Rd29A启动子驱动<em>AtCDPK1</em>基因转化马铃薯的研究

doi:10.13523/j.cb.20151103

中国生物工程杂志

2015, Vol. 35

Issue (11): 13-22 DOI: 10.13523/j.cb.20151103

研究报告

Rd29A启动子驱动AtCDPK1基因转化马铃薯的研究

聂利珍¹, 于肖夏¹, 李国婧¹, 孙杰², 姜超¹, 于卓¹

1. 内蒙古农业大学农学院呼和浩特 010019;
2. 内蒙古农牧业科学院呼和浩特 010031

Study on Transgenic Potato Contained AtCDPK1 Gene Drived by Rd29A Promoter

NIE Li-zhen¹, YU Xiao-xia¹, LI Guo-jing¹, SUN Jie², JIANG Chao¹, YU Zhuo¹

1. Agronomy College, Inner Mongolia Agricultural University, Huhhot 010019, China;
2. Inner Mongolia Academy of Agricultural AnimalHusbandry Sciences, Hohhot 010031, China

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摘要：

为获得抗旱性强、生长正常的转基因马铃薯植株,以野生拟南芥生态型(Col-0)为材料,利用PCR和DNA重组技术,克隆了拟南芥 Rd29A (responsive to dehydration)基因ATG上游+83bp至-1 441bp共1 524bp的启动子区域,其DNA序列与已知拟南芥Rd29A 5'端启动子序列同源性为100%;构建了Rd29A启动子驱动 AtCDPK1 基因表达的植物表达载体pCHFRd-CDPK1。以马铃薯品种‘费乌瑞它’的试管微型薯为材料,利用农杆菌介导法,将构建成功的pCHFRd-CDPK1载体转入马铃薯中,经筛选与植株再生,获得抗性再生植株。通过PCR和Southern blot检测显示,Rd29A启动子驱动的 AtCDPK1 基因已整合在马铃薯的基因组中。利用PEG模拟干旱胁迫后,经RT-PCR分析证实,当用20% 的PEG 胁迫转基因马铃薯植株时,Rd29A启动子驱动 AtCDPK1 基因表达的转基因马铃薯各个株系中 AtCDPK1 基因表达量明显增强,而在无胁迫的条件下,植株中 AtCDPK1 基因基本不表达;同时发现35S 控制 AtCDPK1 转基因植株在PEG 胁迫前后,基因转录未见明显差异。形态学观察还表明,在30% PEG胁迫下,转基因植株能正常生长,其长势优于未转基因的对照,且对照植株略有萎焉。该结果可为进一步利用逆境诱导型启动子驱动抗逆基因在农作物中的表达研究及其遗传改良提供依据。

关键词： AtCDPK1基因; 转基因; 干旱胁迫; 马铃薯; 拟南芥Rd29A启动子

Abstract:

In order to obtain normal growth and resistant drought transgenic potato, using Arabidopsis thaliana (Columbia ecotype) as material, DNA sequence of AtRd29A gene ATG upstream from +83bp to -1 441bp is cloned with PCR and recombinant DNA technologies. The sequence of bases was entirely consistent with that of promoter of the known gene. The plant expression vector pCHFRd-CDPK1 of AtCDPK1 gene drived by Rd29A promoter was constructed, and transformed pCHFRd-CDPK1 into virus-free miniature of potato cultivar‘Favorita’using Agrobacterium-mediated method. After plant selection and regeneration, regenerated plants with resistance were obtained successfully. The PCR and Southern blotting analysis proved that the AtCDPK1 gene has been integrated in the genome of the potato. After drought stress using 30% PEG, RT-PCR analysis proved that the transcript levels of AtCDPK1 gene drived by Rd29A promoter in transgenic potato plants were significantly higher. AtCDPK1 gene drived by Rd29A promoter in transgenic potato plants is barely detectable in the water treatment control. However, expression of AtCDPK1 gene drived by 35S promoter did not make significant difference in transgenic potato plants between PEG stress treatment and water treatment. Morphological observation showed that transgenic plants can grow normally after 30% PEG stress, the plants growing was better than non-transgenic plants, and control plants occur slightly wilt. This research will lay the foundation for further improved stress tolerance of crops by expression of resistant gene drived by stress-inducible promoter in the crop.

Key words: AtRd29A promoter Drought stress Potato Transgenic gene AtCDPK1 gene

收稿日期: 2015-06-11 出版日期: 2015-11-24

ZTFLH:

Q785

基金资助:

国家科技支撑计划(2012BAD02B05),内蒙古自然科学基金重大项目(2013ZD03),内蒙古农牧业科技创新项目(2011-CXJJM01-4)资助项目

通讯作者: 于卓 E-mail: yuzhuo58@sina.com

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引用本文:

聂利珍, 于肖夏, 李国婧, 孙杰, 姜超, 于卓. Rd29A启动子驱动AtCDPK1基因转化马铃薯的研究[J]. 中国生物工程杂志, 2015, 35(11): 13-22.

NIE Li-zhen, YU Xiao-xia, LI Guo-jing, SUN Jie, JIANG Chao, YU Zhuo. Study on Transgenic Potato Contained AtCDPK1 Gene Drived by Rd29A Promoter. China Biotechnology, 2015, 35(11): 13-22.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20151103 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I11/13

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