Tea BAC Library Construction And Related Gene Screening And Sequencing

Tea(Camellia sinensis) is a kind of important economic crops. Its economic value is not only reflected on the tea, but also on the tea flowers and fruits. Tea is known as "one of the world’s three largest non-alcoholic beverages", and it has now become the world’s popular health drink. With the development and application of biotechnology, the scientific research of tea is gradually penetrating to the level of molecular biology. Isolation and cloning of tea plant genes have become an important part of the study of tea.In collaboration with the State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, we built a tea BAC(bacterial artificial chromosome) library using tea plant variety "Shu Cha Zao" as the material. This BAC library has a total of 161,280 clones, and the clones are stored in 420 blocks of 384 plates. Insert test results from 450 randomly selected BAC clones showed that the average insert size was about 113 kb with an empty rate of less than 2.5%. According to the reported tea genome size of 2.9 Gb, the BAC library is calculated to cover about 6.2 folds of the entire genome of the tea plant. In order to estimate the rate of organelles contamination, we sequenced the ends of 48 randomly selected BAC clones. The BAC end sequence information was used to search the NCBI database, none of the clones was found to match the organelle genome sequences.In addition, in order to screen for genes that control the important traits of the tea plant, we constructed 420 384-microtiter plate pools of the tea BAC library. By PCR screening, we determined the 384-microtiter plates that contained 7 genes of the tea plant(Csi092H17, Csi020O15, Csi044B12, Csi274K14, Csi271P8, Csi106D7, and Csi047O18). Then we constructed row pools and column pools for each positive 384-microtiter plate and determined the individual positive clones by PCR. Finally, we selected 5 BAC clones, containing Csi092H17, Csi020O15, Csi044B12, Csi271P8 and Csi106D7 gene, respectively. To construct shotgun libraries, we sequenced these five shotgun libraries using first generation DNA sequencing method. The assembled sequences of the five BACs were provided to the State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University for functional studies of tea plant.