甘蓝型油菜COI1的调控功能分析  

Functional Analysis of COI1 Genes in Oilseed Rape(Brassica napus L.)

作　　者：王文静[1] 杨小川[1] 丁永强[1] 尹国英[1] 马浩然[1] 张洁[1] 石小于 张鼎宇[1] 李加纳[1] 张洪博[1] 

机构地区：[1]西南大学农学与生物科技学院/重庆市油菜工程技术研究中心,重庆400716

出　　处：《中国农业科学》2015年第10期1882-1891,共10页Scientia Agricultura Sinica

基　　金：教育部博士点基金项目(20120182120031);高等学校学科创新引智计划(B12006);西南大学"中央高校基本科研业务费专项资金"(XDJK2012C089)

摘　　要：【目的】研究甘蓝型油菜中茉莉素受体复合体核心成员CORONATINE INSENSITIVE 1(COI1)蛋白的基因表达特性及生理调控功能。【方法】利用基因组数据分析甘蓝型油菜及其亲本种白菜和甘蓝基因组的COI1构成情况;使用定量和半定量RT-PCR扩增方法检测油菜COI1的组织特异性表达;克隆油菜COI1保守区片段,构建病毒诱导的基因沉默(virus-induced gene silencing,VIGS)载体,以农杆菌侵染方法培育VIGS诱导COI1沉默的油菜植株,然后,用鉴定出的COI1沉默植株研究油菜COI1在育性调控及虫害抗性等方面的调控功能。【结果】基因组数据分析表明,甘蓝型油菜亲本种白菜和甘蓝共有7个高度同源的COI1,依序列同源性可分为4类:COI1a、COI1b.1、COI1b.2和COI1c;甘蓝型油菜有8个COI1,也由上述几类同源基因组成。半定量和定量RT-PCR扩增试验表明甘蓝型油菜COI1在茎中的表达水平最低。从培育的VIGS诱导COI1沉默油菜中,共鉴定出COI1表达水平被抑制超过70%的株系25个,其中抑制效果最明显的10个株系被用于育性调控及蚜虫抗性试验。育性分析结果证明,VIGS沉默COI1油菜的结实能力受到严重影响,其角果在授粉20 d以后的长度只达到对照油菜角果授粉5 d后的长度,且角果中无正常种子;沉默COI1油菜的雄蕊长度短于相同花期的对照油菜雄蕊,其花药开裂延迟;显微观察发现,沉默COI1油菜的花粉中有超过80%呈不正常形态。在相同处理条件下进行的蚜虫抗性比较试验中,沉默COI1油菜叶片上的蚜虫数量超过对照油菜1倍多,而且有很多小蚜虫围绕大蚜虫的虫落,繁殖态势比对照油菜叶片上的蚜虫旺盛。【结论】甘蓝型油菜COI1具有组织特异表达特性,沉默COI1会导致油菜雄性不育性状发生,并降低油菜植株对蚜虫的抗性。[Objective]CORONATINE INSENSITIVE 1 （COI1） is a critical component of jasmonate （JA） receptor complex. The objective of this study is to investigate the spatial expression pattern and regulatory roles of COI1 gene in oilseed rape （Brassica napus）, a staple oil crop in the world. [Method] The COI1 genes in oilseed rape （B. napus） and its parental species B. rapa and B. oleracea were analyzed based on the genomic data. The spatial transcription pattern of COI1 in oilseed rape was analyzed by RT-PCR with specific primers according to the conserved region of COI1 gene homologs. Then, a cDNA fragment of the conserved region was cloned into vector pTRV2 of the tobacco rattle virus-based VIGS （virus-induced gene silencing） system. And, VIGS techniques were applied to silence COI1 genes in oilseed rape. The rape plants shown to have COI1 genes silenced were used to＆amp;nbsp;investigate male fertility and aphid resistance.[Result]Analysis of the genomic data of B. rapa, B. oleracea, and B. napus revealed that the genomes of B. rapa and B. oleracea contained 7 highly homologous COI1 genes, which could be classified into 4 subgroups, i.e., COI1a, COI1b.1, COI1b.2, and COI1c, while 8 COI1 genes were presented in the genome of B. napus. Transcriptional assay revealed that the expression of COI1 in oilseed rape is tissue specific. A 505bp fragment of the conserved region of COI1 was introduced into the VIGS vector pTRV2 to develop COI1-silenced plants via VIGS techniques. Twenty-five plants with the transcripts of COI1 down-regulated by over 70% were identified by transcriptional analyses, from which the ten plants with the lowest expression level of COI1 were selected to investigate male fertility and aphid resistance. The results showed that the fertility of COI1-silenced plants was extremely impaired, with no seeds in their siliques. Furthermore, the filaments of COI1-silenced plants were shorter than those of control plants, and over 80%pollens of the COI1-silenced plants were irregular in shape. Aphi

关 键 词：甘蓝型油菜 COI1 茉莉素 雄性不育 蚜虫抗性 

分 类 号：S565.4[农业科学—作物学]