绿萍LmPDS基因RNAi载体的构建及遗传转化  

Construction and transformation of LmPDS gene RNAi vector in Lemna minor

作　　者：刘扬 方扬[1,2,3] 靳艳玲[1,3] 杜安平 杨贵利[1,2,3] 郭铃 谭力 何开泽 赵海 LIU Yang;FANG Yang;JIN Yanling;DU Anping;YANG Guili;GUO Ling;TAN Li;HE Kaize;ZHAO Hai(Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu 610041,China;University of Chinese Academy of Sciences,Beijing 100049,China;Key Laboratory of Environment and Applied Microbiology,Chinese Academy of Sciences,Chengdu 610041,China)

机构地区：[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

出　　处：《应用与环境生物学报》2020年第2期280-286,共7页Chinese Journal of Applied and Environmental Biology

基　　金：国家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。

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)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.

关 键 词：Lemna minor LmPDS基因 RNAi干扰 遗传转化 

分 类 号：Q943.2[生物学—植物学]