大豆GmbZIP16的抗旱功能验证及分析

doi:10.3864/j.issn.0578-1752.2018.15.001

中国农业科学 ›› 2018, Vol. 51 ›› Issue (15): 2835-2845.doi: 10.3864/j.issn.0578-1752.2018.15.001

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

大豆GmbZIP16的抗旱功能验证及分析

赵婉莹^1,2，于太飞²，杨军峰³，刘沛²，陈隽²，陈明²，周永斌²，马有志²，徐兆师²，闵东红¹

¹西北农林科技大学/旱区作物逆境生物学国家重点实验室，陕西杨凌712100；²中国农业科学院作物科学研究所/农作物基因资源与基因改良国家重大科学工程/农业部麦类生物学与遗传育种重点实验室，北京100081；³河北旺丰种业有限公司，河北邢台054900

收稿日期:2018-04-01 修回日期:2018-05-14 出版日期:2018-08-01 发布日期:2018-08-01
通讯作者: 闵东红，E-mail：mdh2493@126.com。徐兆师，E-mail：xuzhaoshi@caas.cn
作者简介:赵婉莹，E-mail：zhaowanying199406@126.com
基金资助:
国家转基因生物新品种培育科技重大专项（2018ZX08009100，2016ZX08002-002）、西北农林科技大学2017年唐仲英育种基金

Verification and Analyses of Soybean GmbZIP16 Gene Resistance to Drought

ZHAO WanYing^1,2, YU TaiFei², YANG JunFeng³, LIU Pei², CHEN Jun², CHEN Ming², ZHOU YongBin², MA YouZhi², XU ZhaoShi², MIN DongHong¹

¹Northwest A & F University/State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling 712100, Shaanxi; ² Institute of Crop Science, Chinese Academy of Agricultural Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement/ Key Laboratory of Biology and Genetic Improvement of Triticeae Crop, Ministry of Agriculture, Beijing 100081; ³ Hebei Wangfeng Seed Industry Co., Ltd. Xingtai 054900, Hebei

Received:2018-04-01 Revised:2018-05-14 Online:2018-08-01 Published:2018-08-01

摘要/Abstract

摘要： 【目的】通过分析干旱条件下大豆的转录组数据，筛选获得大豆锌指蛋白GmbZIP16，对其进行功能验证，确定GmbZIP16参与大豆抵抗干旱的分子机理。【方法】大豆干旱转录组数据分析得到上调倍数较高的锌指蛋白GmbZIP16，以大豆cDNA为模板克隆获得GmbZIP16。并通过In-Fusion连接酶技术，构建pCAMBIA1302- GmbZIP16和pCAMBIA3301-GmbZIP16表达载体。通过液氮冷冻法将重组载体pCAMBIA1302- GmbZIP16和pCAMBIA3301-GmbZIP16分别转入农杆菌GV3101和大豆发根农杆菌K599的感受态细胞中，通过农杆菌侵染拟南芥花序以及大豆子叶节技术，产生过表达拟南芥植株以及过表达大豆毛状根复合体植株。通过半定量RT-PCR和qRT-PCR分析，确定GmbZIP16在转基因拟南芥和大豆毛状根中能够超表达。分别将正常条件下生长2周龄的转基因和野生型拟南芥植株转移至含有不同PEG浓度（6% PEG和8% PEG）的MS0培养基上继续培养7 d，观察转基因拟南芥和对照野生型拟南芥之间的生物量差异；利用qRT-PCR分析转基因拟南芥和野生型拟南芥植物体中胁迫相关的基因表达情况。将生长良好的转GmbZIP16大豆毛状根复合体施加25% PEG处理1周后，分别采取转GmbZIP16大豆毛状根复合体和转空载体大豆毛状根复合体的叶片，用酶标仪测定植株的脯氨酸、丙二醛和叶绿素的含量。【结果】通过PCR技术扩增得到正确的GmbZIP16序列，通过农杆菌转化技术得到2个稳定过表达的转GmbZIP16拟南芥株系。通过对转基因拟南芥的表型鉴定发现转基因拟南芥在干旱处理下的生物量（鲜重和根长）及存活率比野生型显著提高。在过表达GmbZIP16拟南芥植株中，一些与胁迫相关的基因的表达要高于在野生型，如RD29B、DREB2A和P5CS。转GmbZIP16大豆毛状根复合体植株在25% PEG处理1周后，大豆毛状根复合体叶片中叶绿素和脯氨酸的含量要显著高于转空载体大豆毛状根复合体叶片中叶绿素和脯氨酸的含量，而转GmbZIP16大豆毛状根复合体叶片中丙二醛的含量显著低于转空载体大豆毛状根复合体叶片中丙二醛的含量。【结论】在拟南芥中过表达大豆GmbZIP16提高了转基因拟南芥的抗旱性。过表达GmbZIP16可以提高转基因大豆毛状根复合体对干旱的抗性。GmbZIP16提高植物的抗旱性主要是通过影响与抗逆相关基因的表达来实现的。

关键词: 大豆, GmbZIP16, 抗旱性, 实时荧光定量PCR

Abstract: 【Objective】Soybean GmbZIP16 protein was screened by analyzing soybean drought transcriptome. Functions of soybean GmbZIP16 were verified by analyzing the phenotypic characterization of transgenic GmbZIP16 Arabidopsis and soybean hairy root complexes. On the basis of experimental result analyses above-mentioned, we could determine that GmbZIP16 was involved in the drought resistance process.【Method】soybean GmbZIP16 was found out by analyzing soybean drought transcriptome and cloned by PCR, which soybean cDNA as a template, and then ligated the fragment to pCAMBIA1302 and pCAMBIA3301 expression vectors by in-fusion ligase. The recombinant pCAMBIA1302-GmbZIP16 and pCAMBIA3301-GmbZIP16 vectors were transferred into Agrobacterium GV3101 and K599 competent cells by liquid nitrogen freeze-melt method, respectively. The transgenic Arabidopsis plants and transgenic soybean hairy root complexes were generated by Agrobacterium-mediated transformation method. The transgenic GmbZIP16 Arabidopsis plants were identified by semi quantitative PCR and quantitative real time PCR. Which demonstrated GmbZIP16 could over express in transgenic Arabidopsis and transgenic soybean hairy root complex plants. 2 weeks old transgenic Arabidopsis and WT plants grew under normal conditions were transferred into MS0 solid medium supplement with 6% and 8% PEG for 7d. Biomass differences between transgenic Arabidopsis and WT plants were investigated and analyzed. The different expression of stress- related genes between Arabidopsis and WT plants were analyzed by qRT-PCR. The transgenic GmbZIP16 soybean hairy root complexes and control group plants were treated with 25% PEG for 7 days, and then the leaf samples of transgenic GmbZIP16 soybean hairy root complex and control group plants were taken. The proline content, MDA content and chlorophyll content of leaf samples were measured by Multiskan Spectrum Microplate Spectrophotometer.【Result】The GmbZIP16 gene was isolated by PCR technology. The two transgenic GmbZIP16 Arabidopsis lines were obtained by Agrobacterium-mediated transformation method. Compared with the WT plants, the transgenic GmbZIP16 Arabidopsis lines had higher biomass (the fresh weight and the root length) and survival rate under drought stress by phenotypic characterization experiment. The expression levels of some relative genes such as RD29B, DREB2A and P5CS were improved in transgenic GmbZIP16 Arabidopsis, compared with the WT plants. The leaves of transgenic GmbZIP16 soybean hairy root complex plants had the higher proline and chlorophyll content and a lower MDA content than that of the control group plants under the deal with 25% PEG. 【Conclusion】The drought resistance of transgenic Arabidopsis thaliana was improved by expressing soybean GmbZIP16 in Arabidopsis thaliana. Over expression of GmbZIP16 could enhance the drought resistance of transgenic soybean hairy root complex. GmbZIP16 can improve the drought resistance of plants mainly by affecting the expression of genes related to stress tolerance.

Key words: soybean, GmbZIP16, drought resistance, qRT-PCR

赵婉莹，于太飞，杨军峰，刘沛，陈隽，陈明，周永斌，马有志，徐兆师，闵东红. 大豆GmbZIP16的抗旱功能验证及分析[J]. 中国农业科学, 2018, 51(15): 2835-2845.

ZHAO WanYing, YU TaiFei, YANG JunFeng, LIU Pei, CHEN Jun, CHEN Ming, ZHOU YongBin, MA YouZhi, XU ZhaoShi, MIN DongHong. Verification and Analyses of Soybean GmbZIP16 Gene Resistance to Drought[J]. Scientia Agricultura Sinica, 2018, 51(15): 2835-2845.

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大豆GmbZIP16的抗旱功能验证及分析

Verification and Analyses of Soybean GmbZIP16 Gene Resistance to Drought

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