强优势玉米杂交种雌穗均一化cDNA文库的构建与鉴定

以强优势玉米杂交种豫玉22发育时期的雌穗为材料构建cDNA文库,为进一步研究和分离杂种优势相关基因构建技术平台。选择玉米雌穗发育过程中的4个时期(生长锥伸长、小穗分化、小花分化和花丝始伸期)的雌穗组织分别构建了4个定向cDNA文库,依据基因组DNA饱和杂交的原理,将从4个独立的cDNA文库中提取的质粒混合,通过基因组DNA亲和体系饱和杂交,获得雌穗全发育时期均一化cDNA文库。该文库平均插入片段1.3kb,含有3×104个克隆。对文库中随机的150个克隆进行测序,81.6%的序列为非冗余序列,11.2%的序列含有两个或两个以上的拷贝,并且主要来自多基因家族转录本,说明此文库的均一化有效。对测序结果进行功能聚类分析,发现文库序列涉及水解酶、转移酶、核苷酸结合蛋白、蛋白激酶、转录因子等基因,涉及环境应答、蛋白代谢、物质转运、个体发育等多个生化途径,说明该均一化文库基因覆盖度较高。该文库还包含有大量的未知基因,有待进一步的发掘和研究。

Construction and Identification of a Normalized Maize Ear cDNA Library of Hybrid Yuyu22

In this study the total RNAs of maize hybrid Yuyu No.22 ears that are during the important period-ear development period were isolated using Trizol Reagent, and then we synthesized the cDNA strands, and constructed the normalized cDNA library which is the basis for the study of genes about heterosis at last. 4 separated cDNA libraries were constructed and then normalized by saturated hybridization with two complemented genomic DNA from maize. After retransformation to E.coli, we got a normalized cDNA library with 3×10⁴ clones and had an average insert length of 1.3 kb. Random selected 150 clones were sequenced and 81.6% of clones were unique ESTs, 11.2% of clones had repetitive frequency ranging from 2 - 3. These results suggested that the cDNA library was well normalized.