CDNA-AFLP Analysis Of Pepper Cold Resistence And Research On CaAQP Gene's Cloning And Expression

Chilling injury is a prominent problem suffered in protected farming in winter or early spring. In order to study the molecular mechanism of chilling resistance, the variety of P70 was used to invetigate the differential gene expression of leaves induced by cold stress at 4℃with different time with cDNA-AFLP method. Then the method of RACE was useed to clone the full length of the interesting fagments. The expression of full length gene was detected and analyzed by real-time quantitative PCR and semi-quantitative RT-PCR. The result showed as follows:1.256 pairs of AFLP amplification primers were used to analyze the cDNA of the treated and control, there are totally 120 differential expression fragments between treated and control plants, of which 80 were re-amplified and cloned. 30 positive TDFs were collected and 12 fragments were sequenced , only 4 of which were successfully sequenced. Bioinformatic analysis of these sequences indicated that the 4 TDFs (KH-1, KH-2, KH-3 and KH-4) related with cold resistance have high homology to ascorbic acid peroxidase, cytochrome p450, bovine serum albumin (BSA) and water-hole channel protein (AQP) respectively .2.Using one TDF with reference values as candidates TDF to get full-length and as templates to design gene specific primers, The full length of CaAQP was acquired by the method of RACE (rapid amplification of cDNA ends). Bioinformatic analysis showed that CaAQP had over 96% comparability to cDNA gene that encoded water channel protein.3.The expression of CaAQP was detected by semi-quantitative RT-PCR and real-time quantitative PCR. The results showed that the gene CaAQP expression show the trend of downregulation with various treatment time.