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[1]刘扬 方扬 靳艳玲 杜安平 杨贵利 郭铃 谭力 何开泽 赵海**.绿萍LmPDS基因RNAi载体的构建及遗传转化[J].应用与环境生物学报,2020,26(03):1-12.[doi:10.19675/j.cnki.1006-687x.2019.04004]
 LIU Yang,FANG Yang,JIN Yanling,et al.Construct and Transformation of LmPDS Gene RNAi Vector in Lemna minor[J].Chinese Journal of Applied & Environmental Biology,2020,26(03):1-12.[doi:10.19675/j.cnki.1006-687x.2019.04004]
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绿萍LmPDS基因RNAi载体的构建及遗传转化()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
26卷
期数:
2020年03期
页码:
1-12
栏目:
研究论文
出版日期:
2020-06-25

文章信息/Info

Title:
Construct and Transformation of LmPDS Gene RNAi Vector in Lemna minor
作者:
刘扬123 方扬123 靳艳玲13 杜安平13 杨贵利123 郭铃1 谭力13 何开泽13 赵海13**
1中国科学院成都生物研究所 成都 610041
2中国科学院大学 北京 100049
3中国科学院环境与应用微生物重点实验室 成都 610041
Author(s):
LIU Yang123 FANG Yang123 JIN Yanling13 DU Anping13 YANG Guili123 GUO Ling1 TAN Li1 3 HE Kaize1 3 & ZHAO Hai 1 3**
1Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
2University of Chinese Academy of Sciences, Beijing 100049, China
3Key Laboratory of Environment and Applied Microbiology, Chinese Academy of Sciences, Chengdu 610041, China
关键词:
Lemna minorLmPDS基因RNAi干扰遗传转化
Keywords:
Lemna minor LmPDS gene RNA interference genetic transformation
DOI:
10.19675/j.cnki.1006-687x.2019.04004
摘要:
PDS基因编码控制类胡萝卜素生物合成过程中的关键酶,其被沉默或抑制性表达会使叶片组织出现斑驳、黄化、白化的现象。构建绿萍(Lemna minor)LmPDS基因RNAi载体及完成遗传转化研究,对于实现RNAi技术在绿萍中的应用是具有重要意义的。本研究选取595bp LmPDS 基因片段为干涉片段,利用中间载体pUCCRNAi将克隆的LmPDS干涉片段正反向插入植物表达载体pCAMBIA2300-35S-OCST中。将成功构建的RNAi载体通过农杆菌介导法转入绿萍愈伤,经愈伤抗性筛选、植株再生、植株扩培、标记基因NPTII检测等过程,获得植株70株,其中4株为转基因阳性植株。再经荧光定量PCR分析、番茄红素含量检测、表型观察转基因植株,结果表明:转基因阳性植株LmPDS相对表达量明显下降,为对照的28.8%-40.5%,其番茄红素含量也明显下降,且叶片出现了明显的白化表型。综上表明,已成功构建绿萍LmPDS基因RNAi载体,实现了RNAi在绿萍中的应用,可以为进一步的绿萍基因功能研究提供材料和方法。图11 表1 参30
Abstract:
Phytoene desaturase ( PDS) gene encodes the key enzyme of carotenoid biosynthesis. The silenced or inhibited expression of PDS gene will lead to the appearance of mottle, chlorosis or albino at leaves. It is of great significance to use LmPDS gene to construct and transform of LmPDS Gene RNAi Vector for realizing the application of RNA interference in Lemna minor . In this study, 595bp LmPDS gene fragment was selected as interference fragment, and the cloned LmPDS interference fragment was inserted into plant expression vector pCAMBIA2300-35S-OCST by usi ng intermediate vector pUCCRNAi. The constructed RNAi vector was transformed into Lemna minor callus by Agrobacterium-mediated transformation, then through the callus resistance screen, plant regeneration, plant culture and marker genes NPTII detection processes, 70 plants were gotten, included 4 of them were transgenic positive plants . After the fluorescence quantitative PCR analysis , lycopene content detection and phenotype observation, the results showed that the relative expression of LmPDS in the transgenic positive plants decreased significantly, compared with 28.8% - 40.5% of the control plants, t he lycopene content in the transgenic positive plants also decreased significantly, and the transgenic positive plants showed obvious albino phenotype in their leaves . In conclusion, we have successfully constructed the LmPDS gene RNAi vector in Lemna minor , and the realized application of RNAi can provide materials and methods for further studies in gene function analysis of Lemna minor.

备注/Memo

备注/Memo:
收稿日期 Received: 2019-04-03 接受日期 Accepted: 2019-10-10
*自然科学基金面上项目(31770395)、中国科学院种子创新研究院和中国科学院重点部署项目(ZDRW-ZS-2017-2-1)、中国科学院“西部之光”项目(2017XBZG_XBQNXZ_B_012,2018XBZG_XBQNXZ_B_007)、四川省科技计划项目(2017NZ0018,2017HH0077)资助
**通讯作者 Corresponding author (E-mail: Zhaohai@cib.ac.cn)
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更新日期/Last Update: 2019-12-04