《植物生理学报》 2018, 54(2): 291-296

大白菜雄性不育花蕾均一化cDNA文库的构建及评价

李真真, 武佩琪, 程亚雄, 宋红霞, 李梅兰, 侯雷平^*, 许小勇^*

山西农业大学园艺学院, 山西太谷030801

通信作者：侯雷平；E-mail: sxndhlp@126.com; xuxy7926@163.com

摘　要：

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3.2×10⁶ cfu∙mL^-1, 库容量约为1.28×10⁷ 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。

关键词：大白菜; 细胞质雄性不育; pol型; cDNA文库; 均一化

收稿：2017-11-02   修定：2018-01-10

资助：国家自然科学基金(31401885)和山西省自然科学基金(201701D121101)。

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

LI Zhen-Zhen, WU Pei-Qi, CHENG Ya-Xiong, SONG Hong-Xia, LI Mei-Lan, HOU Lei-Ping^*, XU Xiao-Yong^*

College of Horticulture, Shanxi Agricultural University, Taigu, Shanxi 030801, China

Corresponding author: HOU Lei-Ping; E-mail: sxndhlp@126.com; xuxy7926@163.com

Abstract:

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 pDONR^TM/Zeo vector and transformed into E. coli DH10B. The titer of the library was about 3.2×10⁶ cfu∙mL^-1, the total CFU was about 1.28×10⁷ 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 cDNA 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.

Key words: Chinese cabbage; cytoplasmic male sterility; pol type; cDNA library; normalization