Cloning And Expression Analysis Of Fertility Related Gene CYP704C In Tea(Camellia Sinensis)

Tea(Camellia sinensis(L.)O.Kuntze)is one of the most important economic crop in China.But the limitations of conventional breeding techniques,such as difficulties in segregation of genetic character and in distant hybridization,beneficial mutation frequency is too low,it costs too much time in selecting excellent varieties of consumptions,etc.All these restrict the use of high quality germplasm resources of tea,which make it difficult to make breakthrough in variety breeding,while the emergence of genetic engineering breeding provides a wide application prospects.In recent years,the rapid development of modern molecular biotechnology has provided important technical support for the selection of tea tree varieties and the orientation improvement of traits[1].The researchers have gained lots of genes expressed in different period of pollen development based on the analysis of Arabidopsis,Rice,Petunias male sterile mutants,some experiments have verified the possible role of these genes in the pollen development way.Based on these researches,this study isolated CsCYP704 C gene from different organizations of ‘tieguanyin’ tea,study the expression of CsCYP704 C genes in different tissues and organs of three different tea trees;Construct CsCYP704 C gene over-expression vector and transformed it into arabidopsis thaliana to verify its function.The main results are as follows:(1)Amplifying the CsCYP704 C gene: The coding sequence of CsCYP704 C gene was isolated from the leaf of ’tieguanyin’.The length of CsCYP704 C gene is 1527 bp in coding sequence,and translated into 508 amino acids.Sequence alignment showed that the sequence similarity between CsCYP704 C and the CYP704 C gene from other species is 74.25% to the population.CsCYP704 C has the highest sequence similarity(76%)with durian,grape and rubberand the the lowest similarity(62.03%)with papaya and sesame,.(2)The analysis on tissue and organ expression pattern of CsCYP704 C genes: In this study,the transcription level of CsCYP704 C was analyzed in different tissues from three cultivars including ’tie guanyin’,’zhu yeqi 12’ and ’fuding white’ The results showed that the trancripts of CsCYP704 C genes were detected in all tissues,including sepals,filaments,stamens,petals,anthers and mature leaves in three cultivars ’tieguanyin’,’niche Ye Ji 12’ and ’fuding white’ trees and the highest transcript level in three cultivars were all detected in filaments Compared with ’tieguanyin’ and ’fuding white’,CsCYP704 C gene has a higher transcript level in ’niche Ye Ji 12’.(3)Vector construction and verification of gene function : constructing the p Cambio1301::CsCYP704 C vector successfully,transformed the vector into wild-type Arabidopsis by the method of agrobacterium-mediated,obtained transgenic plants,and observed the morphology and cytology of transgenic plants.In addition,transformes the p Cambio1301::CsCYP704 C vector into MS2 and CYP703 Arabidopsis mutants,and screening the positive plants were under way.