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p GADT7-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CHROMA SPIN+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 rRNA和β-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1.26×10~6 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。
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 C HROMA SPIN＋TE-400 column to remove short cDNA fragments. The purified cDNA and linear vector p GADT7-Rec were co-transformed into competent Y187 yeast cell to generate a normalized yeast two-hybrid cDNA library. The results show that th e 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 hi gh abundance of cDNA had been reduced significantly. Moreover, RT-PCR revealed that the transcripts of two ho usekeeping genes 18 S rRNA and β-actin were decreased significantly after normalization. The harvested library had 1.26×10^6 independent clones. The titer of the library was up to 3.23×10^7 cfu·mL^-1, the re combination 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 pr ovides a reference for studying natural rubber biosynthetic pathway and its molecular regulation mechanism in rubber tree.
出处
《植物生理学报》
CAS
CSCD
北大核心
2016年第3期312-316,共5页
Plant Physiology Journal
基金
国家自然科学基金(31300503和31460197)~~
