紫花苜蓿MsZIP基因超表达载体的构建及转基因苜蓿检测

草业学报 ›› 2012, Vol. 21 ›› Issue (6): 182-189.

紫花苜蓿MsZIP基因超表达载体的构建及转基因苜蓿检测

李燕¹,孙彦²,杨青川^1*,康俊梅¹,张铁军¹,房锋³

1.中国农业科学院北京畜牧兽医研究所,北京100193;
2.中国农业大学动物科技学院,北京 100191;
3.中国农业科学院植物保护研究所,北京 100193

收稿日期:2011-12-05 出版日期:2012-06-25 发布日期:2012-12-20
通讯作者: E-mail:qchyang66@yahoo.com.cn
作者简介:李燕(1984-),女,蒙古族,内蒙古乌兰察布人,博士。E-mail:caasliyan@yahoo.com.cn
基金资助:
“十二五"国家科技支撑计划课题(2011BAD17B01-01-3)和中央级公益性科研院所专项资金项目(2011cj-14)资助。

Construction and transformation of an over-expression plasmid of the MsZIP gene from Medicago sativa

LI Yan¹, SUN Yan², YANG Qing-chuan¹, KANG Jun-mei¹, ZHANG Tie-jun¹, FANG Feng³

1.Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
2.College of Animal Science and Technology, China Agriculture University, Beijing 100191, China;
3.Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received:2011-12-05 Online:2012-06-25 Published:2012-12-20

摘要/Abstract

摘要： 根据已经克隆得到的MsZIP基因(GenBank序列号:HQ911778),扩增编码区cDNA,构建植物超表达载体PBI-MsZIP。酶切鉴定表明,目的基因已经正确的插入到载体中,超表达载体构建成功。采用CaCl₂冻融法将其转入农杆菌菌株中,然后采用农杆菌介导的方法,转化紫花苜蓿,共得到11株抗性苗,对其中的4株进行卡那霉素基因PCR检测,均得到了目的条带。同时对这4株抗性苗进行目的基因的RT-PCR检测,均得到了目的条带。说明MsZIP基因已经成功在苜蓿中超表达。为了进一步验证该基因的功能,分别用200 mmol/L NaCl和25 μmol/L PEG-6000处理转基因苜蓿,3 d后进行生理指标的测定。结果表明,MsZIP基因在苜蓿中超表达可以提高苜蓿的耐盐性和耐旱性。

Abstract: Based on the MsZIP gene sequence (GenBank No. HQ911778), a cDNA fragment was cloned and connected to the PMD18-T vector to construct PMD-MsZIP. The target fragment and linear plasmid were obtained from the cloning vector PMD-MsZIP and from the plant expression vector PBI121 using dual digestion with XbaI and BamHI. The plant over-expression vector PBI-MsZIP was built through directional connections using T₄ DNA ligase. The plasmid was transferred to Agrobacterium LBA4404 by the CaCl₂ freeze-thaw method and was then transferred into alfalfa by an Agrobacterium-mediated transformation system. Eleven kanamycin-resistant plants were obtained. Four of them were sampled to detect target fragments and PCR identification showed that the recombinant plasmid had been transferred into alfalfa. The MsZIP gene was successfully over-expressed in transgenic Medicago sativa CV. Zhongmu No.1. To test the function of this gene, the transgenic alfalfa was treated with 200 mmol/L NaCl and 25 μmol/L PEG-6000 for three days, and some physiological parameters were measured. The contents of soluble sugar, soluble protein and proline significantly increased, while the MDA content declined. Over-expressed MsZIP gene enhanced the salt and drought tolerance of alfalfa.

中图分类号:

李燕,孙彦,杨青川,康俊梅,张铁军,房锋. 紫花苜蓿MsZIP基因超表达载体的构建及转基因苜蓿检测[J]. 草业学报, 2012, 21(6): 182-189.

LI Yan, SUN Yan, YANG Qing-chuan, KANG Jun-mei, ZHANG Tie-jun, FANG Feng. Construction and transformation of an over-expression plasmid of the MsZIP gene from Medicago sativa[J]. Acta Prataculturae Sinica, 2012, 21(6): 182-189.

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