激活标签法构建油菜突变体库及油菜BnLACS9基因的功能研究

发布时间：2018-08-11 16:02

【摘要】：油菜突变体库的构建对研究油菜功能基因组学具有重要的意义。本研究通过农杆菌浸花法将激活标签载体pCB260转化到甘蓝型油菜中双11中,来构建油菜激活标签T-DNA插入突变体库。在筛选过程中,共尝试了三种筛选方法以获得快速高通量的筛选T-DNA插入突变体的方法。首先,利用除草剂Basta筛选T-DNA插入突变体。在统一规格的托盘(28×20cm)中均匀地洒上1000粒经农杆菌转化的油菜种子,放进培养箱中生长约10-15天,等长出真叶后开始喷洒5 ml/盘0.01%Basta。每隔一天喷洒一次,直到大部分非转化株死亡。将存活的转化植株转移到盆中继续生长。经PCR鉴定,在存活的34株抗性植株中,有33株为阳性植株,阳性率高达97%。其次,利用绿色荧光筛选T-DNA插入突变体。在直径为150 mm的培养皿中放入等面积的滤纸,用水将滤纸完全润湿。取1000粒经农杆菌转化后的T1代种子均匀的洒在滤纸上面,室温条件下放置12-24 h,至种芽长约3 mm左右后使用Tanon全自动化学发光/荧光图像分析系统进行荧光筛选。将筛选出的146粒带有绿色荧光的种子转移到培养箱中,提叶片基因组,PCR鉴定,有101株为阳性植株,阳性率达到69.18%。最后,改造激活标签载体pCB260,将绿色荧光蛋白基因换成红色荧光蛋白基因。因为已有实验室证明,在亚麻芥中将红色荧光蛋白作为筛选标记时,可以简单的使用绿色LED手电筒和红色太阳眼镜就能筛选出转基因的亚麻芥种子。克隆pCX-DR载体上的红色荧光蛋白基因,通过酶切连接的方法将其连接到激活标签载体pCB260上,通过农杆菌浸花法将改造后的激活标签载体转化到拟南芥中进行验证,但并没有观察到带有红色荧光的拟南芥种子。我们后续将加大转化拟南芥的数量进行验证和完整地克隆了红色荧光蛋白基因的表达框进行连接。我们将获得的转化油菜的T1代种子进行了含油量的检测,发现了3株转化株的种子含油量高于野生型和2株转化株的种子含油量低于野生型的突变体。在对T2代油菜进行观察时,我们发现了诸如少分枝、多头、早花等具有明显表型的突变体。同时,对长链酯酰辅酶A合成酶9(BnLACS9)基因的功能进行了部分研究。将BnLACS9基因的启动子克隆到含有GUS报告基因的载体pBI121上,转化拟南芥发现在根、茎、嫩叶、老叶、花和角果中均有表达,在嫩叶和花中表达最高。BnLACS9基因RNAi抑制表达植株的种子含油量降低,叶片叶绿素和淀粉的含量降低,表明了BnLACS9基因与油菜油脂和叶绿素合成相关。
[Abstract]:The construction of rapeseed mutant library is of great significance to the study of rapeseed functional genomics. In this study, the activated label vector pCB260 was transformed into Shuang11 in Brassica napus by Agrobacterium tumefaciens soaking method to construct the mutant library of T-DNA insertion in Brassica napus. In the process of screening, three screening methods were tried to obtain the fast and high-throughput method for screening T-DNA inserted mutants. First, the herbicide Basta was used to screen T-DNA inserted mutants. 1000 seeds of rape transformed by Agrobacterium tumefaciens were evenly sprinkled on a uniform size tray (28 脳 20cm) and grown in incubator for about 10-15 days. After growing true leaves, the seeds were sprayed on 5 ml/ plates of 0.01sta. Spray every other day until most of the non-transformed plants die. The surviving transformed plants were transferred to the pot and continued to grow. Among the 34 surviving resistant plants, 33 were positive, and the positive rate was as high as 97. Secondly, T-DNA inserted mutants were screened by green fluorescence. The filter paper is wetted completely with water in a 150 mm culture dish with the same area of filter paper. 1000 seeds of T1 generation transformed by Agrobacterium tumefaciens were evenly sprinkled on the filter paper and stored at room temperature for 12 to 24 hours. The seed length was about 3 mm. Then fluorescence screening was carried out by Tanon automatic chemiluminescence / fluorescence image analysis system. The 146 seeds with green fluorescence were transferred to the incubator and identified by PCR. 101 plants were positive, and the positive rate was 69.18%. Finally, the activation label vector pCB260 was modified and the green fluorescent protein gene was transformed into red fluorescent protein gene. Because it has been proved in the laboratory that when the red fluorescent protein is used as screening marker in flax mustard, the transgenic seeds can be easily screened by using green LED flashlight and red sunglasses. The red fluorescent protein gene of pCX-DR vector was cloned and ligated to the activated label vector pCB260 by enzyme digestion. The modified active label vector was transformed into Arabidopsis thaliana by Agrobacterium tumefaciens. However, no red fluorescent Arabidopsis seeds were observed. We then confirmed the transformed Arabidopsis thaliana and cloned the red fluorescent protein gene expression frame for ligation. The oil content of the T1 generation of transformed rapeseed was detected. It was found that the oil content of the three transformed plants was higher than that of the wild type and the oil content of the two transformed plants was lower than that of the wild type mutants. In the observation of T2 generation rapeseed, we found mutants with obvious phenotype, such as less branching, many heads and early flowering. At the same time, the function of long chain ester acyl coenzyme A synthase 9 (BnLACS9) gene was studied. The promoter of BnLACS9 gene was cloned into the vector pBI121 containing GUS reporter gene and expressed in root, stem, tender leaf, old leaf, flower and pod. The highest expression of .BnLACS9 gene RNAi in young leaves and flowers inhibited the decrease of oil content in seeds and the content of chlorophyll and starch in leaves, indicating that BnLACS9 gene was related to oil and chlorophyll synthesis in rape.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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