豌豆清蛋白1(PA1)基因的克隆及对苜蓿的转化

草业学报 ›› 2009, Vol. 18 ›› Issue (3): 117-125.

豌豆清蛋白1(PA1)基因的克隆及对苜蓿的转化

张改娜^1,2,贾敬芬^2*

1.河南科技大学农学院,河南洛阳 471000;
2.西北大学生命科学学院,陕西西安 710069

收稿日期:2009-01-14 出版日期:2009-06-20 发布日期:2009-06-20
作者简介:张改娜(1977-),女,河南许昌人,讲师,博士。E-mail: zgnluck1@163.com
基金资助:
国家自然科学基金项目(30671082)资助。

Cloning of PA1 gene from Pisum sativum and genetic transformation on Medicago sativa

ZHANG Gai-na^1,2, JIA Jing-fen²

1.College of Agriculture of Henan University of Science and Technology, Luoyang 471000, China;

2.School of Life Science, Northwest University, Xi’an 710069, China

Received:2009-01-14 Online:2009-06-20 Published:2009-06-20

摘要/Abstract

摘要： 本研究用PCR方法从豌豆(食荚大菜豌)克隆出富含硫氨基酸、同时具有抗虫作用的双功能的蛋白质基因-豌豆清蛋白1(PA1)基因,并构建了植物表达载体pCAMBIAl301-PA1。采用农杆菌介导法转化了紫花苜蓿,并对其转化体系进行了优化,得到了多个转基因胚性愈伤组织及其再生植株。PCR和Southern杂交检测表明,PA1基因和潮霉素抗性基因已被整合到了宿主细胞。SDS-PAGE分析表明该基因在再生植株中有一定表达。游离氨基酸分析表明,PA1基因的表达转基因苜蓿中蛋氨酸和半胱氨酸的含量从0.1%提高到0.4%。

Abstract: A gene, a sulphur-rich and insect-toxic seed albumin-PA1 gene, was cloned by PCR method from Pisum sativum (cultivar shijiadacaiwan). In order to characterize the function of this gene, a plant expression vector, pCAMBIA1301-PA1, was constructed. This vector was transformed into M. sativa via Agrobacterium-mediated method. The conditions for transformation were optimized. The several transgenic embryogenic calli and regenerated plantlets were obtained. PCR and Southern blotting identification confirmed that PA1 gene and hygromycin-B phosphotransferase-resistant gene has been integrated into the genome of M. sativa, SDS-PAGE analysis indicated that PA1 gene expressed in the transgenic lines. Free amino acid analysis showed that the levels of methionine and cysteine in tansgenic M. sativa. Plantlets improved from 0.1% to 0.4%.

中图分类号:

张改娜,贾敬芬. 豌豆清蛋白1(PA1)基因的克隆及对苜蓿的转化[J]. 草业学报, 2009, 18(3): 117-125.

ZHANG Gai-na, JIA Jing-fen. Cloning of PA1 gene from Pisum sativum and genetic transformation on Medicago sativa[J]. Acta Prataculturae Sinica, 2009, 18(3): 117-125.

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