Analysis On The Transcription Profiles Of Pepper (Capsicum Annuum) Infected By Phytophthora Capsici And Isolation Of CDNA Of 3 Candidate Resistant Genes

Phytophthora blight caused by Phytophthora capsici.Lone..is one of the fatal diseases in pepper production.Transcription profiles analysis of pepper against P.capsici infection and identification of candidate resistant genes were important fundermental works for the molecular elucidation of pepper disease resistance and its genetic improvement.In this paper,the transcription profile of pepper against P.capsici infection was analysis with cDNA-AFLP,and 363 TDFs were sequenced and functional annotated,and 3 full length cDNAs were screened from the cDNA library constructed with total RNA isolated from leaves infected by P.capsici.,whose expression was analysized by Rael-Time PCR.The main results were as followings:1.The transcription profile of pepper against P.capsici infection was analyzed by cDNA-AFLP, and 363 differentially expressed TDFs(transcript-derived fragments)were sequenced and functional annotated by Blast in NCBI.Homologous sequences were found for 246 TDFs and no homologous sequences were found for the other 117 TDFs.And the TDFs were further classified into 5 subgroups:â… .unchanged(172),â…¡.down-regulated(79),â…¢,early up-regulated(28),â…£.late up-regulated(7),â…¤.others(77).Their are small GTP-binding protein,calcium-dependent protein kinase,chloroplast ferredoxin,dehydroascorbate reductase,osmotin-like protein, spermidine synthase,cytochrome p450,oleate desaturase,peroxidase,protein disulfide isomerase.2.The total RNA was isolated from leaves infected by P.capsici,with the total RNA,a cDNA library was constructed with SMART^TMcDNA library kit,the primary cDNA library contained a total of 1×10⁶ individual pfu,and the recombinating rate of the cDNA library was about 90%.3.By the PCR Based 96-hole screening method,the cDNA library was screened for the candidate genes with specific primers.3 full length cDNAs correspending to antifungal protein, rubisco activase and thionin-like protein were screened out and sequenced.A possible full length cDNA of antifungal protein,was 645bp in length harbored a open reading frame of 85 amino acids in length.By sequence aligment analysis,It was found that EU401721 shared 98%,57%,60%,53%homology with chitinase,AAL73184(Capsicum annuum),ABD66068 (Momordica charantia)ABU25226(Oryza sativa(indica cultivar-group)),AAM49597 (Leucaena leucocephala);A possible full length cDNA of rubisco activase was 1741 bp in length harbored a open reading frame of 439 amino acids in length.By sequence aligment analysis,It was found that EU401722 shared 92%,81%homology with rubisco activase,AAC 15236(Lycopersicon pennellii),ABX84141(Ipomoea batatas);A possible full length cDNA of thionin-like protein was 608bp in length harbored a open reading frame of 91 amino acids in length.By sequence aligment analysis,It was found that EU367112 shared 71%,71%,,homology with thionin-like protein,AAF16413(Capsicum annuum),AF112443(Capsicum annuum),.4.The expression of the 3 candidate genes in pepper seedling under treatment of UV-B,P. capsici,JA,SA,JA+SA,NaCl,low temperature(4℃)and mechanical darnage was analyzed by SYBR Green Real-Time PCR.The result indicated that the same tendency was found in the result of Real-Time PCR and cDNA-AFLP,the antifungal protein was expressed more notable by NaCl treatment for 12 hour and by mechanical damage for 24 hour,and expressed most after P.capsici infection for 60 hour.The results showed that the expression of rubisco activase was the most evident under uv lights,thionin-like protein was related closely with pepper hormone of response and expressed gradually after P.capsici infection.The results of this research favored the molecular mechanism dissection of pepper Phytophthora blight resistance and laid a important basis for the further functional identification of the candidate genes responding significantly to P.capsici.