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【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】 The objective of this study was to investigate the gene expression and physiological regulation of CORONATINE INSENSITIVE 1 (COI1), a member of the jasmine receptor complex in Brassica napus. 【Method】 The composition of COI1 in Brassica napus and its parental cabbage and cabbage genome was analyzed by using genomic data. Tissue-specific expression of COI1 in rapeseed was detected by quantitative and semi-quantitative RT-PCR amplification. The virus-induced gene silencing (VIGS) vector was constructed and Agrobacterium-mediated inoculation of COI1-induced COI1-silenced Brassica napus plants was performed. Then COI1-silenced plants were used to study the effects of COI1 on fertility regulation and pest Resistance and other aspects of the regulatory function. 【Result】 Genomic data analysis showed that there are seven highly homologous COI1 genes in Brassica napus parental cabbage and cabbage, which can be divided into four groups according to sequence homology: COI1a, COI1b.1, COI1b.2 and COI1c; Rape has 8 COI1, but also by several types of homologous genes. Semi-quantitative and quantitative RT-PCR amplification experiments showed that the expression level of COI1 in Brassica napus was the lowest. From the cultivated VIGS-induced COI1-silenced canola, 25 lines with COI1 expression level over 70% were identified. Among them, 10 lines with the most obvious inhibitory effect were used in fertility control and aphid resistance test. The result of fertility analysis showed that the ability of VIGS to silence COI1 rapeseed was severely affected. The length of its fruit after 20 days of pollination reached the length of 5 days after pollination, and there was no normal seed in the fruit. The silence of COI1 rape The length of stamens was shorter than that of control rape stamens at the same flowering stage. The anther dehiscence was delayed. Microscopic observation showed that more than 80% of the pollens in silenced COI1 rape were abnormal. Under the same treatment conditions, the number of aphids on the COI1-rape leaves was more than double that of the control rape, and many small aphids larvae around the aphids. Compared with the aphids on the rape leaves, exuberant. 【Conclusion】 COI1 in Brassica napus has tissue-specific expression characteristics. Silencing COI1 can lead to male sterility traits in rapeseed and reduce the resistance to aphid in rape plants.