A Botrytis cinerea gene bcmbf1 related to pathogenicity and its application
A botrytis cinerea and gene technology, which is applied in the application field of discovery and its encoded protein, and can solve the problems of undiscovered homologous genes and the like
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Embodiment 1B
[0029] Example 1 BcMBF1 Gene correlation analysis
[0030] Gene BcMBF1 It is a gene identified by our R&D team by analyzing T-DNA-marked Botrytis cinerea pathogenicity-deficient mutants. Botrytis cinerea BcMBF1 The open reading frame of the gene consists of 1398 nucleotides without introns. The encoded protein product consists of 465 amino acids. Domain analysis revealed that the BcMbf1 protein has no conserved functional domains and is an unknown protein; the sequence contains a transmembrane region and is predicted to be a transmembrane protein (see figure 1 ).
Embodiment 2B
[0031] Example 2 BcMBF1 gene knockout
[0032] 1) Construction of knockout vector
[0033] Using primer P1 (5'-ACTAGTCTGTGCCTCGTGGTGAAGAT-3') with
[0034] P2 (5'-GGTACCTGATGGCAATCCGTTGAAGT -3'), amplified using genomic DNA of Botrytis cinerea strain B05.10 as template BcMBF1 The 604bp fragment upstream of the gene, using
[0035] P5 (5'-GTCGACATGGGTGGTTTATTCTATTTGC-3') with
[0036] P6 (5'-CTGCAGGAGTGGATGGCTCGTGAAG-3') amplified Botrytis cinerea BcMBF1655 bp fragment downstream of the gene, the reaction system is: 10 mmol / L dNTP Mixture, 0.5 µL; 10×PCR buffer, 2.5 µL; each 1 µL of upstream and downstream primers (10 µmol / mL); template DNA, 1 µL; Ex-Taq, 0.2 µL (5 U); ddH 2 O, 18.8 µL; Amplification program: 94°C pre-denaturation for 3 minutes, then (1) 94°C, denaturation for 50 seconds; (2) 58°C, annealing for 50 seconds; (3) 72°C, extension for 60 seconds; (4) ) cycle 30 times; (5) 72°C extension for 10 minutes. The above two DNA amplification products were respec...
Embodiment 3B
[0053] Example 3 BcMBF1 Genetic complementation of gene deletion mutants
[0054] Botrytis cinerea was amplified using primers C-F (5'- AAAGATCAAAGGATCGAATTCAGAAAGAGGCGATTGTGAAGT-3') and C-R (5'- CCGGGTACCGAGCTCGAATTCATATGTTAGCGAACGAAGCAG-3') BcMBF1 The full length of the gene is 3208 bp (including promoter, open reading frame and terminator), first cloned into the pMD18-t vector, and then subcloned into the pXEB vector (containing the glufosinate-ammonium resistance gene) Eco The R I site was constructed into a genetic complement vector pMBF1-ko-c. The vector was verified by sequencing to confirm that there were no amino acid mutations. Using the Agrobacterium-mediated transformation method as described above, use 200 μg / mL glufosinate-ammonium for screening, and transfer the complementary fragment into BcMBF1 In the gene deletion mutant M1 genome, a genetically complemented strain ∆ Bcmbf1- c. The primers P7 and P8, P3 and P4 used in the mutant verification were se...
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