Construction Of CDNA Library And Expressed Sequence Tags(EST) Analysis Of Tripterygium Wilfordii Leaf

Tripterygium wilfordii Hook.f. is a climbing vine plants, which belongs to Tripteryguim genus of Celastraceae. As a traditional medicine, Tripterygiumilfordii has been widely used in medicament, clinical study and pest control. This research compared the effects of six method with RNA extraction, constructed a full-leng thcDNA library with EST function analysis. All the study mentioned above were relied on "The national11th Five-Year technology based plan topic". The main results were as follows:1. Tissue culture seedling and triennial seedling of Tripterygium wilfordii were used to extract total RNA with CTAB、CTAB-LiCl、CTAB-Trizol、Trizol、TIANGEN RNAprep Pure Plant Kit and Bio Teke RNApure Total RNA Plant kit method. It showed that the tissue culture seedling is more clear than the triennial seedling with fewer information. The CTAB and CTAB-LiCl method were better than any others. The CTAB-LiCl method could provide a pure total RNA, and increase the yield with concentration. Meanwhile, the CTAB method could provide a high yield with purification of LiCl. Either CTAB-LiCl or CTAB could meet the requirement of cDNA library construction, with high integrity, high yield, good stability and no DNA pollution.2. A full-length cDNA library was constructed. The factors influencing the quality of library were studied, including the concentration of starting materials,ds cDNA LD-PCR cycles and Connected proportion. The optimal system is built with2μL ss cDNA stock solution,16cycles of LD-PCR and a proportion in vector and ligation product of1:1.84. The plasmid Libraries capacity was1.12×107pfu-mL-1with a recombinant rate of94.7%. And the inserted cDNA fragments ranged from0.3to1.5kb. The titer of the amplified cDNA library was1.86×108pfu·mL-1.3.230clones were sequenced, and214ESTs were obtained. The average rate of recombination is465bp, GC content of the sequenced ESTs has a percetage of47.1%.67unigenes were generated after clustering, including9singletons and58contigs with redundancy at68.69%.Among the67contigs, there were9high abundant genes,39middle abundant genes and19low abundant gene which which account for13.43%,58.21%,28.38%of the total contigs.4. The result of EST blasted against the non-redundant nucleotide and protein database of NCBI. There were60sequences with89.55%of the total ESTs had homology,38sequences with56.72%of the total ESTs had significant homology, and7of the total ESTs were new.40ESTs had been matched with protein database. Among them,2ESTs were unknown protein,38of them were known protein or predict protein. The gene functional annotation showed that the38ESTs were divided into9functions, and unknown function protein was25.0%, the function of metabolism, energy and transport made a contribution with37.5%.The succeed in cDNA library construction provide conditions for SSR, SNP markers development. The large number of ESTs will be beneficial to find key synthase of secondary metabolism, and will be a useful resource for future research on molecular biology study of Tripterygium wilfordii.