激活標簽法構建油菜突變體庫及油菜BnLACS9基因的功能研究
發(fā)布時間：2018-08-11 16:02
【摘要】：油菜突變體庫的構建對研究油菜功能基因組學具有重要的意義。本研究通過農桿菌浸花法將激活標簽載體pCB260轉化到甘藍型油菜中雙11中,來構建油菜激活標簽T-DNA插入突變體庫。在篩選過程中,共嘗試了三種篩選方法以獲得快速高通量的篩選T-DNA插入突變體的方法。首先,利用除草劑Basta篩選T-DNA插入突變體。在統(tǒng)一規(guī)格的托盤(28×20cm)中均勻地灑上1000粒經農桿菌轉化的油菜種子,放進培養(yǎng)箱中生長約10-15天,等長出真葉后開始噴灑5 ml/盤0.01%Basta。每隔一天噴灑一次,直到大部分非轉化株死亡。將存活的轉化植株轉移到盆中繼續(xù)生長。經PCR鑒定,在存活的34株抗性植株中,有33株為陽性植株,陽性率高達97%。其次,利用綠色熒光篩選T-DNA插入突變體。在直徑為150 mm的培養(yǎng)皿中放入等面積的濾紙,用水將濾紙完全潤濕。取1000粒經農桿菌轉化后的T1代種子均勻的灑在濾紙上面,室溫條件下放置12-24 h,至種芽長約3 mm左右后使用Tanon全自動化學發(fā)光/熒光圖像分析系統(tǒng)進行熒光篩選。將篩選出的146粒帶有綠色熒光的種子轉移到培養(yǎng)箱中,提葉片基因組,PCR鑒定,有101株為陽性植株,陽性率達到69.18%。最后,改造激活標簽載體pCB260,將綠色熒光蛋白基因換成紅色熒光蛋白基因。因為已有實驗室證明,在亞麻芥中將紅色熒光蛋白作為篩選標記時,可以簡單的使用綠色LED手電筒和紅色太陽眼鏡就能篩選出轉基因的亞麻芥種子。克隆pCX-DR載體上的紅色熒光蛋白基因,通過酶切連接的方法將其連接到激活標簽載體pCB260上,通過農桿菌浸花法將改造后的激活標簽載體轉化到擬南芥中進行驗證,但并沒有觀察到帶有紅色熒光的擬南芥種子。我們后續(xù)將加大轉化擬南芥的數量進行驗證和完整地克隆了紅色熒光蛋白基因的表達框進行連接。我們將獲得的轉化油菜的T1代種子進行了含油量的檢測,發(fā)現了3株轉化株的種子含油量高于野生型和2株轉化株的種子含油量低于野生型的突變體。在對T2代油菜進行觀察時,我們發(fā)現了諸如少分枝、多頭、早花等具有明顯表型的突變體。同時,對長鏈酯酰輔酶A合成酶9(BnLACS9)基因的功能進行了部分研究。將BnLACS9基因的啟動子克隆到含有GUS報告基因的載體pBI121上,轉化擬南芥發(fā)現在根、莖、嫩葉、老葉、花和角果中均有表達,在嫩葉和花中表達最高。BnLACS9基因RNAi抑制表達植株的種子含油量降低,葉片葉綠素和淀粉的含量降低,表明了BnLACS9基因與油菜油脂和葉綠素合成相關。
[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.
【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2
本文編號：2177478
[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.
【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2
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