Clone And Sequence Analysis Of Capsaicin Synthase And Its Promoter In Pepper (Capsicum Annuum.)

Pepper is a kind of popular vegetables.It can be eaten freshly or processed to pepper powder and pepper jam etc.Capsaicin is the pungency factor,a bioactive molecule of food and of medicinal importance.Capsaicin is useful as a counterirritant,antiarthritic,analgesic,antioxidant,and anti-cancer agent.Capsaicin is biosynthesized by capsaicin synthase(CS) through the condensation of vaniltylamine,a phenyl propanoid pathway intermediate,and fatty acid moieties in placental tissues of Capsicum fruits.CS is specifically localized to the placental tissues of Capsicum fruits and its activity correlated with genotype-specific capsaicin levets.Capsaicin belongs to secondary metabolite in pepper tissue,so there is low accumulation in pepper fruit.People could not obtain capsaicin easily and destroy it during extraction.We hope clone capsaicin synthase and its 5'-upstream regulatory sequence and use genetic engineering to improve the activity and content of CS.So we can improve the capsaicin content in pepper fruit at last.It is very important in both theory and application to clone and analyze their functions of the promoters of the genes related to capsaicin biosynthesis,which will be helpful to the further understanding of the course in capsaicin biosynthesis and provide necessary genes for improving the capsaicin biosynthesis by gene engineering.In this study,we use the material of south Korea pepper fruit to extract and purify the total RNA.Then the ds-cDNA was synthesed by reverse transcriptase.The primers was designed by sequence of CS cDNA reported in the NCBI database.By the RT-PCR method,a fragment about 1000bp was cloned and sequenced.The sequence result showed that the fragment was 981bp,named NCS-1,containing complete opening reading frame,homology analysis showed that the sequence was over 96% homology to the reported sequence.The result showed that we got the right CS cDNA sequence.By using the restriction enzyme to digest the total DNA of south Korea pepper fruit as template,we obtained the 5'-upstream regulatory sequence of CS.1347bp was cloned,sequenced and named CSPRO-1.Homology analysis Showed that the sequence was different from other sequences in the database.So the CSPRO-1 was a new sequence.By using the promoter predictions software to analysis the CSPRO-1,the result showed that the CSPRO-1 contains three presumed promoter region.The result of plant CARE database showed that the CSPRO-1 has the feature of promoter:CAAT-Box,TATA-Box,W-Box and I-Box involved in the transcriptional start and the translation respectively,and regulatory sequences like CACT-Box and GATA-Box,which has a important role in tissue specific promoter.Besides,it has other regulatory sequences.So the CSPRO-1 is regarded as the 5'-upstream regulatory sequence of CS.