巴西橡胶树胶乳均一化酵母双杂交cDNA文库构建

余海洋1, 张宇1, 王萌1,*, 覃碧2,*
1海南大学环境与植物保护学院, 海口570228; 2中国热带农业科学院橡胶研究所, 海南儋州571737

通信作者:王萌;E-mail: wangmeng@hainu.edu.cn; qinbi126@163.com

摘 要:

以巴西橡胶树无性系‘热研7-33-97’胶乳为材料, 采用SMART® cDNA合成技术反转录合成cDNA第一链, 并通过LDPCR合成双链cDNA (ds-cDNA), 采用双链特异性核酸酶(DSN)对ds-cDNA进行均一化处理, 并经过CHROMA SPIN+TE-400柱子去除短片段的cDNA, 纯化后的cDNA和线性化载体pGADT7-Rec共转化酵母Y187感受态细胞构建均一化酵母双杂交cDNA文库。结果显示: 均一化之前橡胶树胶乳cDNA片段分布较广, 丰度分布不均匀, 经DSN均一化处理和CHROMASPIN+TE-400柱纯化后, 小于500 bp的片段得到有效去除, 高丰度cDNA明显下降, 而且RT-PCR扩增结果显示, 均一化处理后, 18S rRNAβ-actin两个管家基因的表达丰度均明显降低。最终获得初始文库的独立克隆为1.26×106 cfu, 文库滴度达到3.23×107 cfu•mL-1, 重组率为87%, 平均插入片段大于1.0 kb。本研究构建了一个橡胶树胶乳均一化酵母双杂交cDNA文库, 为进一步研究天然橡胶生物合成途径及其调控的分子机制提供借鉴。

关键词:巴西橡胶树; 胶乳; 酵母双杂交cDNA文库; 均一化

收稿:2015-12-04   修定:2015-12-29

资助:国家自然科学基金(31300503和31460197)。 致谢 中国热带农业科学院橡胶研究所的康桂娟博士在文库构建过程中给予的指导和帮助。

Construction of a normalized yeast two-hybrid cDNA library of the latex of rubber tree (Hevea brasiliensis Müll. Arg.)

YU Hai-Yang1, ZHANG Yu1, WANG Meng1,*, QIN Bi2,*
1College of Environment and Plant Protection, Hainan University, Haikou 570228, China; 2Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan 571737, China

Corresponding author: WANG Meng; E-mail: wangmeng@hainu.edu.cn; qinbi126@163.com

Abstract:

In this study, the latex of rubber tree (Hevea brasiliensis) clone ‘Reyan 7-33-97’ was used as plant material. SMART® cDNA synthesis technology was used to generate the first strand cDNA, and then long distance PCR (LD-PCR) was used to amplify double-strand cDNA (ds-cDNA). The ds-cDNA was normalized by duplex-specific nuclease (DSN). The normalized cDNA was purified by running CHROMA SPIN+TE-400 column to remove short cDNA fragments. The purified cDNA and linear vector pGADT7-Rec were co-transformed into competent Y187 yeast cell to generate a normalized yeast two-hybrid cDNA library. The results show that the cDNA of the latex of rubber tree was wide range of fragment sizes and with uneven abundance before normalization. After normalized by DSN and purified using CHROMA SPIN+TE-400 column, cDNA below 500 bp had been removed efficiently, and high abundance of cDNA had been reduced significantly. Moreover, RT-PCR revealed that the transcripts of two housekeeping genes 18S rRNA and β-actin were decreased significantly after normalization. The harvested library had 1.26×106 independent clones. The titer of the library was up to 3.23×107 cfu•mL-1, the recombination rate was 87%, and the average insert size was more than 1.0 kb. In this study, a normalized yeast two-hybrid cDNA library from the latex of rubber tree has been successfully established, which provides a reference for studying natural rubber biosynthetic pathway and its molecular regulation mechanism in rubber tree.

Key words: Hevea brasiliensis; latex; yeast two-hybrid cDNA library; normalization

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