Transcription Profiles Analysis Of Pepper To The Treatment Of Stresses And CDNA Isolation And Identification Of Several Candidate Genes

Plants are frequently exposed to abiotic stresses and biotic stresses, and their growth and yield often reduce due to the damage of various stresses. However, prior challenge by various stresses also can induce resistance to subsequent challenge, and this resistance must be orchestrated within a larger and physiological context. In the past decade, intensive molecular biological study on the plant stresses response have been undertaken and it was found that the defense reaction and the corresponding defense signal cascades,which is mediated by phytohormones, interacted each other,but the molecular mechanism mediating by different phytohormones in the stresses defense remains mostly unclear. In this study, the transcription profiles of pepper againt UV-B, P. capsici as well as different exogenous phytohormones treatment were analyzed and the differential expressed genes were isolated by SSH, which were further verfied by real time PCR, isolation and sequencing of the the candidate genes. The main results were as followings:1) The transcription profile of pepper elicited by exogenous salicylic acid (SA), methyl jasmonate (JA), ethylene (ET), abscisic acid (ABA), UV-B, Phytophthora capsici were analyzed by cDNA-AFLP. About 5000 transcript-derived fragments (TDFs) were showed and 363 TDFs were sequenced and functional annotated by Blast and GO. Homologous sequences were found for 246 TDFs and no homologous sequences were found for the other 117 TDFs. And the expression patterns of TDFs were further classified into 5 subgroups: decrease, unchanged, early up-regulated, late up-regulated, increase without early and late up-regulated. Furthermore, every one of the 363 TDFs can be compared their expression patterns in the 30 treatments.2) UV-B induced and Phytophthora capsici induced genes of pepper were studied by using suppression subtractive hybridization (SSH) technique. Two subtractive libraries of pepper including 500 cDNA clones were constructed in the study. And some inserts cDNA clones were sequenced. 14 cDNA sequences of UV-B SSH library are catalase, cpn60b, wrky family transcription factor, protein phosphatase 2c, ycf2, potassium channel, leucine zipper protein, small nuclear ribonucleoprotein, photosystem i p700 apoprotein a2, reverse transcriptase-like protein, electron carrier iron ion binding. 12 cDNA sequences of Phytophthora capsici SSH library are chloroplast clp subunit of peptidase complex, nac2-like protein, methionine synthase, phosphoribulokinase precursor, ribulose bisphosphate carboxylase, light harvesting chlorophyll a/b-binding protein, 40s ribosomal protein s11, ribosomal protein 132, pstvd RNA-biding virp1, cis-zeatin o-glucosyltransferase, 60s ribosomal protein 17, leucine-rich repeat transmembrane protein.3) The three total RNAs were extracted from the leaves of pepper cultivar 'L11', separately treated by JA+SA, UV-B and Phytophthora capsici. With the total RNAs, three cDNA libraries were constructed with SMART^TM cDNA library kit, titer of the three cDNA libraries were about 1.2-2.0×10⁶ pfu/ml, and the recombination rates of the three cDNA libraries were all more than 90%. They are all good enough for screening genes.4) Eight differential expression TDFs were designed primers, and these primers were used to gain the eight full-length cDNAs of the differential expression genes by the PCR based 96 wells cDNA library screen method. There are the differential expression genes: pectin methyl esterase inhibitor (EU380203), thionin-like protein (EU367112), antifungal protein (EU401721), rubisco activase (EU401722), cyclophilin-like protein (EU401723), chlorophyll a/b binding protein (EU401724), non-specific lipid transfer protein (EU401725), ferredoxin (EU401726).5) The expression of the eight candidate genes in pepper seedling under treatment of JA, JA+SA, SA, UV-B, Phytophthora capsici, NaCl, low temperature (4℃), mechanical damage and aphides eating were analyzed by Real-Time PCR.6) Eight entry vectors and eight destination vectors were constructed by Gateway BP and LR reactions. There are the seven genes which had been screened, pectin methyl esterase inhibitor, thionin-like protein, antifungal protein, rubisco activase, cyclophilin-like protein, ferredoxin, ethylene responsive element binding protein, resveratrol synthase.7) A efficiency Agrobacterium mediated transformation system for tobacco K326 was established, and Agrobacterium mediated tobacco transformations were conducted by this system to acquire the eight genes transgenic tobacco plants for further use.