大白菜雄性不育花蕾均一化cDNA文库的构建及评价

Construction and evaluation of flower bud normalized cDNA library of male sterile of Chinese cabbage (Brassica rapa L. pekinensis)

以大白菜pol型细胞质雄性不育系16C14花蕾为试验材料,采用SMART(switching mechanism at 5′end of RNA transcript)技术合成cDNA,利用DSN(duplex-specific nuclease)对所得cDNA进行均一化处理,之后与pDONR^(TM)/Zeo载体重组并转化DH10B大肠杆菌菌株,构建了大白菜pol型雄性不育花蕾均一化cDNA文库。经检测,文库滴度为3.2×10~6 cfu?mL^(-1),库容量约为1.28×10~7 cfu,重组率97%,插入片段平均大小1.2 kb左右。随机挑选100个阳性菌斑进行测序,冗余度仅为3.7%,文库质量较高。本研究成功构建了大白菜胞质雄性不育系花蕾均一化cDNA文库,为进一步研究大白菜pol雄性不育相关分子机制奠定基础。

To obtain normalized cDNA library of Chinese cabbage buds with pol cytoplasmic male sterile, the library was constructed by DSN（duplex-specific nuclease） normalization method in combination with SMART（switching mechanism at 5＇ end of RNA transcript） technology. Then the purified cDNA was recombined with p DONRTM/Zeo vector and transformed into E. coli DH10 B. The titer of the library was about 3.2×10~6 cfu？mL^（-1）, the total CFU was about 1.28×10~7 cfu, and the average size of the inserted fragments was about 1.2 kb with a recombinant about 97%. The redundancy of the library which tested by 100 randomly sequenced clones was only 3.7%. These results indicated that this normalized c DNA library had been successfully established with high quality, which laid the foundation for further research on the molecular mechanism of pol male sterility of Chinese cabbage.