摘要
为克隆筛选棉花腺体形成相关的基因,运用SMART技术构建cDNA文库.首先抽提棉花腺体形成时期的mRNA,mRNA逆转录后合成cDNA,经均一化处理后,SfiⅠ酶切,连接质粒载体,电转化,成功构建了棉花腺体形成时期的cDNA文库.经鉴定原始文库滴度为5.8×105cfu/mL,其重组率高达94%,插入片段的平均长度约为1.4 kb.
A Normalized cDNA library was constructed from upland cotton during its pigments gland forming age with SMART（switching mechanism at 5 end of RNA transcript） technique using Creator SMART cDNA library construction kit. mRNAs were isolated from upland cotton using Reagent; Double-stranded cDNAs were synthesized from mRNAs and were processed by normalization and Sfi I and then the cDNAs were ligated to pDNR-LIB vector. The ligation mixture was transformed into E. coli JM109 by electroporation. Counting the number of colonies, the titer was 5.86 × 10^5 cfu/mL in this library. Electrophoresis gel results fragments ranged from 800 bp to 2 kb, with an average size of 1400 bp. Random picking clones showed that the recombination rate was 94 %. Our successfully constructed cDNA library is a full-length library with high quality, and can be used to screen the genes related to development of pigments gland cottons.
出处
《四川大学学报（自然科学版）》
CAS
CSCD
北大核心
2007年第6期1321-1325,共5页
Journal of Sichuan University(Natural Science Edition)
基金
国家自然科学基金(3044003230771311)
重庆市自然基金(cstc2007BB1328)
