甘蓝型油菜尿卟啉原Ⅲ合成酶基因BnHemd的克隆和功能分析  

Cloning and functional analysis of uroporphyrinogenⅢSynthase gene BnHemd from Brassica napus

作　　者：郑潇潇 朱瑶瑶 梁华兵 詹杰鹏[1] 师家勤[1] 王新发[1] ZHENG Xiao-xiao;ZHU Yao-yao;LIANG Hua-bing;ZHAN Jie-peng;SHI Jia-qin;WANG Xin-fa(Oil Crops Research Institute of Chinese Academy of Agricultural Sciences,Key Laboratory of Biology and Genetic Im⁃provement of Oil Crops,Ministry of Agriculture and Rural Affairs,Wuhan 430062,China)

机构地区：[1]中国农业科学院油料作物研究所/农业农村部油料作物生物学与遗传育种重点实验室,湖北武汉430062

出　　处：《中国油料作物学报》2020年第3期380-389,共10页Chinese Journal of Oil Crop Sciences

基　　金：中央级公益性科研院所基本科研业务费专项(1610172017001);现代农业产业技术体系建设专项(CARS-13);中国农业科学院科技创新工程(CAAS-ASTIP-2013-OCRI)。

摘　　要：为探讨油菜尿卟啉原Ⅲ合成酶基因Bn Hemd的功能及其与叶绿素合成的关系,促进光合效率和产量的提升,利用基因编辑技术对甘蓝型油菜中双11号中该基因在A9和C8染色体上的两个拷贝进行编辑。从中双11号中克隆到基因Bn A09.Hemd和Bn C08.Hemd,CDS序列分别为885 bp和876 bp,均由9个外显子构成,各编码294和291个氨基酸残基。Bn Hemd蛋白性质与结构分析结果表明,甘蓝型油菜中基因Bn Hemd的两个拷贝的蛋白理化性质十分相似,均为不稳定蛋白,相对分子质量分别为31.97 k D和31.40 k D。系统进化树分析表明,Bn A09.Hemd和Bn C08.Hemd分别与白菜和甘蓝中的同源基因亲缘关系最近。荧光定量PCR结果表明,Bn Hemd在中双11号的根、茎、叶、花蕾、种子和角果皮中均有表达,在角果皮和花蕾中的表达量远高于根、茎、叶。初步的功能分析结果表明,中双11号中该基因两个拷贝同时编辑的植株表现为:叶绿素含量显著减少、光合速率降低、生长迟缓,表明甘蓝型油菜BnHemd对叶绿素合成起重要作用,同时影响光合效率和生长发育。The object of this study was to analyse the function of gene Bn Hemd encoding uroporphyrinogen Ⅲ synthase(UROS) in rapeseed, and to better understand its relationship with chlorophyll synthesis for higher photosyn-thetic efficiency and yield. Gene editing technology was used to edit 2 copies of the gene on A9 and C8 chromosomesin Brassica napus cv Zhongshuang 11. The 2 genes were named Bn A09.Hemd and Bn C08.Hemd and cloned from thecultivar. Their CDS sequences were 885 bp and 876 bp respectively, composed of 9 exons, each encoding 294 and291 amino acid residues. Protein analysis showed that the 2 copies had similar physicochemical properties, and bothwere unstable and hydrophobins. Phylogenetic tree showed that Bn A09.Hemd and Bn C08.Hemd were recently relatedto B. rapa and B. oleracea respectively, which is consistent with genetic evolution of B. napus. RT-PCR resultsshowed that Bn Hemd was expressed in roots, stems, leaves, flower buds, seeds, and keratin peels of Zhongshuang 11.The expression levels in keratin peels and flower buds were much higher than those in roots, stems and leaves. Prelim-inary functional analysis showed that plants with 2 copies of the gene double-simultaneously edited at the same timeshowed a significant decrease in chlorophyll content, lower photosynthetic rate and delayed growth.

关 键 词：甘蓝型油菜 尿卟啉原Ⅲ合成酶 基因编辑 叶绿素 光合效率 

分 类 号：S565.4[农业科学—作物学]