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作 者 ：贺超, 肖小军, 章智, 彭琦, 黄英金, 廖江林

期 刊 ：核农学报 2015年 29卷 8期 页码：1437-1443

关键词：水稻；灌浆期；籽粒；高温胁迫；全长cDNA文库；

Keywords:rice, early grain-filling stage, grain, high temperature stress, full-length cDNA library construct,

摘 要 ：

为克隆水稻灌浆期耐热性相关基因的全长cDNA序列,本研究以水稻灌浆期耐热纯系XN0437T和热敏感纯系XN0437S为材料,于灌浆初期(扬花后第8 天)进行38.0/33.0 ± 0.5℃(白天/夜间)温度处理,设3次高温处理重复;高温处理结束时,取耐热和热敏感纯系籽粒提取总RNA,3次高温处理样品的总RNA等量混合后,利用SMART技术合成cDNA双链,经琼脂糖凝胶电泳分离后,分5个区段回收大小不同的cDNA,分别克隆到pGEM-T Easy Vector载体,构建了5个全长cDNA亚文库并分析了这些亚文库的质量。结果表明:5个全长cDNA亚文库共获得7974个阳性克隆,平均插入片段大于1.0 kb,整个文库的全长率约为75%;插入片段大于0.5 kb的亚克隆文库的滴度均大于4.6 × 10⁶ pfu·mL^-1、重组率大于98.1% ± 0.79%。为获得全长cDNA文库克隆目的基因的全长奠定了基础。

Abstract:A full-length cDNA library was constructed to clone the full-length sequence of the genes related to heat tolerance during rice grain filling stage using the heat-tolerant line XN0437T and the heat-sensitive line XN0437S as plant materials. Two rice lines were treated by 38.0/33.0 ± 0.5℃ (day/night) high temperature at early milky stage (on the 8^th day after flowering) with three replicates. Total RNAs were extracted respectively from the grains and then mixed equally. Double strand cDNA of the mixed RNAs was synthesized by SMART PCR cDNA Kit produced by Takara Clontech Co., Ltd., then the synthesized cDNAs were separated through agarose gel electrophoresis, collected according to fragment size and ligated to pGEM-T Easy vector respectively to construct full-length cDNA sub-clone libraries. To identify the quality of the library, the insert fragments length of the clones, library titer and recombinant rate were determined and analyzed, and the full-length rate of library was analyzed by random cloning sequencing, besides that, the sequences from the library was further blasted in GenBank database. The results showed that, 7 974 positive clones were included in the library, the average size of the inserted fragment of the clones were longer than 1.0 kb, the rate of full-length gene in the whole library was about 75%, the titer of sub-clone libraries which the size of insert fragments were more than 0.5 kb was more than 4.6 × 10⁶ pfu·mL^-1, and the recombinant rate of these sub-clone libraries was more than 98.1% ± 0.79%. The constructed full-length cDNA library supported a foundation for the full-length cloning of the object genes.

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