Plant tmk1 gene and its application against Sclerotinia sclerotiorum
A sclerotinia and gene technology, which is applied in the field of plant disease resistance of the plant receptor transmembrane protein TMK1 gene and its encoded protein, can solve problems affecting national economy and people's livelihood, national food security, crop disasters, and affecting plant growth, etc.
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Embodiment 1
[0036] Example 1 After inoculation of sclerotiorum oxalate deletion mutant (S. sclerotiorum A2) hyphae, tmk1 Tolerance phenotypic analysis of mutant (SALK_016360).
[0037] In vitro Arabidopsis leaves grown for approximately 4 weeks were inoculated with sclerotiorum oxalate deletion mutant (S. sclerotiorumA2) hyphae. 36h after inoculation, observation of leaf disease phenotype and statistical analysis of leaf plaque area were performed. Taking leaf disease spots as the object of investigation, tmk1 The overall disease state of the mutant was weaker than that of the wild type, and the phenotype could be complemented by TMK1, which was consistent with the results of trypan blue staining ( figure 1 ).
[0038] tmk1 The dead cell area of the mutants infected by S. sclerotiorum was significantly smaller than that of the wild type. The statistical results of leaf damage area also showed that, tmk1 Mutants have enhanced resistance to the fungus S. sclerotiorum A2 ( fig...
Embodiment 2
[0039] Example 2 Construction of pTMK1:TMK1 vector
[0040] For further verification tmk1 For the resistance phenotype of mutants to Sclerotinia sclerotiorum, we cloned the TMK1 gene (shown in SEQ ID NO. 1) including its upstream 3000bp promoter sequence from the wild-type Arabidopsis genome (see image 3 ); and connect the amplified gene sequence to the expression vector pCAMBIA1300 containing the FLAG tag, and perform sequencing for final detection and verification, and finally construct the pTMK1:TMK1-FLAG vector.
[0041] The primer sequences used are:
[0042] Upstream primer F: 5-CTTATTTTTTGCTTTTTGATTGTTT-3 (SEQ ID NO. 3),
[0043] Downstream primer R: 5-TCGTCCATCTACTGAAGTGAATG-3 (SEQ ID NO. 4).
Embodiment 3
[0044] Example 3 Construction and detection of transgenic plants
[0045] In Example 2 gTMK1-FLAG The pCAMBIA1300 vector of the gene was transformed into Agrobacterium GV3101, and then transformed by Arabidopsis flower dip method tmk1 Homozygous mutants, obtained by resistance screening tmk1 Complementary lines of mutants. The specific method is as follows: inoculate the Agrobacterium containing the target vector in 80mL of LB liquid medium (containing 50ug / mL Kan, 50ug / mL Rif), shake and culture at 28°C overnight, and wait for its OD 600 When it is about 1.6, centrifuge at 5000rpm for 10min; remove the supernatant, add 30mL of 5wt.% sucrose to suspend the bacterial cells, and add 10uL of Silwet to mix to obtain a transformation solution. The buds of Arabidopsis thaliana were soaked in the transformation solution for 1 min, taken out and put on a black fresh-keeping bag and placed in the dark for 12 hours; the fresh-keeping bag was removed the next day, and the plants we...
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