Construction and Characterization of Full-Length cDNA Library and Expressed Sequence Tags Analysis in Developing Seeds of Vegetable Soybean

서지정보

· 발행기관 : 한국원예학회
· 수록지 정보 : Horticulture, Environment, and Biotechnology / 50권 / 1호 / 51 ~ 56페이지
· 저자명 : Li-Fei Yang, Jun-Yi Gai, Yue-Lin Zhu, Gang Chen, Guo-Ping Wei, Cong Wang, Qian-Qian Liu

초록

The objectives of this paper were to construct a full-length cDNA library and identify genes encoding high quality proteins in developing seeds of vegetable soybean [Glycine max (L.) Merr.]. The construction of full-length cDNA library was essential for screening and cloning of novel genes. Using vegetable soybean variety ‘NN99-10’ as an experimental material, a full-length cDNA library from the developing seeds was constructed by switching mechanism at 5’-end of RNA transcript (SMART) approach. Total RNA from different developmental stages of seeds was purified. Double strand cDNA was synthesized by SMART method and the cDNA fragments were ligated to the pBluescript II SK vector. The recombinant plasmids were transformed into E. coli by electroporation and a cDNA library of developing seeds was successfully constructed. By counting the number of colonies, the titer of the primary cDNA library was 7.6×106 cfu･mL-1, and that of the amplified library was 2.4×1012 cfu･mL-1. Electrophoresis gel results indicated that most of the cDNA inserts were ranged from 0.40 to 2.0 kb, with an average size of larger than 800 bp. Randomly picked clones showed that the recombination rate was above 99%. These evidences showed that the cDNA library constructed was a full-length library with high quality, and could be used to screen the genes encoding high quality proteins. A total of 206 randomly selected positive clones were successfully sequenced, and 73% of them contained full-length coding regions. Based on bioinformatics analysis of the sequences, we obtained 192 expressed sequence tags (ESTs) from 206 cDNA positive clones. Eight important genes which might be involved in seed quality were identified, and two of their genes were identified to be related to the allergenicity of seed proteins. The full-length cDNA library constructed in this research has laid the foundation for the further cloning of novel genes and the revealed EST sequences have enriched our knowledge of the function genes of vegetable soybean seeds.

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