Application of sesame SiWAKL6 gene and its encoded protein in disease resistance breeding

Abstract

The present invention relates to an application of a sesame SiWAKL6 gene and a protein encoded by the gene in disease-resistant breeding, wherein the coding sequence of the sesame SiWAKL6 gene is shown in SEQ ID NO.1. The sesame SiWAKL6 gene can be induced by Phaseolus vulgaris and salicylic acid, and is involved in the resistance of sesame to stem blight. The disease index and the colonization amount of M.phaseolina in transgenic Arabidopsis thaliana heterologously overexpressing SiWAKL6 are significantly reduced. Further studies show that Arabidopsis thaliana overexpressing SiWAKL6 can activate the high-level expression of genes related to the SA signaling pathway and actively regulate the homeostasis of reactive oxygen species in the body, thereby improving the resistance of Arabidopsis thaliana to M.phaseolina. The new function of the SiWAKL6 gene of the present invention provides a new method for sesame disease-resistant breeding, and has very important value in sesame production.

Description

Technical Field

[0001] The invention relates to application of a sesame SiWAKL6 gene and a protein encoded therein in disease-resistant breeding, and belongs to the field of plant genetic engineering. Background Art

[0002] Sesame (Sesamum indicum L.) belongs to the genus Sesamum Linn of the family Pedaliaceae. Its oil content exceeds that of crops such as peanuts, soybeans and sunflowers, making it one of the most nutritious oil crops. Stem blight caused by Macrophomina phaseolina is one of the most serious fungal diseases in sesame production, posing a serious threat to the yield and quality of sesame. Plant WAK like genes (WAKLs) can generally confer plant resistance to pathogens by regulating processes including cell wall strengthening, activation of disease resistance-related genes, accumulation of salicylic acid (SA) or jasmonic acid (JA), peroxidase (POD) and superoxide dismutase (SOD) activities, and ROS homeostasis.

[0003] The applicant initially screened SiWAKL6 as a candidate gene for sesame stem blight resistance through comparative transcriptomic analysis. Within 48 hours of M. phaseolina stress, SiWAKL6 was continuously induced to express in ZZ13 (a disease-resistant variety), but not in J9014 (a disease-susceptible variety). Therefore, identifying the function of the sesame SiWAKL6 gene is of great significance for sesame disease-resistant breeding. Summary of the invention

[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide an application of a sesame SiWAKL6 gene and a protein encoded therein in disease-resistant breeding.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A sesame SiWAKL6 gene, the coding sequence of the sesame SiWAKL6 gene is shown as SEQ ID NO.1.

[0007] The protein encoded by the sesame SiWAKL6 gene.

[0008] The protein sequence encoded by the sesame SiWAKL6 gene is shown in SEQ ID NO.2.

[0009] The application of the sesame SiWAKL6 gene in disease resistance breeding.

[0010] The application of the protein encoded by the sesame SiWAKL6 gene in disease-resistant breeding.

[0011] Beneficial effects of the present invention:

[0012] The present invention found that the sesame SiWAKL6 gene can be induced by Macrophomina phaseolina (M.phaseolina) and salicylic acid (SA), and is involved in the resistance of sesame to stem blight. The disease index and M.phaseolina colonization of transgenic Arabidopsis thaliana heterologously overexpressing SiWAKL6 were significantly reduced. Further studies showed that Arabidopsis thaliana overexpressing SiWAKL6 can activate the high-level expression of genes related to the SA signaling pathway and actively regulate the homeostasis of reactive oxygen species in the body, thereby improving the resistance of Arabidopsis thaliana to M.phaseolina.

[0013] The new function of the SiWAKL6 gene of the present invention provides a new method for sesame disease-resistant breeding and has very important value in sesame production. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Expression analysis of SiWAKL6 gene under treatment of M. phaseolina (A), salicylic acid (B) and methyl jasmonate (C).

[0015] Figure 2 Schematic diagram of the structure of the SiWAKL6 gene.

[0016] Among them, A. Chromosomal localization of SiWAKL6; B. Gene structure of SiWAKL6; C. Conserved domain of SiWAKL6 protein; D. Signal peptide and transmembrane domain of SiWAKL6 protein.

[0017] Figure 3 Schematic diagram of the overexpression pCAMBIA2301-KY vector.

[0018] Figure 4 Evaluation of disease resistance in Arabidopsis thaliana overexpressing the SiWAKL6 gene.

[0019] Among them, A. Symptoms of WT and OE-SiWAKL6 plants 14 days after inoculation; B. PCR positive verification of overexpression lines; C. Disease index of WT and OE-SiWAKL6 plants 14 days after inoculation; D. Relative abundance of biomass of M. phaseolina and Arabidopsis thaliana genomic DNA in WT and OE-SiWAKL6 plants 14 days after inoculation.

[0020] Figure 5 Expression patterns of marker genes of SA signaling pathway (A) and JA signaling pathway (B) in WT and OE-SiWAKL6 after 12 h of M. phaseolina stress.

[0021] Figure 6 H2O2 (A) and MDA (B) levels and CAT (C) and SOD (D) enzyme activities in WT and OE-SiWAKL6 plants 14 days after inoculation with bacteria. DETAILED DESCRIPTION

[0022] The specific implementation modes of the present invention are further described in detail below in conjunction with the embodiments.

[0023] Example 1 Expression of SiWAKL6 induced by M. phaseolina and exogenous SA hormone

[0024] 1. Sesame materials and culture conditions

[0025] Sesame varieties resistant to stem blight: Zhengzhi 13 (ZZ13); susceptible varieties: Ji 9014 (J9014).

[0026] Sesame seeds of Zhengzhi 13 and Ji 9014 were sterilized in a 5% (wt) sodium hypochlorite (available chlorine) solution for 10 minutes, then sterilized with 70% (v / v) alcohol for 30 seconds, and finally rinsed with sterile water for 3-4 times. After drying, the sesame seeds were planted in mixed nutrient soil (sterile soil: nutrient soil: sterile vermiculite volume ratio = 3:1:1) and cultured in an artificial climate chamber. The culture conditions were: temperature 29±1°C, relative humidity 80%, and photoperiod of 16h light and 8h dark.

[0027] 2. Inoculation and exogenous hormone treatment of sesame seedlings

[0028] The pathogen of stem blight is Phaseolina (M. phaseolina), strain number Mp2010028C. The strain was isolated and preserved by the Biological Control Laboratory of the Institute of Plant Protection, Henan Academy of Agricultural Sciences.

[0029] After the preserved strain is activated, it is inoculated into PDA solid culture medium, cultured in a 30°C incubator for 7 days until the mycelium fills the culture dish, then the culture medium filled with mycelium is divided into bacterial blocks, and the bacterial blocks of each 1 / 2 culture dish are evenly inoculated into 300mL of PD liquid culture medium, and then cultured for 5 days under shaking conditions of 30°C and 200r / min. Subsequently, the mycelium is crushed with a tissue crusher to obtain a mycelium suspension. 100mL of distilled water and 200g of sterilized mixed nutrient soil (the volume ratio of peat soil: vermiculite = 3:1) are added to each 100mL of mycelium suspension and stirred evenly to obtain the contaminated soil.

[0030] Inoculation treatment: When Zhengzhi 13 and Ji9014 grew to the stage of sesame with three leaves and one heart, they were transplanted into the prepared soil with bacteria. Sesame root tissues were collected at 0h, 3h, 6h, 12h, 24h and 36h after inoculation and stored at -80℃ for later use.

[0031] Hormone treatment: When Zhengzhi 13 sesame grows to the three-leaf and one-heart stage, 167mL of 200umol / L methyl jasmonate and 167mL of 2umol / L salicylic acid solution were applied for root irrigation, and the same volume of water was used as a control. The sesame root tissues were collected at 0h, 3h, 6h, 12h, 24h and 36h after treatment and stored at -80℃ for later use.

[0032] 3. qPCR analysis

[0033] Using sesame SiUBQ5 gene as internal reference gene, RNA extraction, reverse transcription and fluorescence quantitative PCR were performed on sesame root tissues of Zhengzhi 13 and Ji9014 after inoculation with M.phaseolina, and sesame root tissues of Zhengzhi 13 after treatment with salicylic acid (SA) and methyl jasmonate (MeJA) hormones to detect the expression of SiWAKL6 gene. The primers are as follows:

[0034] Gene Forward Primer Reverse primer SiUQ5 TCTCGCCGACTACAACATTCA TGGACACTCTTTCCTCAACCTCT SiWAKL6 CAGTTATTTGTTAGATCCATTTGAC CACAGTAGCATACTTTACTTAATGT

[0035] The qPCR system is as follows:

[0036] Reagents 10μL system 2×ChamQ Universal SYBR qPCR Master Mix 5μL Forward Primer 0.25μL Reverse primer 0.25μL cDNA template 1μL <![CDATA[ddH2O]]> 3.5μL

[0037] Note: ChamQ Universal SYBR qPCR Master Mix is ​​a universal high-sensitivity dye-based quantitative PCR detection kit from Vazyme.

[0038] The qPCR program was as follows: 94°C for 2 min, followed by 40 cycles of 94°C for 15 s, 55°C for 15 s, and 72°C for 20 s. -△△C Methods Analysis of qPCR data.

[0039] The expression levels of SiWAKL6 gene in the roots of Zhengzhi 13 (ZZ13) and the susceptible variety Ji 9014 (J9014) at different time points after inoculation with M.phaseolina were examined by qPCR technology. The results showed that SiWAKL6 gene could be significantly induced by M.phaseolina in ZZ13, reaching a peak after 3 hours, but was not induced in J9014 ( Figure 1 A).

[0040] Plant hormones often regulate the expression of plant disease-related proteins. To explore the hormone signaling pathway associated with SiWAKL6, the expression pattern of SiWAKL6 in ZZ13 after SA and MeJA hormone treatment was detected, with water treatment as a control (Mock). The results showed that plant hormones had different effects on the expression of SiWAKL6. After the application of exogenous SA, SiWAKL6 could be rapidly induced to express, but it was not induced to express in the control treatment ( Figure 1 B). After MeJA treatment, the expression level of SiWAKL6 was close to that after water treatment ( Figure 1 C).

[0041] The above results collectively indicate that SiWAKL6 is induced by M. phaseolina and SA, and may enhance sesame resistance to stem blight through SA-mediated signaling pathways.

[0042] 4. SiWAKL6 gene cloning

[0043] Primers for the SiWAKL6 gene were designed based on the sequence splicing results, and the cDNA from the roots of the disease-resistant variety Zhengzhi 13 was used to amplify the full length of the coding sequence of the SiWAKL6 gene in sesame. The reaction procedure was: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 1 minute, 35 cycles; 72°C final extension for 10 minutes and then stored at 4°C. Then the PCR product of cloned SiWAKL6 was recovered to obtain the purified target DNA. The primer information is as follows:

[0044] SiWAKL6-F:ATGAGGCTGCCCTTGATACTCC

[0045] SiWAKL6-R: TCAAGCAACTAATGTTCGACTGTCG

[0046] Then the target gene SiWAKL6 was ligated and transformed, and the ligation system was as follows:

[0047] Reagents System (10μL) DNA 5μL Solution I 4μL pMD19-T simple victor 1μL

[0048] The above system was connected at 16°C for 4 h.

[0049] Note: pMD19-T simple victor is a linearized vector with "T" added to the 3' ends on both sides, Solution I is the buffer in the ligation system, and both pMD19-T simple victor and Solution I are gene cloning vector kits, TAKARA.

[0050] The successfully connected vector was then transformed with DH5α Escherichia coli, and a single colony with regular growth, fullness and roundness was picked and inoculated into a 2mL tube of LB medium containing 100mg / mL ampicillin, and placed in a shaker at 37°C and 200rpm for 12h. The bacterial liquid was then tested by PCR, and samples containing the correct bands were selected and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing to obtain the SiWAKL6 sequence information.

[0051] The results showed that the SiWAKL6 gene was located on chromosome 5 ( Figure 2 A), it contains two exons and one intron, and the gene length is 2924bp ( Figure 2 B), encoding a 729-residue WAKLs, whose N-terminal domain is a GUB-WAK domain (aa 30 to aa 89), which is the characteristic domain of the WAKL family, and the C-terminal domain contains a PKinase domain (aa 398 to aa 664), which is the core of its kinase activity ( Figure 2 C). A 22aa signal peptide (SP) was detected at the N-terminus of SiWAKL6 protein. In addition, the protein has a predicted transmembrane (TM) structure between aa 323 and aa 342 ( Figure 2 D) These features indicate that SiWAKL6 encodes a typical WAKL protein.

[0052] Coding sequence of SiWAKL6 gene (SEQ ID NO.1):

[0053] ATGAGGCTGTCCGTAATATACCTTTTCGTTTCCACCCTTTGCCTAATGACCTCTGCTGCTGTGGCTTTATCTTTGGCAAAGCGTGGATGTCAAGACACTTGTGGAAATGTCACCATTCCATATCCTTTTGGAATTGGTTCTGAATGTTATGCAAATTCGTCATTCGCAGTGGTCTGTGACAATTCAACCAATCCAGC AAGGCTGCACTTGAGCAGCATCCAATTGGAAATACTAGATATTTCGTTGCATGGTACAGTTAGAGTTGTGCAGCGTGTTTTCCCTATGACTTGCTCTGATGAACAGAAAACACAATCTTTTGGGGAAATCACTTGTTGGAAGTCCTTTTACCATCTCATCGATTCAAAATTCATTGACTGTTTTGGGCTGCAGAAATTC

[0054] TGTCTGGTTGCGCGCCAACAAGACGACAATAACTGGTGGATGCATGGCTCTTTGTGATTACAACTCTAGG

[0055] GATACAAGTTGTAATGGTATAAACTGCTGCAAGATAACAATTCCTATGGGCCTTCAAGAGCTTCAAGTA

[0056] CTTACCAAAGCATTGCAAATGACAAAAATAATACTCCCTTCTGTGGATATGCCTTCCTTGCTGACATGAA

[0057] ATGGTTTAGGAAGATTACGGGAAATATATGGCCTGCGCAGTTATTTGTTAGATCCATTTGACGGAGAA

[0058] TTCGGAAATGCAAGTATGGTGCTTGAGTGGGAATACGCCCGCAGTGTGAAGTTTATCCGTTCCTATTTCT

[0059] GTGTGTATCCGGGGTATACAAGCTTTGCAGCTTTTAATTATGAAACATTAAGTAAAGTATGCTACTGTGA

[0060] CTTCGGGTTTGAAGGAAATCCCTATCTACCTGAAGGATGCCAAGATATTGATGAGTGTGGCAATTCGACA

[0061] ACTAATTCCTGTTCTGAGGGAGATACTTGTGTCAACAGAATAGGATCTTACGAATGTCAGAAGCCGAAAT

[0062] CTCGGCTGAAAATAGCATTTATCGTCATTGGTTCTGTGCTTGGTGCACTAATTTTGCTCCTGGGAGCATG

[0063] GAGGTCCACCAAGCTTATTAGAAAGAGAATCAAGGGTATTCGAAAACGAAAGTTTTTTAAGCGAAATGGA

[0064] GGCCTATTATTGGAACAGCAATTGTCTTCAACTGATAATGGCCTAGAGAAAACTAAGTTGTTTACTTCCA

[0065] AAGAGTTGGCAGCTGCTACCGACCATTATAATGAGAATCGTATACTTGGTCATGGCGGCCAAGGTACCGT

[0066] CTACAAAGGCATGTTGGCAGATGGAAGAATTGTGGCTGTCAAGAAGTCCAAAAGGGTAGATGAAGATGAT

[0067] CTTGAAGTCTTCATTAATGAGATCGTTATTCTATCTCAGATAAACCATAGAAATGTAGTAAAGTTACACG

[0068] GATGTTGTCTAGAGAACGAAGTTCCTCTTCTTGTCTACGAATTCATCCCAAATGGCACACTTTTCCGACA

[0069] CATCCATGAACCAAACGAGGACTTCCCGTTATCTTGGGAAATGCGTACGCGAATTGCTAGAGACGTAGCT

[0070] GGAGCACTTTCTTACTTGCACTCTGCTGCATATGCACCAATTTATCATAGGGATATCAAGTCAACAAACA

[0071] TACTATTGGATGAAAAGTATGGTGCCAAAGTTTCAGACTTCGGGACGTCAAAGTCAGTTGCCATTGATCA

[0072] AACTCACCTGACCACAAGAGTATTAGGCACCTTCGGATACTTGGATCCAGAGTACTTCCAATCGAGCCAG

[0073] TTCACAGAAAAGAGCGATGTCTACAGTTTCGGCGTTGTTATGGTTGAGCTTTTAACTGGGGAGAAAGCAA

[0074] TTTCATCAGTTAGAGCTGAAGTGGGAAAGAGTTTGGCCACACATTTCTTGCATTCAATGGAGGAAGATCA

[0075] TTTATTCGATATTCTTGACCCGAGGGTCCTCAAAGAGGGCAGTAGGGAAGAAGTTGTGGCTATTGCTGAA

[0076] CTTGCTAGAAGATGTCTGCATTTGAATGGAAAGAGAAGGCCAACAATGAAGGAAGTTGCCGTTGAGTTGG

[0077] AAGGAATTCAACTGCTGAAAGAAGGGTCGGTGGTGGTGCAAAATCATGATAATAACAGAGAATACCATTC

[0078] AGTTCATGAGTTTGCTGAGTGCTATGACTTCTCTGCCATGTCAGGAAGCATGCATTTCCACACAATTACT

[0079] GCATCCTCAGCCGAAGTCGAGCGTCCGTTGCTTGATGAGCCGTGA

[0080] Protein sequence encoded by SiWAKL6 gene (SEQ ID NO.2):

[0081] MRLSVIYLFVSTLCLMTSAAVALSLAKRGCQDTCGNVTIPYPFGIGSECYANSSFAVVCD

[0082] NSTNPARLHLSSIQLEILDISLHGTVRVVQRVFPMTCSDEQKTQSLGKSLVGSPFTISSI

[0083] QNSLTVLGCRNSVWLRANKTTITGGCMALCDYNSRDTSCNGINCCKITIPMGLQELQVTY

[0084] QSIANDKNNTPFCGYAFLADMKWFREDYGKYNGLRSYLLDPFDGEFGNASMVLEWEYARS

[0085] VKFIRSSFCVYPGDTSFAAFNYETLSKVCYCDFGFEGNPYLPEGCQDIDECGNSTTNSCS

[0086] EGDTCVNRIGSYECQKPKSRLKIAFIVIGSVLGALILLLGAWRSTKLIRKRIKGIRKRKF

[0087] FKRNGGLLLEQQLSSTDNGLEKTKLFTSKELAAATDHYNENRILGHGGQGTVYKGMLADG

[0088] RIVAVKKSKRVDEDDLEVFINEIVILSQINHRNVVKLHGCCLENEVPLLVYEFIPNGTLF

[0089] RHIHEPNEDFPLSWEMRTRIARDVAGALSYLHSAAYAPIYHRDIKSTNILLDEKYGAKVS

[0090] DFGTSKSVAIDQTHLTTRVLGTFGYLDPEYFQSSQFTEKSDVYSFGVVMVELLTGEKAIS

[0091] SVRAEVGKSLATHFLHSMEEDHLFDILDPRVLKEGSREEVVAIAELARRCLHLNGKRRPT

[0092] MKEVAVELEGIQLLKEGSVVVQNHDNNREYHSVHEFAECYDFSAMSGSMHFHTITASSAEVERPLLDEP*

[0093] Among them, * represents a terminator.

[0094] Example 2 Study on the resistance of SiWAKL6 to M.phaseolina via SA signaling pathway

[0095] 1. Arabidopsis materials and culture conditions

[0096] Arabidopsis seeds (Columbia wild type) were sterilized with 5% (wt) sodium hypochlorite solution for 10 minutes, washed repeatedly with sterile water 3 to 5 times, and then sown on 1 / 2MS medium, placed in a 4°C refrigerator for low temperature treatment for 4 days, and after the treatment, cultured in an incubator at 22±1°C, 16h light, 8h dark conditions. When Arabidopsis grew to two true leaves, the seedlings were transferred to mixed nutrient soil (nutrient soil: vermiculite volume ratio = 3:1) and continued to be cultured under the above conditions.

[0097] 2. Construction of vector for overexpression of SiWAKL6 gene and genetic transformation of Arabidopsis thaliana

[0098] 1) Construction of SiWAKL6 overexpression vector

[0099] The overexpression vector of SiWAKL6 was constructed by homologous recombination. The plasmid vector number is pCAMBIA2301-KY, the promoter is a strong 35S promoter, and it contains a kanamycin resistance gene. The vector structure is as follows Figure 3 As shown. In this study, the plasmid vector was double digested and recombined using XbaⅠ and BamHⅠ restriction endonucleases, and the SiWAKL6 gene was inserted into the multiple cloning site of the vector to obtain the recombinant plasmid pCAMBIA-SiWAKL6. The recombinant plasmid pCAMBIA-SiWAKL6 was then transformed into DH5α Escherichia coli for propagation and detection. Then, a single colony with regular growth and full roundness was picked and inoculated into a 2mL tube of LB medium containing 40mg / mL ampicillin, placed in a shaker at 37℃ and 200rpm for 12h, and then the bacterial liquid was PCR tested, and the samples containing the correct bands were sent to Shanghai Shenggong Biological Company for sequencing.

[0100] The detection sequence of the obtained recombinant plasmid (SEQ ID NO.3):

[0101] GACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATAAGGAAGTTCATTTCATTTGGAGAGGA

[0102] GCTCCCGCGGGTCGACGGTACCCATATGTCTAGACTCGAGATGAGGCTGTCCGTAATATACCTTTTCGTT

[0103] TCCACCCTTTGCCTAATGACCTCTGCTGCTGTGGCTTTATCTTTGGCAAAGCGTGGATGTCAAGACACTT

[0104] GTGGAAATGTCACCATTCCATATCCTTTTGGAATTGGTTCTGAATGTTATGCAAATTCGTCATTCGCAGT

[0105] GGTCTGTGACAATTCAACCAATCCAGCAAGGCTGCACTTGAGCAGCATCCAATTGGAAATACTAGATATT

[0106] TCGTTGCATGGTACAGTTAGAGTTGTGCAGCGTGTTTTCCCTATGACTTGCTCTGATGAACAGAAAACAC

[0107] AATCTTTGGGGAAATCACTTGTTGGAAGTCCTTTTACCATCTCATCGATTCAAAATTCATTGACTGTTTT

[0108] GGGCTGCAGAAATTCTGTCTGGTTGCGCGCCAACAAGACGACAATAACTGGTGGATGCATGGCTCTTTGT

[0109] GATTACAACTCTAGGGATACAAGTTGTAATGGTATAAACTGCTGCAAGATAACAATTCCTATGGGCCTTC

[0110] AAGAGCTTCAAGTTACTTACCAAAGCATTGCAAATGACAAAAATAATACTCCCTTCTGTGGATATGCCTT

[0111] CCTTGCTGACATGAAATGGTTTAGGGAAGATTACGGGAAATATAATGGCCTGCGCAGTTATTTGTTAGAT

[0112] CCATTTGACGGAGAATTCGGAAATGCAAGTATGGTGCTTGAGTGGGAATACGCCCGCAGTGTGAAGTTTA

[0113] TCCGTTCTAGTTTCTGTGTGTATCCGGGTGATACAAGCTTTGCAGCTTTTAATTATGAAACATTAAGTAA

[0114] AGTATGCTACTGTGACTTCGGGTTTGAAGGAAATCCCTATCTACCTGAAGGATGCCAAGATATTGATGAG

[0115] TGTGGCAATTCGACAACTAATTCCTGTTCTGAGGGAGATACTTGTGTCAACAGAATAGGATCTTACGAAT

[0116] GTCAGAAGCCGAAATCTCGGCTGAAAATAGCATTTATCGTCATTGGTTCTGTGCTTGGTGCACTAATTTT

[0117] GCTCCTGGGAGCATGGAGGTCCACCAAGCTTATTAGAAAGAGAATCAAGGGTATTCGAAAACGAAAGTTT

[0118] TTTAAGCGAAATGGAGGCCTATTATTGGAACAGCAATTGTCTTCAACTGATAATGGCCTAGAGAAAACTA

[0119] AGTTGTTTACTTCCAAAGAGTTGGCAGCTGCTACCGACCATTATAATGAGAATCGTATACTTGGTCATGG

[0120] CGGCCAAGGTACCGTCTACAAAGGCATGTTGGCAGATGGAAGAATTGTGGCTGTCAAGAAGTCCAAAAGG

[0121] GTAGATGAAGATGATCTTGAAGTCTTCATTAATGAGATCGTTATTCTATCTCAGATAAACCATAGAAATG

[0122] TAGTAAAGTTACACGGATGTTGTCTAGAGAACGAAGTTCCTCTTCTTGTCTACGAATTCATCCCAAATGG

[0123] CACACTTTTCCGACACATCCATGAACCAA

[0124] The detection primers were designed based on the vector sequence at the upstream of its promoter and the downstream of the target gene to perform PCR identification on the transgenic plants. The sequences are as follows:

[0125] 35S-F:GACGCACAATCCCACTATCC

[0126] SiWAKL6-R:TTGGTTCATGGATGTGTCGG

[0127] The recombinant plasmid extraction operation was performed using the TAKARA plasmid extraction kit. The recombinant plasmid pCAMBIA-SiWAKL6 was transformed into Agrobacterium GV3101 competent cells (Agrobacterium GV3101 belongs to Agrobacterium tumefaciens), and the colonies were tested again for the presence of the correct bands. The single colony solution with the correct sequencing was added to a conical flask containing 100 mL of YEB liquid culture medium for expansion culture. When the bacterial solution OD 600 = 1.7, it was centrifuged at 5000 rpm for 5 minutes to collect the bacteria. Then the bacteria were resuspended with a resuspension solution to a concentration of OD 600 = 0.8 for standby use.

[0128] 2) Genetic transformation of SiWAKL6

[0129] The genetic transformation of sesame stem blight resistance-related gene SiWAKL6 was carried out using Agrobacterium-mediated in situ transformation of Arabidopsis floral organs.

[0130] When the Arabidopsis thaliana (Columbia wild type) in the incubator grows the main inflorescence, cut off the fully opened inflorescence and fruit pods with scissors. After a week, when more secondary inflorescences are produced on the side branches, you can prepare for infection. Transfer the prepared Agrobacterium GV3101 solution to a large beaker prepared in advance, turn the Arabidopsis to be transformed upside down so that the inflorescence is immersed in the beaker containing the infiltration culture solution, and gently shake the inflorescence during the process to ensure full contact between the inflorescence and the infiltration culture solution to improve the infection efficiency. Then cover the infected Arabidopsis plants with a hood sprayed with water, lay them flat in the dark for 16 to 24 hours, and then resume normal light culture. When the Arabidopsis thaliana pods mature and dry yellow, harvest the seeds in time and place them in a culture dish with a desiccant to dry. After drying, collect the seeds and store them at 4°C for short-term storage or -20°C for long-term storage for later use.

[0131] 3) Screening and molecular detection of Arabidopsis positive plants

[0132] After the harvested Arabidopsis T0 seeds were disinfected, they were spread on 1 / 2MS culture plates containing kanamycin (40 mg / L) for germination and resistance screening. The resistant seedlings that grew true leaves and could take root normally were selected for transplanting and growing. When the seedlings grew true leaves, appropriate amount of leaves were taken to extract genomic DNA, which was amplified by PCR using detection primers (35S-F and SiWAKL6-R), and the recombinant plasmid was used as a positive control to verify whether the Arabidopsis plants were transferred into SiWAKL6. The seeds of the positive plants were harvested and marked by individual plants, and the offspring of each individual plant was a strain. The T3 generation SiWAKL6 gene homozygous Arabidopsis (i.e. transgenic Arabidopsis) was screened by the same method for subsequent experiments.

[0133] Construction of Arabidopsis plants overexpressing SiWAKL6 (OE-SiWAKL6) Figure 4 A). PCR verification was performed using DNA from three overexpressing transgenic Arabidopsis lines OE-SiWAKL6 (OE-1, OE-2 and OE-3) and Columbia wild type (WT) as templates ( Figure 4 B).

[0134] It can be seen from the above figure that SiWAKL6 was successfully transferred into Arabidopsis thaliana, and three homozygous transgenic Arabidopsis thaliana overexpressing SiWAKL6 were obtained, namely OE-1, OE-2 and OE-3.

[0135] 3. Verification of SiWAKL6 gene function

[0136] 1) Evaluation of disease resistance in Arabidopsis

[0137] The preserved pathogen M.phaseolina was inoculated into PDA solid culture medium and cultured in a 30°C incubator for 7 days until the mycelium filled the culture dish. Then the culture medium filled with mycelium was divided into bacterial blocks. The bacterial blocks of each 1 / 2 culture dish were evenly inoculated into 300mL of PD liquid culture medium, and then cultured at 30°C and 200r / min for 5 days. Subsequently, the bacterial liquid was crushed with a tissue crusher to obtain a mycelium suspension. 100mL of distilled water and 120g of sterilized mixed nutrient soil (peat soil: vermiculite volume ratio = 3:1) were added to each 20mL of mycelium suspension and stirred evenly to obtain the bacterial soil. When the Columbia wild type and transgenic Arabidopsis thaliana grew to eight weeks old, they were transplanted into the bacterial soil. The leaf tissue was taken 12h after inoculation and stored at -80°C for later use.

[0138] The disease resistance phenotype of Arabidopsis thaliana was identified 14 days after inoculation, and the plant disease level was classified using a 0-5 grading standard based on the phenotypes of leaf chlorosis, leaf necrosis and death.

[0139] Level 0: Plant health (no symptoms)

[0140] Level 1: Damage to plant growth

[0141] Level 2: 0-30% of plant tissues are chlorotic or necrotic

[0142] Level 3: 30% to 60% of plant tissues become chlorotic or necrotic

[0143] Level 4: 60% to 90% of plant tissues become chlorotic or necrotic

[0144] Level 5: More than 90% of plant tissues become chlorotic or necrotic or the plant dies

[0145] Disease index = 100 × ∑ (number of diseased plants at each level × representative value of each level) / (total number of plants surveyed × highest representative value).

[0146] The results showed that 14 days after inoculation, the leaves of Columbia wild-type Arabidopsis (WT) inoculated with Phaseolus vulgaris showed obvious yellowing and necrosis, and growth was delayed. However, the three strains overexpressing the SiWAKL6 gene (OE1, OE2 and OE3) showed significantly reduced leaf yellowing and necrosis symptoms, and the growth delay phenomenon was also improved ( Figure 4 A). Further statistical analysis of the disease index of Arabidopsis thaliana revealed that the disease indexes of OE-1, OE-2, and OE-3 were 33.3, 40, and 37.8, respectively, which were significantly lower than the disease index of 71.1 in WT ( Figure 4 C).

[0147] 2) M. phaseolina colonization experiment

[0148] The aboveground parts of Arabidopsis plants in each treatment group were collected 14 days after inoculation, and genomic DNA was extracted. The relative expression levels of the sequence feature amplification regions of M.phaseolina (MpSyk) and Arabidopsis (AtSK11) were quantitatively analyzed by qPCR to determine the relative abundance of M.phaseolina and Arabidopsis genomic DNA. The primers are as follows:

[0149] Gene Forward Primer Reverse primer Mk ATCCTGTCGGACTGTTCCAG CTGTCGGAGAAACCGAAGAC AtSK11 CTTATCGGATTTCTCTATGTTTGGC GAGCTCCTGTTTATTTAACTTGTACATACC

[0150] qPCR reactions were performed on species-specific sequence characterized amplified regions of M.phaseolina (MpSyk) and Arabidopsis (AtSK11) DNA, and the relative abundance of DNA between M.phaseolina and Arabidopsis 14 days after inoculation was compared. The results showed that the relative abundance of M.phaseolina in transgenic plants was significantly reduced compared with Columbia wild-type Arabidopsis (WT) ( Figure 4D) The above results collectively indicate that SiWAKL6 can enhance the resistance of transgenic Arabidopsis to M. phaseolina.

[0151] 3) qPCR analysis

[0152] When pathogens invade, we speculate that SiWAKL6 may improve the disease resistance of transgenic Arabidopsis by regulating the expression levels of stress marker genes downstream of Arabidopsis. To verify this hypothesis, we detected the expression of marker genes of SA and JA hormone signaling pathways in Arabidopsis (OE-1) 12 hours after pathogen inoculation, including AtNPR1, AtPR1 and AtPR5 genes of salicylic acid signaling pathway and AtVSP2 and AtPDF1.2 genes of jasmonic acid signaling pathway, with water treatment as control (Mock).

[0153] In Arabidopsis, the Arabidopsis gene AtUBQ10 was used as an internal reference gene to perform RNA extraction, reverse transcription, and qPCR experiments on leaf samples treated with M.phaseolina. The primer sequences are as follows:

[0154] Gene Forward Primer Reverse primer AtUBQ10 AACTTTGGTTTGTGTTTTGG TCGACTTGTCATTAGAAAGAAAGAGATAA AtNPR1 GGCTTGCGGAGAAGACGAC ACGACGATGAGAGAGTTTACGG AtPR1 GCTACGCAGAACAACTAAGAGGC CCAGACAAGTCACCGCTAC AtPR5 ACTCCAGGTGCTTCCCGACAG GAACTCCGCCGCCGTTACATC AtVSP2 CTTTCACTTCTCTTGCTCTTGGC GCAGTTGGCGTAGTTGATGGA AtPDF1.2 ACCCTTATCTTCGCTGCTCTTG ATGTCCCACTTGGCTTCTCG

[0155] Among them, AtNPR1, AtPR1 and AtPR5 are marker genes in the SA signaling pathway, and AtVSP2 and AtPDF1.2 are marker genes in the JA signaling pathway.

[0156] The results showed that after inoculation with pathogens, the expression levels of SA-related marker genes in OE-SiWAKL6 (OE-1) were higher than those in WT ( Figure 5 A), however, the expression levels of JA-related marker genes in OE-SiWAKL6(OE-1) and WT were generally similar ( Figure 5 B). This shows that SiWAKL6 can improve the resistance of Arabidopsis to M. phaseolina by regulating genes in the SA signaling pathway.

[0157] Example 3 SiWAKL6 regulates ROS homeostasis and participates in immune response

[0158] In plants, biotic stress can cause reactive oxygen species to be produced in the body, and excessive accumulation of reactive oxygen species can cause oxidative damage. At this time, the antioxidant enzyme defense system in the plant will start the expression of SOD, CAT, POD, etc. to remove reactive oxygen species (ROS) in the plant, thereby inhibiting cell death. In order to study whether the resistance mediated by the SiWAKL6 gene is related to ROS homeostasis, we detected the reactive oxygen levels (H2O2 and MDA) and the activities of antioxidant enzymes (SOD and CAT) in transgenic plants (OE-1) and wild-type plants after inoculation.

[0159] 1. Arabidopsis materials and culture conditions

[0160] The Arabidopsis thaliana culture conditions and inoculation method were the same as those in Example 2.

[0161] 2. Detection of Arabidopsis physiological indicators

[0162] Fourteen days after inoculation with M. phaseolina, the aerial parts of Arabidopsis plants were taken and the contents of hydrogen peroxide (H2O2), malondialdehyde (MDA) and the activities of two enzymes, catalase (CAT) and superoxide dismutase (SOD), were detected using detection kits (Suzhou Gres Biotechnology Co., Ltd.).

[0163] The results showed that after inoculation with pathogens, the contents of H2O2 and MDA increased significantly, but compared with OE-SiWAKL6 (OE-1), WT accumulated more H2O2 and MDA ( Figure 6 In addition, after inoculation, the SOD and CAT activities in OE-SiWAKL6 (OE-1) were significantly higher than those in WT ( Figure 6 C, 6D). This indicates that SiWAKL6 participates in regulating the ROS homeostasis of plants by regulating the activity of CAT and SOD, thereby enhancing resistance to M. phaseolina.

Claims

1. An application of sesame SiWAKL6 gene in disease resistance breeding, characterized in that: The coding sequence of the sesame SiWAKL6 gene is shown in SEQ ID NO.1, and is applied to stem blight caused by Macrophomi-na phaseolina.

2. An application of the protein encoded by the sesame SiWAKL6 gene as claimed in claim 1 in disease resistance breeding, characterized in that: The protein sequence encoded by the sesame SiWAKL6 gene is shown in SEQ ID NO.2, and is applied to stem blight caused by Macrophomina phaseolina.

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