AtSUS3基因的克隆及对棉花的遗传转化研究
发布时间：2018-07-18 13:02
【摘要】：棉花,锦葵科棉属植物,是世界上主要的经济作物之一。棉纤维由胚珠部分表皮细胞发育形成,涉及许多基因的表达调控。UDPG(uridine 5'-diphosphoglucose)是纤维素合成的底物,蔗糖合成酶(Sucrose Synthase, SuSy)可作用于表皮细胞产生UDPG,同时降低细胞中UDP (uridine diphosphate)的含量,提高细胞膨压,促进细胞伸长及纤维素合成。棉花无绒毛突变体和野生型0 DPA (day post anthesis)的胚珠比较,表皮细胞突起锐减,同时未检测到SuSy的转录及酶活性。棉花中蔗糖合成酶3(SUS3)基因的低表达,会导致0 DPA胚珠表面突起减少,3 DPA的棉纤维变短。本实验室前期工作发现干涉拟南芥AtSUS3基因的表达,引起其它SUS成员代偿性增加,进而促进转基因拟南芥抽苔、果实成熟,其角果产率也均高于野生型植株。在此基础上,本研究克隆了AtSUS3基因,并将此基因连接植物表达载体pBI121,转化农杆菌LBA4404,利用两种方法对棉花进行遗传转化,以期获得转AtSUS3棉。已取得的结果如下：1.克隆AtSUS3基因。从拟南芥中克隆AtSUS3,该基因cDNA全长2430 bp,编码809个氨基酸,AtSUS3的分子量为92.00 kDa,理论等电点为5.85,疏水性平均值为-0.276,含量最丰富的氨基酸为Leu,Glu,Val,Gly。蛋白结构预测分析表明AtSUS3蛋白属于糖基转移酶家族,含有2个功能域,即N端的蔗糖合成结构域和C端的糖基转移结构域。在AtSUS3的二级结构中,α-螺旋占34.49%,随机卷曲占48.95%,延伸链占16.56%。其中,a-螺旋和随机卷曲是AtSUS3最主要的结构元件。2.构建表达载体。将AtSUS3基因连入表达载体pBI121,命名为:pBI121-AtSUS3,并转入农杆菌LBA4404中。利用农杆菌介导棉花下胚轴转化法对棉花进行转化。选取饱满、脱绒的棉种无菌培养,将生长7天后的棉花子叶下胚轴切段,通过农杆菌侵染3分钟,漂洗后共培养48小时,在抗性培养基上诱导形成愈伤组织,再进一步诱导形成胚性愈伤组织,通过胚胎发生等过程获得再生植株。3.分析嫁接方法。分别对劈接法,合接法,贴接法等方法进行分析,结果表明：嫁接土培苗时合接法成活率最高,劈接法嫁接后棉苗生长最好。无菌苗嫁接中,劈接法成活率最高,棉苗生长良好。4.获得转AtSUS3棉花。转AtSUS3基因的再生植株通过劈接法嫁接获得19株转AtSUS3棉花。通过ELISA方法研究了转基因棉花中蔗糖合成酶(SuSy),蔗糖转化酶(INV),蔗糖磷酸合成酶(SPS)的含量及酶活性,结果表明：转基因棉花普遍高于野生型棉花,但各阳性株系间存在差异。5.探索棉花活体胚转化外源基因的方法。用含外源基因GFP的农杆菌侵染去掉半个子叶的棉花种子胚,农杆菌OD600为0.4-0.6,侵染3小时,共培养48小时,用含100 mg/L卡那霉素的MS基础培养基筛选,生长30天后的棉苗在卡那霉素敏感性实验具有抗性,在DNA水平,转录水平上均可检测到GFP,棉花叶片也可检测到绿色荧光,但40天后外源基因表达减弱,60天后丢失,表明该转基因植物为嵌合体。
[Abstract]:Cotton is one of the major cash crops in the world. Cotton fiber is formed by ovule partial epidermal cells. UDPG (uridine 5'-diphosphoglucose) is the substrate of cellulose synthesis. Sucrose Synthase (SuSy) can act on the epidermal cells to produce UDPGs and decrease the content of (uridine diphosphate) in the cells. Increase cell pressure, promote cell elongation and cellulose synthesis. Compared with the ovule of wild-type 0DPA (day post anthesis), the epidermal processes of cotton without villous mutants decreased sharply, and the transcription and enzyme activity of SuSy were not detected. The low expression of sucrose synthase 3 (SUS3) gene in cotton resulted in the reduction of cotton fiber from the surface of 0 DPA ovules. Our previous work found that interfering with the expression of AtSUS3 gene in Arabidopsis thaliana caused compensatory increase of other SUS members, and then promoted transgenic Arabidopsis thaliana to sprout coating and mature fruit, and the rate of keratin production was also higher than that of wild-type plants. On this basis, AtSUS3 gene was cloned and ligated into plant expression vector pBI121to transform Agrobacterium tumefaciens LBA4404. Two methods were used to carry out genetic transformation of cotton in order to obtain transgenic cotton. The results achieved are as follows: 1. AtSUS3 gene was cloned. AtSUS3 was cloned from Arabidopsis thaliana. The total length of AtSUS3 gene was 2430 BP, encoding 809 amino acids, the molecular weight of AtSUS3 was 92.00 kDa, the theoretical isoelectric point was 5.85, and the hydrophobic average was -0.276. Protein structure prediction analysis showed that AtSUS3 protein belongs to the glycosyltransferase family and contains two functional domains: N-terminal sucrose biosynthesis domain and C-terminal glycosyltransferase domain. In the secondary structure of AtSUS3, 伪 -helix is 34.49, random crimp is 48.95 and extension chain is 16.56. The main structural components of AtSUS3, I. e., A helix and random crimp, are. 2. 2. Construct expression vector. AtSUS3 gene was inserted into the expression vector pBI121and named as: pBI121-AtSUS3, and transferred into Agrobacterium tumefaciens LBA4404. Cotton was transformed by Agrobacterium tumefaciens mediated Hypocotyl transformation. After 7 days of growth, cotton cotyledon Hypocotyl was cut into segments, infected with Agrobacterium tumefaciens for 3 minutes, then rinsed for 48 hours, then induced to form callus on resistant medium. Embryogenic calli were further induced and regenerated plants were obtained by embryogenesis. The grafting method was analyzed. The results showed that the survival rate of the grafting method was the highest, and the cotton seedling growth was the best after the split grafting method. In the grafting of sterile seedlings, the survival rate of split grafting was the highest, and cotton seedlings grew well. 4. The cotton was transferred to AtSUS3. Nineteen transgenic plants of AtSUS3 were obtained by split-grafting. The content and activity of sucrose synthase (SuSy), sucrose invertase (INV) and sucrose phosphate synthase (SPS) in transgenic cotton were studied by Elisa. The results showed that the content and activity of sucrose synthase (SuSy), sucrose invertase (INV) and sucrose phosphate synthase (SPS) in transgenic cotton were generally higher than those in wild type cotton. To explore the method of transformation of foreign gene from cotton in vivo embryo. Agrobacterium tumefaciens containing foreign gene GFP was used to infect and remove half cotyledon of cotton seed embryo. Agrobacterium-OD600 was 0.4-0.6, infected for 3 hours, co-cultured for 48 hours, and screened by MS basic medium containing 100 mg / L kanamycin. After 30 days of growth, cotton seedlings were resistant to kanamycin, GFP could be detected at DNA level and transcription level, green fluorescence could also be detected in cotton leaves, but after 40 days, the expression of exogenous genes was decreased and lost after 60 days. The results showed that the transgenic plants were chimerism.
【学位授予单位】：陕西师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2;S562
本文编号：2131981
[Abstract]:Cotton is one of the major cash crops in the world. Cotton fiber is formed by ovule partial epidermal cells. UDPG (uridine 5'-diphosphoglucose) is the substrate of cellulose synthesis. Sucrose Synthase (SuSy) can act on the epidermal cells to produce UDPGs and decrease the content of (uridine diphosphate) in the cells. Increase cell pressure, promote cell elongation and cellulose synthesis. Compared with the ovule of wild-type 0DPA (day post anthesis), the epidermal processes of cotton without villous mutants decreased sharply, and the transcription and enzyme activity of SuSy were not detected. The low expression of sucrose synthase 3 (SUS3) gene in cotton resulted in the reduction of cotton fiber from the surface of 0 DPA ovules. Our previous work found that interfering with the expression of AtSUS3 gene in Arabidopsis thaliana caused compensatory increase of other SUS members, and then promoted transgenic Arabidopsis thaliana to sprout coating and mature fruit, and the rate of keratin production was also higher than that of wild-type plants. On this basis, AtSUS3 gene was cloned and ligated into plant expression vector pBI121to transform Agrobacterium tumefaciens LBA4404. Two methods were used to carry out genetic transformation of cotton in order to obtain transgenic cotton. The results achieved are as follows: 1. AtSUS3 gene was cloned. AtSUS3 was cloned from Arabidopsis thaliana. The total length of AtSUS3 gene was 2430 BP, encoding 809 amino acids, the molecular weight of AtSUS3 was 92.00 kDa, the theoretical isoelectric point was 5.85, and the hydrophobic average was -0.276. Protein structure prediction analysis showed that AtSUS3 protein belongs to the glycosyltransferase family and contains two functional domains: N-terminal sucrose biosynthesis domain and C-terminal glycosyltransferase domain. In the secondary structure of AtSUS3, 伪 -helix is 34.49, random crimp is 48.95 and extension chain is 16.56. The main structural components of AtSUS3, I. e., A helix and random crimp, are. 2. 2. Construct expression vector. AtSUS3 gene was inserted into the expression vector pBI121and named as: pBI121-AtSUS3, and transferred into Agrobacterium tumefaciens LBA4404. Cotton was transformed by Agrobacterium tumefaciens mediated Hypocotyl transformation. After 7 days of growth, cotton cotyledon Hypocotyl was cut into segments, infected with Agrobacterium tumefaciens for 3 minutes, then rinsed for 48 hours, then induced to form callus on resistant medium. Embryogenic calli were further induced and regenerated plants were obtained by embryogenesis. The grafting method was analyzed. The results showed that the survival rate of the grafting method was the highest, and the cotton seedling growth was the best after the split grafting method. In the grafting of sterile seedlings, the survival rate of split grafting was the highest, and cotton seedlings grew well. 4. The cotton was transferred to AtSUS3. Nineteen transgenic plants of AtSUS3 were obtained by split-grafting. The content and activity of sucrose synthase (SuSy), sucrose invertase (INV) and sucrose phosphate synthase (SPS) in transgenic cotton were studied by Elisa. The results showed that the content and activity of sucrose synthase (SuSy), sucrose invertase (INV) and sucrose phosphate synthase (SPS) in transgenic cotton were generally higher than those in wild type cotton. To explore the method of transformation of foreign gene from cotton in vivo embryo. Agrobacterium tumefaciens containing foreign gene GFP was used to infect and remove half cotyledon of cotton seed embryo. Agrobacterium-OD600 was 0.4-0.6, infected for 3 hours, co-cultured for 48 hours, and screened by MS basic medium containing 100 mg / L kanamycin. After 30 days of growth, cotton seedlings were resistant to kanamycin, GFP could be detected at DNA level and transcription level, green fluorescence could also be detected in cotton leaves, but after 40 days, the expression of exogenous genes was decreased and lost after 60 days. The results showed that the transgenic plants were chimerism.
【学位授予单位】：陕西师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2;S562
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1 蔡云巧;AtSUS3基因的克隆及对棉花的遗传转化研究[D];陕西师范大学;2016年
，本文编号：2131981
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