CryⅢA基因植物表达载体构建及马铃薯遗传转化

doi:10.11686/cyxb20140130

草业学报 ›› 2014, Vol. 23 ›› Issue (1): 248-256.DOI: 10.11686/cyxb20140130

CryⅢA基因植物表达载体构建及马铃薯遗传转化

尤佳^1，2，张宁²，文义凯²，吴家和³，司怀军^1，2*，王蒂^1*

1.甘肃省干旱生境作物学省部共建国家重点实验室培育基地甘肃省作物遗传改良与种质创新重点实验室,甘肃兰州730070
2.甘肃农业大学生命科学技术学院,甘肃兰州730070
3.中国科学院微生物研究所植物基因组学国家重点实验室,北京100101

收稿日期:2013-01-07 出版日期:2014-02-20 发布日期:2014-02-20
通讯作者: E-mail:hjsi@gsau.edu.cn,wangd@gsau.edu.cn
作者简介:尤佳(1988-),女,甘肃兰州人,硕士。E-mail:abbyyj@126.com
基金资助:
甘肃省农业科技创新项目(GNCX-2011-49)和国家公益性行业(农业)科研专项(201103026-2)资助

Construction of a plant expression vector of the CryⅢA gene and its genetic transformation in potato

YOU Jia^1,2,ZHANG Ning²,WEN Yi-kai²,WU Jia-he³,SI Huai-jun^1,2,WANG Di¹

1.Gansu Provincial Key Laboratory of Aridland Crop Science,Gansu Key Laboratory of Crop Genetic &Germplasm Enhancement,Lanzhou 730070,China;
2.College of Life Science and Technology,Gansu Agricultural University,Lanzhou 730070,China;
3.State Key Laboratory of Plant Genomic,Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101,China

Received:2013-01-07 Online:2014-02-20 Published:2014-02-20

摘要/Abstract

摘要： 利用In-Fusion技术将酶切位点不匹配、目的基因内部含载体酶切位点的CryⅢA基因和植物表达载体pBI121相连接,分别成功构建了组成型启动子CaMV 35S和马铃薯茎叶特异表达启动子ST-LS1驱动的CryⅢA基因植物表达载体。利用冻融法将2个重组质粒转入根癌农杆菌LBA4404,转化马铃薯栽培品种陇薯3号和甘农薯2号获得了转化植株,经卡那霉素筛选和PCR检测,共有10株阳性植株CryⅢA基因成功整合到马铃薯基因组中。经实时荧光定量PCR检测表明,CryⅢA基因在CaMV 35S驱动的CryⅢA转基因植株的根、茎、叶中均有表达,且在叶中表达量较高,茎和根次之;在马铃薯茎叶特异表达启动子ST-LS1驱动的CryⅢA转基因植株中,在根中未见表达,仅在茎、叶中表达,且在叶中表达量较高。

Abstract: Using In-Fusion technology,plant expression vectors were constructed from the vector pBI121 for transformation of the CryⅢA gene under the control of the constitutive promoter CaMV 35S or the potato leaf and stem-specific promoter ST-LS1. The recombinant plasmids were introduced into Agrobacterium tumefaciens strain LBA4404 by the freeze-thaw method. The putative transgenic plants of potato cultivars Longshu No.3 and Gannongshu No.2 were obtained by the Agrobacterium-mediated transformation system. Results from selection of the transformed plants on culture media containing kanamycin and PCR assay showed that the CryⅢA gene was integrated into the genome of 10 potato plants. Real-time fluorescence quantitative PCR (qRT-PCR) analysis indicated that the CryⅢA gene driven by the promoter CaMV 35S,was expressed in roots,stems and leaves,with a high expression in leaves of the transgenic plants. The CryⅢA gene driven by the promoter ST-LS1 was expressed only in stems and leaves with the highest expression in leaves of the transgenic potato plants. However,no traces of it were found in roots of the transgenic potato plants.

中图分类号:

S435.32

尤佳，张宁，文义凯，吴家和，司怀军，王蒂. CryⅢA基因植物表达载体构建及马铃薯遗传转化[J]. 草业学报, 2014, 23(1): 248-256.

YOU Jia,ZHANG Ning,WEN Yi-kai,WU Jia-he,SI Huai-jun,WANG Di. Construction of a plant expression vector of the CryⅢA gene and its genetic transformation in potato[J]. Acta Prataculturae Sinica, 2014, 23(1): 248-256.

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CryⅢA基因植物表达载体构建及马铃薯遗传转化

Construction of a plant expression vector of the CryⅢA gene and its genetic transformation in potato

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