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[1]刘扬,方扬,靳艳玲,等.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(02):280-286.[doi:10.19675/j.cnki.1006-687x.2019.04004]
 LIU Yang,,et al.Construction and transformation of LmPDS gene RNAi vector in Lemna minor[J].Chinese Journal of Applied & Environmental Biology,2020,26(02):280-286.[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02期
页码:
280-286
栏目:
研究论文
出版日期:
2020-04-25

文章信息/Info

Title:
Construction and transformation of LmPDS gene RNAi vector in Lemna minor
作者:
刘扬方扬靳艳玲杜安平杨贵利郭铃谭力何开泽赵海
刘 扬1, 2, 3 方 扬1, 2, 3 靳艳玲1, 3 杜安平1, 3 杨贵利1, 2, 3 郭 铃1 谭 力1, 3 何开泽1, 3 赵 海1, 3?
Author(s):
LIU Yang1 2 3 FANG Yang1 2 3 JIN Yanling1 3 DU Anping1 3 YANG Guili1 2 3 GUO Ling1 TAN Li1 3 HE Kaize1 3 & ZHAO Hai1 3?
1 Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China 2 University of Chinese Academy of Sciences, Beijing 100049, China 3 Key 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 L. 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具有重要意义. 选取595 bp 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. 结果显示,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. 本研究成功构建绿萍LmPDS基因RNAi载体,实现了RNAi在绿萍中的应用,结果可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:
The phytoene desaturase gene (PDS) encodes the key enzyme for the biosynthesis of carotenoid. Silencing or inhibition of PDS gene expression leads to the appearance of mottled, chlorosis, or albino leaves. It is of great importance to select the LmPDS gene for the construction and transformation of the LmPDS gene RNAi vector for RNA interference in Lemna minor. In this study, the 595-base pair LmPDS gene fragment was selected as the interference fragment and the cloned LmPDS interference fragment was inserted in the plant expression vector pCAMBIA2300-35S-OCST using the intermediate vector pUCCRNAi. The constructed RNAi vector was transformed into the callus of L. minor by Agrobacterium-mediated transformation. Then, using the callus resistance screen, plant regeneration, plant culture, and marker gene NPTII detection processes, 70 plants were obtained, with four being transgenic positive plants. After fluorescence quantitative polymerase chain reaction analysis, lycopene content detection, and phenotype observation, the relative expression of LmPDS in all transgenic positive plants decreased significantly compared with 28.8%-40.5% of the control plants. The lycopene content in the transgenic positive plants also decreased significantly and these plants showed an obvious albino phenotype in their leaves. In conclusion, we successfully constructed the LmPDS gene RNAi vector in L. minor and the application of RNAi can provide materials and methods for further research in gene function analysis of L. minor.

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更新日期/Last Update: 2020-04-25