respond to abscisic acid, and methyl jasmonate and salicylic acid response elements ( Fig.11 A);

[0152] The analysis believes that GbCCD7 / 8b The promoter is likely to respond to the regulation of hormone signals. At the same time, statistical analysis found that the most cis-acting elements responded to ABA, followed by JA and SA ( Fig.11 B); This suggests that these hormones are involved in regulating GbCCD7 / 8b The expression of SLs in turn affects the synthesis of SLs, thereby finely regulating the balance of hormones in the plant.

[0153] In addition, due to GbCCD7 In cotton, there are two copies. GbCCD8In cotton, there are four copies. In order to understand the differences in promoter cis-acting elements between homologous genes or alleles, the inventors further compared and analyzed the cis-acting elements of their promoters. The comparison results of the cis-acting elements that respond to ABA, biological stress, JA, SA, MYB and MYC showed that these cis-acting elements have certain similarities in gene structure between homologous genes or alleles, which also indicates that the cis-acting elements of these genes have a high degree of homology and a close relationship in evolutionary relationship, and they may be subject to similar regulatory effects or perform similar functions ( Fig.11 C).

[0154] 2. Protein 3D structure prediction and subcellular localization

[0155] As the key rate-limiting enzymes in SLs biosynthesis, CCD7 / 8b, their protein three-dimensional structures were predicted to have two forms, monomer and dimer, and each form contained Fe 2+ The enzyme active center ( Fig.12 A), which is a typical feature of carotenoid-cleaving dioxygenases and also indicates that CCD7 and CCD8b do function as enzymes.

[0156] GbCCD7 and GbCCD8b belong to carotenoid cleavage dioxygenases, whose substrates are 9- cis - β Carotenoids, for a better understanding of cotton GbCCD7 and GbC The inventors further performed subcellular localization to determine the site where the gene exerts its function.

[0157] During the experiment, referring to the above operations, we first used the Gateway method to build GbCCD7 and GbC and GFP fusion expression vector, and then transform the recombinant correct plasmid into Agrobacterium to prepare the infection solution, which was injected on the back of tobacco leaves. After culturing in the dark for 72 hours, the localization of green fluorescence was observed under a fluorescence microscope.

[0158] The subcellular localization results showed that: Gb CCD7 and Gb CCD8b is localized in chloroplasts ( Fig.12 B and C), indicating that they are indeed in the chloroplast with carotenoids as substrates for the process of cleavage and dioxygenation, and finally generate SLs precursor monochlorohydrin (CL) and release it into the cytoplasm for the next step of synthesis.

[0159] 3. Effects of different hormone treatments on cotton GbCCD7 / 8b Effects on gene expression

[0160] Combining the above GbCCD7 / 8b The analysis results of cis-acting elements in gene promoters show that GbCCD7 and GbC The expression of is likely to be induced by ABA, JA and SA three plant hormones up-regulated or down-regulated. The inventors quantitatively detected the expression of GbCCD7 and GbC The expression levels of genes were used to verify the regulatory relationship between ABA, JA and SA on SLs.

[0161] The specific experimental operation was as follows: cotton plants grown to two leaves and one heart were taken, and hormone solutions of corresponding designed concentrations (100 µM ABA, 2.5 mM SA, 100 µM MeJA) were irrigated on the leaves or roots of the cotton plants according to the hormone types. Samples were taken at different time points (0 h, 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h), and the concentrations of hormones in the samples were measured. GbCCD7 and GbC The gene expression levels were detected and quantified.

[0162] The results showed that after JA treatment of cotton plants, GbCCD7 and GbC The transcription levels of genes all showed a downward trend ( Fig.13 A); SA’s treatment GbCCD7 and GbC There was no significant effect on the transcription level of the gene ( Fig.13 B); After ABA treated cotton plants, GbCCD7 and GbC The transcription levels of genes all showed an upward trend ( Fig.13 C).

[0163] Based on the above results, it can be preliminarily concluded that the biosynthesis of SLs in cotton plants may be regulated by both ABA and JA, and this regulation may change the disease resistance of cotton to Verticillium wilt.

[0164] (IV) GbMYC2 GbCCD7 / 8b Expression of influence

[0165] The above experimental results show that SLs analogs rac -GR24 treatment of cotton plants after JA downstream gene expression patterns and SLs synthesis genes after JA treatment of cotton plants GbCCD7 and GbC The expression of JA signaling pathway is completely opposite, which may be a negative feedback regulation mode. To verify this regulatory relationship, the inventors selected JA signaling pathway transcription factor MYC2 to study its effect on GbCCD7 and GbC The relevant experimental results are briefly described as follows.

[0166] First, for the target gene, the primer sequences for PCR amplification are designed as follows:

[0167] pAbAi-ProGbCCD7-F:5'-gaaaagcttgaattcgagctcGTAGCAAATGCAGACACATGGTCC-3',

[0168] pAbAi-ProGbCCD7-R: 5'-agcacatgcctcgaggtcgacGATGGTTGATGTAGGTAAGAAGGGA-3';

[0169] pAbAi-ProGbCCD8b-F: 5'-gaaaagcttgaattcgagctcACAAGGTGCATCATGCTAAAGACT-3',

[0170] pAbAi-ProGbCCD8b-R: 5'-agcacatgcctcgaggtcgacTTGAAATCCAACATGGTTATGGG-3';

[0171] pGADT7-GbMYC2-F: 5'-gtaccagattacgctcatatgATGAAGGACTATAGGTTAGCACCGA-3',

[0172] pGADT7-GbMYC2-R: 5'-attcatctgcagctcgagctcTTATCTTGCATCTCCAACTTTGGA-3';

[0173] Subsequently, PCR amplification was performed using the above primers, and the amplified product was used to construct the bait vector pAbAi-PCCD7 / 8b;

[0174] Then, the bait vector pAbAi-PCCD7 / 8b was transferred into the Y1Hgold yeast strain for self-activation detection (during the detection, the successfully transformed Y1H yeast solution was spread on SD / -Ura plates containing different concentrations to detect the self-activation activity of pAbAi-PCCD7 / 8b and the lowest AbA inhibition concentration).

[0175] For related operations, please refer to the existing technology and will not be described in detail.

[0176] The results are as follows Fig.14It can be seen that the Y1H yeast strain successfully transformed with pAbAi-PCCD7 / 8 has self-activation activity, and the minimum inhibitory concentration of AbA is 700 ng / mL, which can be used for screening in subsequent interaction experiments.

[0177] After determining the self-activation activity of pAbAi-PCCD7 / 8b and the minimum AbA screening concentration, the pGADT7-MYC2 plasmid was further transformed into the yeast strain Y1H containing pAbAi-PCCD7 and pAbAi-PCCD8b for mutual verification. The Y1H yeast solution that was successfully co-transformed was gradiently diluted and spotted on SD / -Ura plates containing 700 ng / mL AbA for normal growth. The growth of yeast Y1H was observed after 3-5 days of culture.

[0178] In the yeast one-hybrid interaction experiment: first, the pGADT7-MYC2 vector was transferred into the pAbAi-PCCD7 / 8 Y1Hgold yeast competent cells; then, it was spread on the SD / -Ura plate containing 500 ng / mL AbA and the number of clones was counted; then, the transformant bacterial liquid was diluted according to the gradient of 1:1, 1:10, 1:100, 1:200, and 1:1000, and then inoculated on the SD / -Ura plate containing 500 ng / mL AbA, and cultured at a constant temperature of 30°C for 3-5 days to detect the results of the interaction.

[0179] The results are as follows Fig.15 As shown in the figure, it can be seen that the growth of yeast containing only pAbAi-PCCD7 and pAbAi-PCCD8 is inhibited, while the expression of AbA resistance gene in yeast co-transformed with pGADT7-MYC2 plasmid is activated and can grow on AbA selection medium. This indicates that GbMYC2 can bind to Gb CCD7 and Gb CCD8b The promoter regions of genes regulate their expression.

[0180] The above yeast Y1H experiment can preliminarily determine that the transcription factor GbMYC2 can GbCCD7 and GbC The promoter of GbMYC2 interacts with that of MYC2, but the regulatory relationship between them cannot be accurately determined at this time. GbCCD7 and GbC The specific experimental results are as follows.

[0181] The P35s-GbMYC2 overexpression vector (Effector), ProCCD7-LUC and ProCCD8b-LUC luciferase reporter vectors (Reporter) were constructed and transformed into Agrobacterium for transient expression in tobacco leaves (the vector structure is shown in Fig.16 A).

[0182] The related operations can refer to the existing technology. The primer design (5' to 3') used in the related PCR amplification process is described as follows: ProGbCCD7-LUC-F:ctatagggcgaattgggtaccGTAGCAAATGCAGACACATGGTCC,

[0183] ProGbCCD7-LUC-R: cgctctagaactagtggatccGATGGTTGATGTAGGTAAGAAGGGA;

[0184] ProGbCCD8b-LUC-F:ctatagggcgaattgggtaccACAAGGTGCATCATGCTAAAGACT,ProGbCCD8b-LUC-R:cgctctagaactagtggatccTTGAAATCCAACATGGTTATGGG;

[0185] Pro35S-GbMYC2-F:cgctctagaactagtggatccATGAAGGACTATAGGTTAGCACCGA,

[0186] Pro35S-GbMYC2-R: tcagcgtaccgaattggtaccTTATCTTGCATCTCCAACTTTGGA.

[0187] After the Effector and Reporter plasmid Agrobacterium were mixed in a ratio of 1:1 and injected into tobacco leaves, tobacco leaves injected with Reporter Agrobacterium alone were used as controls. After appropriate cultivation (24 h of dark cultivation, followed by 16 h light / 8 h dark cultivation for 48 h), detached leaves were evenly sprayed with 0.2 mg / mL luciferase substrate. After 8-10 min of dark treatment, the fluorescence intensity was detected using a live imager.

[0188] The results are as follows Fig.16As shown in B, it can be seen that when ProCCD7-LUC and ProCCD8b-LUC were expressed separately in tobacco leaves, stronger fluorescence signals could be detected; while when P35s-GbMYC2 was co-expressed with ProCCD7-LUC and ProCCD8b-LUC, the intensity of the LUC fluorescence signal was weakened. This result shows that MYC2 GbCCD7 / 8b The binding of the gene promoter produces an inhibitory effect, that is, MYC2 acts as a repressor to regulate GbCCD7 / 8b This result is consistent with the results of cotton plants treated with JA. GbCCD7 / 8b The results of downregulation of gene transcription levels are consistent, and also verify a negative feedback regulatory relationship between GbMYC2 and the biosynthesis of SLs.

[0189] Example 5

[0190] Since the regulation between plant hormones is often mutual, in order to further study the regulatory effect of hormone SLs on other defense pathways, the inventors detected and analyzed the expression and activity burst of defense genes in the JA and SA signaling pathways. The specific process is briefly described as follows.

[0191] 1. Effects of hormone SLs on the expression of plant defense-related genes

[0192] Studies have shown that plant hormones such as JA, SA and ET are hormones that can regulate plant stress. Their signal transduction pathways can participate in regulating the expression of some defense genes and thus regulate the adaptability of plants to stress. For example: genes regulated by the JA signaling pathway MYC2 and PDF1.2 Often positively regulates plant stress resistance; genes regulated by the SA signaling pathway NPR1 and PR1 It can also regulate the stress resistance of plants. To this end, the inventors conducted a preliminary study on whether SLs can also change the disease resistance of cotton Verticillium wilt by affecting the expression of these hormone signaling pathway genes.

[0193] During the experiment, cotton plants grown to two leaves and one heart were treated with 3 µM SL GR24, and samples were taken at 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h. qPCR technology was used to quantitatively detect MYC2 , PDF1.2, NPR1 and PR1 The expression level of the gene. During the detection process, the relevant primers were designed as follows:

[0194] qPCR-PDF1.2-F: 5'-GATATGCGAGTCCCCAAGCA-3',

[0195] qPCR-PDF1.2-R: 5'-ACGCATTTGCGAAGGAAACC-3';

[0196] qPCR-PR1-F: 5'-GGCACAGAACTACGCTAATCAAC-3',

[0197] qPCR-PR1-R: 5'-GCTTTACCCTCTCACTAACCCACAT-3';

[0198] qPCR-NPR1- F: 5'-GTCTGGCTGATGTCAATCTGCG-3',

[0199] qPCR-NPR1-R: 5'-TCCTTCCCTTGCTCTGTCTTGG-3';

[0200] qPCR-MYC2-F: 5'-GGAGGCTAGATCTGCAGTAAGG-3',

[0201] qPCR-MYC2-R: 5'-AAGCAGTGCAGAACCCTCTC-3';

[0202] qPCR-VSP2-F: 5'-TTGTTCGATGAGTGGGTAATGG-3',

[0203] qPCR-VSP2-R: 5'-CCTGAATACTTTGATGGTTCCTTG-3'.

[0204] The results show that rac -GR24 treatment of cotton plants also resulted in corresponding changes in the transcription levels of JA and SA signaling pathway genes. Fig.17 As shown. It can be seen that: JA signaling pathway defense-related genes MYC2 and PDF1.2 The expression of rac -GR24 treatment showed an up-regulation trend, while the defense genes of the SA signaling pathway NPR1 and PR1 The expression of SLs showed a downward trend. This indicates that SLs enhance cotton Verticillium wilt resistance probably through the up-regulation and down-regulation of defense gene expression in JA and SA signaling pathways.

[0205] In addition, it is worth noting that rac- Expression patterns of JA and SA signaling pathway genes in cotton plants treated with GR24 and in cotton plants treated with JA and SA GbCCD7 and GbCCD8The situation is completely opposite. The initial speculation is that this is a negative feedback regulation mode.

[0206] To further verify the quantitative results of the above genes, referring to the above experimental operation, the inventors also detected the expression of 3μM SL synthesis inhibitor TIS108 in cotton plants. MYC2 , PDF1.2 , NPR1 and PR1 expression level.

[0207] The results are as follows Fig.18 As shown in Figure 2, it can be seen that when TIS108, an inhibitor of SLs synthesis, is applied externally, MYC2 , PDF1.2 It shows a downward trend, while NPR1 and PR1 Similarly, when interfering with SLs synthase genes GbCCD7 and GbCCD8 In the case of MYC2 , PDF1.2 It also shows a downward trend, and NPR1 and PR1 It shows an upward trend ( Fig.19 ), the results and rac -The results after GR24 treatment were consistent.

[0208] (II) SLs and reactive oxygen species (ROS) V. dahliae Relationship between resistance

[0209] Previous studies have shown that when plants respond to pathogens, they often produce reactive oxygen species to enhance resistance to pathogens. Due to the mutual regulation between hormones, the accumulation of ROS may also change accordingly. Therefore, the inventors used DAB staining to analyze the accumulation of SLs and ROS, as well as the changes in cotton Verticillium wilt resistance. The specific experimental results are briefly described below.

[0210] During the experiment, the cotton leaves were first treated with DAB staining solution: the cotton leaves were placed in DAB staining solution (2 mg / mL), vacuumed for 10 min, and shaken at 60 rpm overnight; then, decolorized with anhydrous ethanol for 15 min under boiling water bath conditions. After decolorization, they were immersed in ddH2O containing 20% ​​glycerol and preserved, and photographed and observed after 24 h.

[0211] In terms of specific experimental results: GbCCD7 and GbC After gene, DAB staining results showed that V. dahliae The accumulation of reactive oxygen in cotton leaves was weakened 2 days after infection ( Fig. 20A); The test results of hydrogen peroxide (H2O2) content also show that GbCCD7 and GbC The H2O2 content of the intervention plants decreased ( Fig. 20 B).

[0212] Based on these results, it can be preliminarily concluded that the accumulation of ROS in cotton at the early stage of Verticillium wilt infection may be one of the ways in which SLs enhance cotton resistance to Verticillium wilt.

[0213] In order to further verify the effect of ROS accumulation on cotton resistance to Verticillium wilt, the inventors observed the number of dead cells in the leaves of interfering plants by trypan blue staining of cotton leaves.

[0214] During the experiment: place the true leaves of cotton in trypan blue dye solution, vacuum for 30 minutes, and dye for 8 hours in a 100℃ water bath for 8 minutes; decolorize in 50% alcohol and 85℃ water bath for 15 minutes, then decolorize in 75% alcohol and 85℃ water bath for 15 minutes, and then decolorize completely in 100% alcohol at room temperature, and store in ddH2O.

[0215] The results are as follows Fig. 20 C. It can be seen that: GbCCD7 and GbC The number of dead cells in the leaves of the gene-interferenced plants was higher, indicating that the number of dead cells in the leaves of cotton with reduced ROS accumulation was increased, which again indicated that SLs changed the resistance of cotton to Verticillium wilt by regulating the accumulation of ROS.

[0216] In summary, it can be preliminarily determined that SLs can enhance the defense-related genes in the JA signaling pathway MYC2 , PDF1.2 and VSP2 The expression of JA signaling pathway enhances resistance to Verticillium wilt; at the same time, the transcription factor MYC2 negatively regulates CCD7 / 8 The expression of SLs forms a negative feedback regulation loop to maintain the balance of SLs; the weakening of hormone SA signal, the enhancement of ABA signal and the burst of ROS are also involved in the cotton wilt resistance regulated by SLs, constructing the regulatory relationship between SLs and the above hormones in cotton wilt resistance.

Claims

1. Cotton GbC The application of the gene in cotton resistance to Verticillium wilt is characterized by: The cotton GbC The genes include two: GbC , GbC ; in GbC The gene sequence is shown in SEQ ID No. 1, GbC The gene sequence is shown in SEQ ID No. 2; The cotton GbC Gene, which is the carotenoid cleavage dioxygenase gene in the SLs synthase of strigolactone.

2. Cotton as claimed in claim 1 GbC The application of the gene in cotton resistance to Verticillium wilt is characterized by: When applied specifically, cotton GbC The expression of the gene was upregulated in response to the induction of Verticillium dahliae.

3. The cotton as claimed in claim 1 GbC The application of the gene in cotton resistance to Verticillium wilt is characterized by: cotton GbC Gene expression is positively correlated with resistance to Verticillium wilt; Silent cotton GbC Gene expression can reduce cotton resistance to Verticillium wilt; overexpression of cotton GbC After the gene is introduced, the resistance of cotton to Verticillium wilt can be enhanced.

4. A method for enhancing cotton resistance to Verticillium wilt, characterized in that: The resistance of cotton to Verticillium wilt can be improved by applying strigolactone SLs or their analogs externally. The SLs analogue of strigolactone is rac-GR24.

5. A method for breeding cotton varieties resistant to Verticillium wilt, characterized in that: Through overexpression GbC and GbC Genetic approaches to improve Verticillium wilt resistance; Said GbC The gene sequence is shown in SEQ ID No. 1; Said GbC The gene sequence is shown in SEQ ID No.2.

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