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作物学报 ›› 2009, Vol. 35 ›› Issue (7): 1253-1360.doi: 10.3724/SP.J.1006.2009.01253

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

叶绿体型转昆虫抗冻蛋白基因烟草的耐寒性

王艳,马纪*,黄薇,邱立明,叶锋,张富春   

  1. 新疆大学生命科学与技术学院/新疆生物资源基因工程重点实验室，新疆乌鲁木齐830046
  • 收稿日期:2008-09-05 修回日期:2009-02-18 出版日期:2009-07-12 网络出版日期:2009-05-19
  • 通讯作者: 马纪, E-mail: majiuci@xju.edu.cn; Tel: 0991-8583259
  • 作者简介:bluelovewy@126.com
  • 基金资助:

    本研究由国家自然科学基金项目（30760056）和教育部2008“促进与美大地区科研合作与高层次人才培养项目”资助。

Codl Tolerance of Transplastomic Tobacco Lines Carrying Insect Antifreeze Protein

WANG Yan,MA Ji*,HUANG Wei,QIU Li-Ming,YE Feng,ZHANG Fu-Chun   

  1. Xinjiang Key Laboratory of Biological Resources and Genetic Engineering,College of Life Science and Technoogy,Xinjiang University,Urumqi 830046,China
  • Received:2008-09-05 Revised:2009-02-18 Published:2009-07-12 Published online:2009-05-19
  • Contact: MA Ji, E-mail: majiuci@xju.edu.cn; Tel: 0991-8583259
  • About author:bluelovewy@126.com

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被引次数

2

摘要：

根据已构建的大豆叶绿体表达载体pJY01，设计特异性引物，将昆虫抗冻蛋白基因MpAFP149插入此载体中构成叶绿体表达载体pJY01-MpAFP149，利用基因枪轰击法转化烟草，经壮观霉素筛选获得4株叶绿体型转抗冻蛋白基因烟草株系。PCR和PCR-Southern结果显示外源基因已整合至烟草叶绿体基因组中但同质化水平不高，RT-PCR结果也表明昆虫抗冻蛋白基因已发生了转录。将野生型烟草、叶绿体型转抗冻蛋白基因烟草及核转化T1代转抗冻蛋白基因烟草(pCAMBIA1302- MpAFP149)于–1℃低温处理3 d，观察耐寒表型及测定相对电导率。结果表明, 叶绿体型转基因烟草的耐寒表型优于野生型烟草，但与核转化的T1代转抗冻蛋白基因烟草无显著差异。处理3 d时，叶绿体型转基因烟草和T1代转抗冻蛋白基因烟草的电导率分别为39.2%和38.2%，而野生型烟草已达73.7%。本实验获得的异质化转叶绿体抗冻蛋白基因烟草与转核基因烟草的耐寒力无差异。

关键词: 抗冻蛋白基因MpAFP149, 叶绿体转化载体, 烟草, 耐寒性

Abstract:

The antifreeze protein gene MpAFP149 from desert insect Microdera punctipennis dzungarica was inserted into soybean chloroplast vector pJY01 to construct recombinant chloroplast vector pJY01-MpAFP149 by designing special primers. The plasmid was then transformed into tobacco by gene gun. Four transplastomic tobacco lines were obtained by spectinomycin screening. PCR and PCR-Southern analysis showed that the MpAFP149 gene was successfully integrated into the tobacco chloroplast genome, but the transgenicplants exhibited low homoplasmy. The result of RT-PCR also validated that MpAFP149 gene was transcribed at mRNA level. The antifreeze effect of transplastomic tobacco with insect antifreeze protein gene in its chloroplasts was evaluated by measuring the relative conductivity and comparing the phenotypes of different plants after cold treatment. Wild-type tobacco, transplastomic tobacco and T1 generation of transgenic tobacco containing pCAMBIA1302-MpAFP149 in its nuclei were subjected to –1℃ for different days (0, 1, 2, and 3 d). The results showed that transgenic plants with insect antifreeze protein gene in chloroplasts or in nuclei performed better phenotype after cold treatment at –1℃ for three days and recovering at room temperature for five days than wild-type tobacco. After three days, the electrolyte leakage reached 73.7% for wild-type tobacco, 39.2% for chloroplast transgenic plants and 38.2% for nuclei transformed T1 generation tobacco. There was no cold tolerance difference between nuclei transgenic tobacco and heterogeneous tansplastomic tobacco in our research.

Key words: Antifreeze protein gene MpAFP149, Chloroplast transformed vector, Tobacco, Cold tolerance


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