Cloning And Functional Analysis Of The Calcineurin B Gene In Setosphaeria Turcica

Calcineurin is a conserved Ca²⁺/calmodulin-activated,serine/threonine-specific protain phosphatase that plays an important role in the regulation of morphogenesis and pathogenesis in various fungi.In this work,CnB gene was cloned and identified from Setosphaeria turcica,then studied and deduced its roles in S.turcica development by advantage of antisense inhibition expression method and bioinformatics analysis.It is the first time for us to isolate the completed cDNA sequence and DNA sequence of CnB gene from S.turcica using RACE method,and predict the corresponding structure and functions by the bioinformatic software.The results showed that CnB gene had 4 exons and 3 introns and the largest open reading frame was 525 bp which encoded a protein of 174 amino acid residues.The CnB gene of S.turcica had four "EF-hand"calcium-binding regions,and showed 90%homology with CnB genes from P. nodorum,B.cinerea and Neurospora crassa.Predicted 3D structure for CnB protein by Swiss-model,was constituted with 8 helixes and 4 turns,forming 4 "helixes-loop-helixes" regions.The structure characteristic was consistent with CnB four "EF-hand"calcium-binding regions.A 2kb gene flank region was obtained by Genomic Walking.Bioinformatic analysis showed that it had a lot of cis-acting elements corresponding G-box,DET1,CDA-1,HSF, but no typical TATA box.We calculated that CnB gene perhaps plays an important role in the regulation of cell morphogenesis,hot tolerance and light signal conduction.The result of Southern blotting showed that CnB was single copy in the genome of S.turcica. The sense or antisense CnB gene expression induction vectors were constructed based on the plasmid pSOI,then transformed into S.turcica protoplast and 5 antisense CnB gene which hph gene inserted were screened from 23 transformants.Two transformants grew less slowly on PDA than wild isolate 01-23.