Study On The Expression And Transformation Of Genes In The Capsaicin Biosynthesis Pathway Of Pepper

Pepper (Capsicum annuum.L), a perennial or annual crops (chromosome 2N=24), is widely cultivated in Asia, Europe and South America areas. As a Solanaceae vegetable crops, pepper contents a variety of essential vitamins, minerals, fiber, carbohydrates and proteins and numerous of anti-aging substances, such as flavonoids, phenols and carotenoids. Nowadays, pepper was extensively used in the fields of food, medical, industry, agriculture, navigation and military. The extensive usage of pepper was owned to the spicy ingredients—capsaicinoids which mainly include of capsaicin and dihydrocapsaicin. The contents of capsaicin have become one of the most important qualities to estimate the quality of pepper, product quality, and drug active ingredients. The current study indicated that the capsaicin accumulation was closely related to the environment, genetype and cultivation condition and had negative correlation to fruit weight, which led to the limitation of capsaicin yield. This yield can't effectively meet the market demand. Besides, with the improving of living standard, the food function of therapeutic health has become the focus of personal's attention, therefore, natural and no pollution capsaicin has broad development prospects. Accordingly, there are requirements to deeply research the mechanism of capsaicin biosynthesis.Based on the homology, PAL, pAMT (GenBank:HM991733), FatA (GenBank:HM126670), Pun1 (GenBank:GU300812) cDNA and Kas (GenBank: HQ229922) DNA sequences were isolated from chilli pepper ?Yidu-Red'(Capsicum annuum), revealing that they encode the putative phenylalanine ammonia-lyase, amino- transferase, acyl-ACP-thioesterase, acyltransferase and 3-oxoacyl-[acyl-carrier- protein] synthase respectively.The gene of phenylalanine ammonia-lyase had 2156bp, encoding 718-amino acid protein. The closest species to Pal was red cluster pepper with the homology of 99%. SDS-PAGE analysis confirmed that the recombinant 6xHis-PAL with the predicted molecular weight of about 93 ku was successfully expressed in E.coli BL21 (DE3) strain with the optimal condition: 0.3 mmol·L-1 IPTG, 29℃, shaking 6 h. The gene of 3-oxoacyl-[acyl-carrier-protein] synthase had 3456bp, consisting of 8 extron area, encoding 488 amino acids. The molecular weight and PI of the putative protein were 52.2 ku and 8.24, respectively, and the putative protein shared high identity to other homologues, especially Capsicum chinense, about 98%. In this research, the expression pattern of Kas at various time-points after pollinated in flesh and placenta was examined by semi-quantity RT-PCR. The mRNA abundance of Kas in placenta was higher than flesh. In addition, the Kas expression level was reduced after pollinated 10 days. Furthermore, we studied the effect of brassinosteroids on Kas expression, and the results showed that the mRNA abundance of Kas was dramatically induced after treated with 24-epibrassinolide (24-epiBR) 6h and reached a peak after 24h and then reduced slowly, indicating that brassinosteroids had a positive effect on Kas expression. The length of FatA was 1213bp and the ORF was from 1 bp to 1116bp, encoding 371-amino acid protein with an estimated molecular mass of 41.93kD and an estimated pI of 6.63. The length of pAMT was 1457bp and the ORF was from 1 bp to 1380bp, encoding 459-amino acid protein with an estimated molecular mass of 50.72kD and an estimated pI of 6.73.The length of Pun1 was 1541bp and the open reading frame (ORF) was from 37bp to 1359bp, encoding 440-amino acid protein with an estimated molecular mass of 49.29kD and an estimated pI of 7.8. The putative protein PUN1 shared high identity to other homologues, especially Capsicum chinense, with the genetic distance of 0.0193. This research revealed that the Pun1 was not expression in the root, stems and leaves by the technique of Semi-quantity RT-PCR. A 63ku protein of PUN1 was induced under the optimal condition: 0.3 mmol·L-1 IPTG, 29℃, shaking 6 h, through SDS-PAGE and Western Blot detected. Subcellular localization analysis shows that the fusion protein of PUN1::GFP was located in the area of membrane and cell wall.The expression patterns of the genes in the capsaicin biosynthesis pathway were studied by the Real-time RT-PCR technique. Through the pepper growth, the expression patterns were classified into four groups: placenta specific expression, consistent expression in flesh and highly expression in placenta, expression with the same steps in flesh and placenta, and expression in the late growth. The pAMT, Pun1, BCAT, FatA, HCT, C4H, 4CL and COMT genes were up-regulation after treated with 24-epibrassinolide (24-BL) and the promotion time in placenta is longer than flesh.An effective pepper genetic transformation prosidure was also built, which found that 12 days cotyledonary node explants of pepper had more effective shoot differentiation rate, about 90%. The optimal transformation time was 15min, which was profit to the cultural of pepper explants differentiation. The plant expression vector of Pun1 gene was constructed and transformed by Agrobacterium-mediated method. At last, three PCR identified positive peppers were acquired.This study would be useful to cultivate high capsaicin content pepper through the isolation of relative genes, function analysis, expression patterns classified and exogenous hormone regulation research and would be meaningful and valuable to promote the added value of pepper.