【作者】 刘蓉；

【导师】 刘春林；

【作者基本信息】 湖南农业大学 ， 作物学， 2024， 硕士

【摘要】 甘蓝型油菜（Brassica napus L.）是世界第二大油料经济作物,在世界范围内广泛种植。低温是一个不可忽略的非生物胁迫因素,常常会影响植物生长发育和限制作物种植区域。油菜是我国重要的油料作物,特别容易受到低温胁迫的不利影响,严重影响其生长发育过程。BnaMYB8（RVE3）为REVEILLE（RVE）转录因子家族成员之一,属于MYB相关（1R）重要亚家族,其特征在于单一MYB结构域中含有高度保守的SHAQK（Y/F）F序列。该家族的生物学功能除参与生物钟调控之外,还在植物生长发育和激素信号传递发挥作用,同时也涉及了生物和非生物胁迫方面。本研究对功能未知的BnaMYB8的功能进行了分析,获得了以下结果:1.在甘蓝型油菜中鉴定得到33个RVE家族成员,对家族成员进行了生物信息学分析,克隆得到了BnaMYB8基因。2.成功构建了RNAi载体和CRISPR/Cas9载体,并将RNAi载体和CRISPR/Cas9载体成功转化,分别获得了4株和6株转基因植株。3.用低温胁迫（-2℃黑暗处理3 h）处理对照植株（77GAO）和BnaMYB8基因沉默的植株,结果表明:BnaMYB8影响油菜耐低温能力,沉默了BnaMYB8会降低甘蓝型油菜耐低温的能力;接着分别测定了在25℃和-2℃时实验植株的脯氨酸（Proline,Pro）、可溶性糖（Soluble sugar,SS）、丙二醛（Malondialdehyde,MDA）、过氧化氢（H₂O₂）和丙酮酸（Pyruvic acid,PA）含量,结果显示,低温胁迫条件下,对照植株中Pro、SS和MDA、H₂O₂以及PA的积累量均显著高于沉默BnaMYB8基因的敏感植株。4.同时分析了25℃和-2℃时实验植株的抗寒标记基因COLD1,CBF4,COR25,ERF15和DREB1c的转录水平,qRT-PCR结果表明,低温胁迫下对照植株中抗寒标记基因的表达量显著高于沉默BnaMYB8基因的敏感植株;推测BnaMYB8是一个正调节因子,BnaMYB8会通过正调节CBF4的表达,进而促使抗冻基因COR25发挥其功能,维持油菜耐低温能力。更多还原

【Abstract】 Brassica napus.L is the world’s second largest oil cash crop and is widely cultivated worldwide.Low temperature is a key abiotic stress factor that affects the growth anddevelopment of plants and limits the growth range of crops.Rapeseed is an important oilcrop in our country,which is often subjected to low temperature stress during its growth and development.BnaMYB8（RVE3）is a member of the REVEILLE（RVE）family oftranscription factors,an important subfamily of MYB correlation（1R）,characterized by a highly conserved SHAQK（Y/F）F sequence in a single MYB domain.In addition tocircadian clock regulation,this family plays important functions in plant growth anddevelopment and hormone signaling,as well as biological and abiotic stresses.In this study,the function of BnaMYB8 whose function is unknown was analyzed,and the followingresults were obtained:1.33 members of the RVE family were identified in B.napus,and the BnaMYB8 gene was cloned by bioinformatics analysis.2.The overexpressed vector,RNAi vector and CRISPR/Cas9 vector were successfully constructed,and the RNAi vector and CRISPR/Cas9 vector were successfully transformed,and 4 and 6 transgenic plants were obtained respectively.3.Plants with wild type and BnaMYB8 gene silencing were treated with lowtemperature stress.The results showed that BnaMYB8 affected the low temperature tolerance of Brassica napus,and the silencing of BnaMYB8 reduced the low temperature tolerance of brassica napus.Therefore,the contents of Proline（Pro）,Soluble sugar（SS）,Malondialdehyde（MDA）,Hydrogen peroxide（H₂O₂）and Pyruvic acid（PA）in experimental plants were determined respectively at 25℃and-2℃.The results showed that under low temperature stress,The accumulation of proline,soluble sugar and membrane lipid oxide in control plants was greater than that in sensitive plants with silenced BnaMYB8 gene.4.At the same time,the transcription levels of cold resistance marker genes COLD1,CBF4,COR25,ERF15 and DREB1c in experimental plants were analyzed at 25℃and-2℃.qRT-PCR results showed that the expression of cold resistance marker genes in control plants under low temperature stress was higher than that in sensitive plants that silenced BnaMYB8 gene.It is speculated that BnaMYB8 is a positive regulator,and by positively regulating the expression of CBF4,BnaMYB8 can promote the function of antifreeze gene COR25 and maintain the low temperature tolerance of rapeseed.更多还原