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2025年8月13日 星期三
  2015, Vol. 23 Issue (10): 1261-1272    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
水稻CDPK12基因启动子的表达模式及其逆境应答元件分析
张利娜1,刘瑜2,林拥军3
1. 华中农业大学生命科学技术学院
2. 华中农业大学 生命科学技术学院/作物遗传改良国家重点实验室及国家植物基因研究中心
3. 华中农业大学
Expression Pattern and Stress Response Elements Analysis of CDPK12 Gene Promoter in Rice (Oryza sativa ssp. japonica)
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摘要 水稻(Oryza sativa)是人类赖以生存的重要粮食作物之一,但是各种不利的环境条件严重影响水稻的生长发育,进而限制水稻的生产。深入研究植物启动子的结构、功能及作用模式,有助于进一步探索植物对其生长环境的适应机制以及创造优良的抗逆转基因水稻。V9703Q8E(calcium-dependent and calmodulin-independent protein kinases, CDPKs)是一类仅在植物和部分原生生物中存在的丝氨酸/苏氨酸蛋白激酶,在介导植物生长发育信号和逆境信号转导中具有重要作用。本研究主要对OsCDPK12的表达及其启动子区域顺式调控元件进行了分析。首先通过PCR技术从粳稻(Oryza sativa ssp. japonica)品种日本晴基因组上克隆到OsCDPK12上游2 109 bp的启动子区域,命名为12P,将其融合报告基因β-glucuronidase(GUS),转化粳稻品种中花11,组织化学染色结果显示主要在根茎过渡区、茎节、花药、种子中表达;之后对该启动子进行5'端缺失分析,构建8个融合报告基因GUS的缺失载体,对其转基因水稻植株的研究发现,除12P687外,各个缺失启动子均能驱动GUS基因在根茎过渡区、幼穗花药、枝梗、茎节、种子中表达,而在叶、叶鞘和根中不表达,12P687却能驱动GUS基因在各个组织中表达,表现为组成型表达的特征。对比分析不同缺失启动子转基因植株各组织的染色结果,可以看出,在-966~-687 bp区域,存在关键的负调控元件;-687~-472 bp区域,具有正向调控的顺式作用元件。GUS活性的定量测定结果显示,12P687启动子在叶和叶鞘组织中的活性约为35S启动子活性的1/2,而根中12P687启动子的活性约为35S启动子活性的1/3。此外,还对不同缺失启动子的转基因植株进行了低温、低氮和高盐胁迫处理,发现12P1828的转基因材料经冷胁迫后在叶片中表达上升,而12P687的转基因材料经高盐胁迫后在根中表达下降。本研究鉴定出12P驱动下游靶基因的表达特征及潜在的重要调控元件,并初步验证OsCDPK12基因的表达受低温和高盐调控,为今后抗逆转基因水稻的培育及基因功能研究工作提供参考。
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张利娜
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关键词 水稻逆境胁迫启动子表达模式逆境应答元件    
Abstract:As is known to all, rice (Oryza sativa) is one of the most important food crops to human beings, but a variety of adverse environmental stress severely affect the growth and development and limits the production of rice. Further study of the structure, function and expression pattern of plant promoter have great significance for further exploring the adaptation mechanism of plants to its growing environment and creating stress-resistance improved transgenic rice. Calcium-dependent and calmodulin-independent protein kinases (CDPKs) are a Ser/Thr protein kinase family which exist only in the plant and parts of protista, and play a very important role in mediating the signal transductions of growth and development, and the abiotic stresses. The research mainly analyzed the expression pattern and cis regulatory elements of OsCDPK12 gene promoter which named 12P. Firstly, 12P with 2 109 bp in length of upstream regions was cloned by PCR from the genomic DNA of rice Nipponbare (Oryza sativa ssp. japonica) and transformed into rice zhonghua11 (Oryza sativa ssp. japonica) after fused with the report gene β-glucuronidas (GUS). The histochemical staining results showed that GUS gene driven by the promoter of 12P mainly express in root-stem transition zone, stem node, anther and seed. Subsequently, eight different length of 12P 5' deletions were fused to GUS gene and transformed into rice zhonghua11, respectively. The result of deletion analysis indicated that GUS gene driven by the deletions of 12P1975, 12P1828, 12P1570, 12P1351, 12P1194, 12P966 and 12P472 expressed in root-stem transition zone, tender panicle, branch, stem node and mature seed, respectively, but not in leaf, sheath and root. 12P687 drived GUS gene expressed in all the tissues above, and showed a constitutive expression pattern. The histochemical staining results of different deleting promoters transgenic plants showed that there are several important negatively regulatory elements locate at the fragments from -966 bp to -687 bp, several positive regulatory elements locate at the fragments from -687 bp to -472 bp. Quantitative analysis of GUS activity showed that GUS activity of the 12P687 promoter in the tissue of leaf and leaf sheath was about half of the 35S promoter, and 12P687 GUS activity in the root was about 1/3 of the 35S promoter. Furthermore, the different deleting promoters transgenic plants undertook low temperature, low nitrogen and high salt stress treatment, respectively, the result showed that GUS gene in12P1828 transgenic rice plants was up-regulated expressed in the leaf after low temperature treatment, while, in 12P687 transgenic rice plants it's down-regulated in the root after salt stress treatment. These results identified the expression characteristics of 12P which can drive downstream target genes expressed and some potentially important regulatory element that existing, and also verified the expression of OsCDPK12 gene is regulated by low temperature and high salt stress preliminarily, so it will be conducive to provide reference for further work of cultivating stress resistant transgenic rice and researching the function of gene.
Key wordsRice    Abiotic stress    Promoter    Expression pattern    Stress-responsive element
收稿日期: 2015-04-21      出版日期: 2015-07-24
基金资助:抗虫转基因水稻新品种培育
通讯作者: 林拥军     E-mail: yongjunlin@mail.hzau.edu.cn
引用本文:   
张利娜 刘瑜 林拥军. 水稻CDPK12基因启动子的表达模式及其逆境应答元件分析[J]. , 2015, 23(10): 1261-1272.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2015/V23/I10/1261
 
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