作物杂志,2016, 第4期: 47–55 doi: 10.16035/j.issn.1001-7283.2016.04.008

• 遗传育种·种质资源·生物科技 • 上一篇    下一篇

玉米抗逆基因ZmqLTG3-1的克隆及功能分析

韩赞平1,2,陈彦惠2,郭书磊2,祖小峰2,王顺喜2,赵西拥2   

  1. 1 河南科技大学农学院,471003,河南洛阳
    2 河南农业大学农学院,450002,河南郑州
  • 收稿日期:2016-04-02 修回日期:2016-05-11 出版日期:2016-08-15 发布日期:2018-08-26
  • 作者简介:作者简介:韩赞平,博士,从事作物遗传育种研究
  • 基金资助:
    国家自然科学基金(U1504315);河南科技大学科研启动基金(1347800)

Cloning and Function Analysis of Resistance Gene ZmqLTG3-1 in Maize

Han Zanping1,2,Chen Yanhui2,Guo Shulei2,Zu Xiaofeng2,Wang Shunxi2,Zhao Xiyong2   

  1. 1 Agricultural College,Henan University of Science and Technology,Luoyang 471003,Henan,China
    2 Agronomy College of Henan Agricultural University,Zhengzhou 450002,Henan,China
  • Received:2016-04-02 Revised:2016-05-11 Online:2016-08-15 Published:2018-08-26

摘要:

以玉米自交系豫82为材料,同源克隆了与水稻qLTG3-1基因同源的玉米ZmqLTG3-1基因。序列分析发现,该基因cDNA序列全长606bp,开放阅读框441bp,编码包含147个氨基酸的蛋白。该蛋白含有AAI_LTSS超蛋白家族的LTP(含8个半胱氨酸)保守结构,分子量为14.16kDa,等电点是8.3,与高粱、水稻的相似度分别为92%和88%。荧光定量RT-PCR分析结果表明,ZmqLTG3-1在玉米胚中的表达量最高,其次是茎尖,在叶、根等器官中的表达量较低;随着胚萌发时间的延长,其表达量逐渐增加,在萌发24h时呈现最高丰度表达,之后表达丰度与萌发时间呈现规律性下降。亚细胞定位结果表明,该基因所编码的产物被定位在细胞膜上,是一跨膜蛋白。通过农杆菌介导的花序侵染法将过表达载体OE-ZmqLTG3-1和空载体pCAMBIA1304转入拟南芥,在诸如干旱、高温和盐等非生物胁迫下,与对照相比,T3转基因植株的成活率显著提高,说明ZmqLTG3-1基因对于抵抗非生物胁迫发挥了重要作用。

关键词: 玉米, ZmqLTG3-1, 克隆, 功能分析

Abstract:

By the method of orthologus gene cloning,the ZmqLTG3-1 gene in maize was successfully cloned, which was homologous to the qLTG3-1 gene in rice. The sequence analysis indicated that the full-length cDNA was composed of 606bp, including an open reading frame (ORF) of 441bp, which encoded 147 amino acids. The protein contained LTP conservative structure of the extra protein family AAI-LTSS, and protein molecular weight and isoelectric point were 14.16kDa and 8.3, respectively, with the similarity of amino acid sequence for ZmqLTG3-1 with sorghum and rice being 92% and 88%. The results of quantitative RT-PCR showed that the highest expression level was in the embryo, then in the stem tip, lower was in the leaf and root. The highest expression abundance at 24h and then regular down along with germination time. The result of subcell location revealed that the product of ZmqLTG3-1 gene encoding is positioned on the cell membrane and it was a trans-membrane protein. To carry the preliminary validation of its function,transgenic T3 generation, trans-empty vector and wild type Arabidopsis thaliana as materials were studied. Results showed that in different abiotic conditions, such as drought, high temperature and salt, compared with control group, the performance and the survival rate of transgenic plant are significantly higher. All these suggest that ZmqLTG3-1 gene plays an important role in resisting abiotic stress.

Key words: Maize (Zea mays L.), ZmqLTG3-1, Cloning, Function analysis

图1

RNA检测 M为DL2000,1-3为样品"

图2

利用18S引物检测反转录cDNA M为DL2000,1-3为样品"

图3

ZmqLTG3-1 cDNA的PCR扩增结果 M:DM2000 plus;1:豫82 Yu 82"

图4

ZmqLTG3-1基因编码的蛋白结构域"

图5

氨基酸序列相似性分析"

图6

ZmqLTG3-1与其他不同物种同源基因编码蛋白的系统发生"

图7

ZmqLTG3-1在不同部位中的表达情况"

图8

ZmqLTG3-1在胚的不同萌发时间的表达2.5 ZmqLTG3-1基因编码产物的亚细胞定位"

图9

pMD18-T-ZmqLTG3-1双酶切 M:DL2000,1:质粒对照Plasmid,2:双酶切Enzyme digestion"

图10

pCAMBIA1304-ZmqLTG3-1-GFP双酶切 M:DM2000 plus,1:质粒对照Plasmid,2:双酶切Enzyme digestion"

图11

OE-ZmqLTG3-1双酶切检测 M:DM2000 plus;1:质粒对照Plasmid;2:双酶切Enzyme digestion"

图12

OE-ZmqLTG3-1转化农杆菌GV3101的PCR检测 M:DL2000,1-10为单克隆,11为重组质粒"

图13

不同非生物胁迫处理下转基因拟南芥的表现 **表示差异极显著(P<0.01)"

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