Cloning And Sequence Analysis Of The Promoter And Calcineurin β Subunit Of Botrytis Cinerea

Signal transduction cascades regulating fungal growth, morphology, and pathogenicity are remarkably conserved between distantly related fungi, such as Ca²⁺ cascades, the cAMP-dependent protein kinase and MAP kinases. In order to identify the relationship between Calcineurinβsubunit(CNB)and the pathogenicity of Botrytis cinerea of tomato, CNB were cloned and the functional analysis were done by bioinformatics analysis, CNB copies' confirmation and constructed the sence and antisence CNB gene expression induction.Homologous cDNA fragments of CNB were obtained by polymerase chain reaction (PCR) amplification from degenerated primer sets designed on the basis of the conserved amino acid regions of CNB from others fungi. Using NCBI blastn, the sequences of CNB showed 100% similarity to two ESTs of B. cinerea. The 525 bp completed cDNA sequence of CNB in B. cinerea was obtained with DNAMAN. The 810 bp completed DNA sequence of CNB in B. cinerea was obtained with primer sets designed on the basis of cDNA sequence of CNB. There is one open reading frame composed of 174 aa and three deduced introns of 40 bp, 120 bp and 84 bp. All introns were accordance with GT-AG rules.Analyzing CNB protein sequences by NCBI, conclusion showed it contained molecular weight of 19.7498 KD and PI of 4.28 and four EF-hand calcium-binding domains, containing 32 aa with negative electric charge and 21 aa with positive electric charge, no containing Cys, His, Trp. The promoter sequence of CNB was amplified using the Genomic walking method. Promoter sequence analysis showed that the promoter sequence contained a nucleus transcription factor NF-Kb union position spot, an activation protein AP-2 union position spot, and other regulatory elements, such as TF II -I, AP-2, BKLF, Sp1. We proofed furtherly if the regulatory elements relating cell multiplication were critical for B. cinerea pathogenicity.The result of Southern blotting showed that CNB was single copy in the genome of B.cinerea, which provided evidence for us to identify furtherly the relationship between CNB and the pathogenicity of B. cinerea.