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African Journal of Biotechnology

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Construction of full-length cDNA library of white flower Salvia miltiorrhiza bge f.alba root and partial EST sequence analysis

G Hao, R Shi, J Wang, B Qi

Abstract


In order to screen and isolate secondary metabolite biosynthesis related gene, we construct a cDNA library of white flower Salvia miltiorrhiza bge. f.alba. High quality of total RNA was successfully isolated from roots of white flower S. miltiorrhiza using modified CTAB method. Double strand cDNA was cloned into pDNR-LIB vector. The number of clones, recombinant rate and length of insert fragments were
determined. Results showed that the capacity of the original library was 1.8×107 with a recombinant rate of 91% and the inserted cDNA fragments ranged from 0.5 to 2.0 kb. Partial cDNAs chosen by random
were sequenced. After BLAST analysis of some cDNAs, their possible functions were predicted. It is found that most of these cDNAs were similar to homological genes of Arabidopsis thaliana, Oryza
sativa, and other plants. Most of the genes were related to cell metabolism, stress resistance, cell growth and development, etc. More importantly, some key enzymes and factors involved in secondary
metabolism of S. miltiorrhiza, such as EST fragments of phenylalanine ammonialyase (SmPAL1), chorismate synthase (SmCHS), 3-hydroxy-3-methylglutaryl CoA reductase (SmHMGR), 4-Coumaratecoenzyme
A ligase (Sm4CL1) and SmMYB90, were found from this library. These results indicated that the library has enough capacity, high recombinant rate and long insert fragment. This study provided a base for further study on the structure and function of these cDNAs.



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