甘蓝型油菜全基因组cDNA FOX-hunting过表达文库构建  

Construction of whole genome full-length cDNA FOX-hunting overexpression library in Brassica napus

作　　者：张凯[1] 魏丝雨 常玮 范永海 卢坤[1] ZHANG Kai;WEI Si-yu;CHANG Wei;FAN Yong-hai;LU Kun(College of Agronomy and Biotechnology,Southwest University/Rapeseed Engineering Technology Research Center,Chongqing 400715,China)

机构地区：[1]西南大学农学与生物科技学院/重庆市油菜工程技术研究中心,重庆400715

出　　处：《中国油料作物学报》2021年第3期435-442,共8页Chinese Journal of Oil Crop Sciences

基　　金：国家重点研发计划(2016YFD0101007);国家自然科学基金(31871653)。

摘　　要：为系统解析甘蓝型油菜基因功能,结合FOX-hunting和Gateway重组技术,以4种激素和4种非生物胁迫处理的甘蓝型油菜幼苗根和叶片及成熟期种子和角果全长cDNA为混样,构建了全长cDNA FOX-hunting过表达文库。经检测,初级文库滴度为3.0×10^(6),库容为1.2×10^(7)CFU;次级FOX-hunting过表达文库滴度为2.6×10^(6),库容为1.04×10^(7)CFU。次级文库菌液PCR鉴定表明,阳性克隆率为96%,扩增均为250~3000 bp随机分布的单一条带,表明初级文库已较好地重组到pJVC55载体上。农杆菌转化后的克隆测序发现,140个克隆中有12个克隆为非特异扩增。GO富集分析发现剩余128个基因中,除21个基因功能富集于光合作用途径外,其余基因在转录因子和激酶中随机分布,且功能涉及器官发育、物质代谢和胁迫响应等多个方面。上述结果表明,本研究构建的甘蓝型油菜FOX-hunting过表达文库无功能偏向性、多态性丰富、完整性高,可作为油菜功能基因组的研究基础。Genome-wide overexpression library is an important basis for systematic characterization of gene function in Brassica napus.Full-length cDNA FOX-hunting overexpression library was constructed by combining FOX-hunting and Gateway recombination techniques with full-length cDNA of mixed samples of seedling roots and leaves treated with 4 hormones and 4 abiotic stresses,and mature seeds and silique pericarps in B.napus.Titer of the primary library was 3.0×10^(6),and total CFU was 1.2×10^(7).The secondary FOX-hunting overexpression library had a titer of 2.6×10^(6)and a total CFU of 1.04×10^(7).PCR identification showed that the positive clone rate was 96%,and the amplificons were all exhibited as single band random distributed within 250-3000 bp,indicating that the primary library had been well recombined into pJVC55 vector.Sequencing results of Agrobacterium-transformed clones showed that 12 of the 140 clones were non-specific.GO enrichment analysis found that,except for 21 genes enriched in photosynthesis,the remaining genes randomly distributed in transcription factors and kinases,and their functions were involved in multiple aspects involved organ development,metabolism and stress response.The results showed that our FOX-hunting overexpression library of B.napus had rich polymorphism and high integrity,but no functional preference,which laid an important foundation for the functional genome study in B.napus.

关 键 词：甘蓝型油菜 全长CDNA FOX-hunting 过表达 文库 

分 类 号：S565.4[农业科学—作物学]