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Journal of Tea Science ›› 2004, Vol. 24 ›› Issue (1): 18-22.doi: 10.13305/j.cnki.jts.2004.01.003

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Construction of Tender Shoots cDNA Library and Preliminary Analysis of Expressed Sequence Tags Sequencing of Tea Plant

CHEN Liang1, ZHAO Li-ping1, 2, GAO Qi-kang3   

  1. 1 Key Laboratory of Tea Chemical Engineering, Ministry of Agriculture; Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310008, China;
    2 Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China;
    3 Biological Macromolecular Research Laboratory of Analyzing and Measurement Center, Zhejiang University, Hangzhou 310029, China
  • Received:2003-07-25 Revised:2003-12-16 Online:2004-02-15 Published:2019-09-11

Abstract: The construction of the first cDNA library of tea plant [Camellia sinensis cv. Longjing 43] in China and the analysis of expressed sequence tags sequencing successful ratio were reported. Total RNA was isolated from tender tea shoots using TRIZOL single-step method, mRNA separated and then the double-strand cDNA amplified by LD-PCR. After size fractionation, the ds-cDNA was ligated to λTripEX2 and recombinant bacteriophages were packaged. The cDNA library was tittered and amplified using XL1-Blue as receptor bacterium. The titer of the original library was 6.8×105pfu/ml with a recombinant rate of 98.05% and 3.5×105 clones in total, the amplified titer was 7.2×109pfu/ml. PCR amplification suggested the inserted cDNA fragments ranged from 0.5 kb to 2 kb, mostly from 1.0 kb to 1.5 kb. The data indicate that the tea cDNA library has high titer, high recombinant percentage and large inserted fragments. Totally, 4320 clones were sequenced, 2963 useful sequences were obtained, corresponding to 68.5%. The first batch of 1687 valid tea plant ESTs were generated.

Key words: Tea science, Tea plant (Camellia sinensis), Tender shoots, mRNA, cDNA library, ESTs, Sequencing

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