黑籽南瓜基因组可转化人工染色体(TAC)文库的构建及分析

黑籽南瓜(Cucurb ita ficifolia)具有很强的抗逆能力,对低温、干旱、贫瘠土壤以及瓜类枯萎病等逆境均具有较强的抗性,这些优良的抗性基因很难通过有性杂交转移到其它瓜类作物上,但可通过可转化人工染色体(TAC)以大片段的形式转化到其它瓜类作物中.本研究利用TAC载体构建了黑籽南瓜基因组DNA文库,该文库包含57 560个克隆,保存在150块384孔板中,其库容量相当于黑籽南瓜基因组的7倍.TAC文库克隆插入片段大小分布范围为20～100 kb,超过40 kb插入片段克隆占40％以上,插入片段平均大小为45kb.在挑取的15个TAC克隆中无空克隆；细菌继代培养30、60和100代后质粒的酶切鉴定表明,TAC克隆可以在大肠杆菌(Escherichia coli) DH 10B中稳定复制和保存,说明本研究构建的文库质量较高.该研究结果表明,TAC文库为黑籽南瓜抗性基因的克隆、功能验证、物理图谱的构建和基因组测序等有关研究提供了基础资料.

The Construction and Analysis of Genomic Library Based on Transformation-competent Artificial Chromosome (TAC) Vector in Cucurbita ficifolia

Cucurbita ficifolia genome contains a large number of beneficial genes of great agronomic value,which confers multiple traits including strong stress tolerance for low temperature,drought,poor soil and fusarium wilt of Cucurbits.However,it is difficult to transform these beneficial genes into other cultivated cucurbit crops by the conventional breeding program,while the transformation by TAC vector library may serve as an alternative.To facilitate the mapping and cloning of these beneficial genes,a genomic transformation-competent artificial chromosome(TAC) library of Cucurbita ficifolia was constructed by using the vector of pYLTAC747NH/sacB in this study.The library consisted of 57 560 individual clones,which was stored in one hundred fifty 384-well plates with an average DNA inserted size of 45 kb ranging from 20~100 kb(more than 40% of the clones were above 40 kb);and the whole library covers 7× genome equivalents.All 15 selected clones carried inserted fragments,and the restriction digestion of the plasmids also indicated that TAC clone could be stably replicated and retained in Escherichia coli DH10B after sub-culturing 30,60 and 100 generations,thus demonstrating that high quality TAC library was created.To conclude,our study suggest that TAC library can provide valuable information for the isolation of genomic clones containing stress tolerance genes,functional validation,the construction of a physical map and the genome sequencing.