PEPc基因RNAi的拟南芥转化及性状分析

范文编号:SW067  范文字数:11820,页数:27

摘 要：利用基因工程技术进行植物高油育种，是当前国际上植物基因工程研究的热点之一。找到控制作物种子含油量的相关基因，通过运用生物技术，抑制或超表达目的基因，探讨该基因在植物中的调控机理，已成为此类研究的主要方法。作物种子中的贮藏物质主要由蛋白质、油脂及糖类组成，在油料作物中蛋白质及油脂构成其干物质的70％以上。研究表明作物种子中磷酸烯醇式丙酮酸羧化酶（phosphoenopyruvate carboxylase,简称PEPc）活性与其蛋白质含量正相关，而与油脂含量呈负相关。为了探讨PEPc基因调控油分/蛋白的分子机理，本实验通过构建好的PEPc基因RNAi的载体（命名为PEPi），通过花粉管浸泡法将PEPi载体导入拟南芥，对转化后的拟南芥进行除草剂抗性筛选、PCR鉴定得到阳性转基因植株，并利用除草剂筛选统计了转基因拟南芥T2代的遗传分离比，大部分符合3:1的经典遗传分离比。利用气相色谱分析测定T3代转基因植株种子含油量，表明转基因可以提高种子含油量，在盐处理的初步实验中还观察到转基因种子的抗盐性有明显降低。
关键词： PEPc基因；拟南芥(Arabidopsis thaliana)；RNAi；含油量

Abstract: The use of genetic engineering technology for high oil plant breeding was the one of the hot international plant genetic engineering at current. Through the use of biotechnology to inhibit gene expression or enhance gene expression in the plant, the relevant gene of the controlling crop seed oil was found. Crop seeds store material mainly from protein, fat and carbohydrate, protein and fat constitute over 70% of the dry matter in Oil-seed composition. Research shows that phosphoenopyruvate carboxylase (PEPc) which activity was relevant with protein content and negatively correlated with fat content in crop seeds. To explore PEPc gene how to regulate oil / protein in molecular mechanism, this experiment has set the PEPc gene of RNAi vector up and carried it into Arabidopsis through tube with the method of tube immersion. Then the herbicide resistance of the transformed Arabidopsis screening was measured. In result, positive strains of transgenic plants were obtained through PCR identification. Statistic from transgenic Arabidopsis T2 generation which screened by herbicide resistance showed that it was segregation ratio most with a 3:1 classic genetic ratio. Using gas chromatographic analysis for T3 transgenic plant seed oil content, it was showed that the transgenic seeds can increase oil content.  In the salt preliminary experiment which also observed that the transgenic seeds were significantly reduced in resistance salt.

Keywords : PEPc gene；Arabidopsis thaliana； RNAi；Oil content

目　录

中文摘要 Ⅰ
英文摘要 Ⅱ
目录 Ⅲ
1. 绪 论 1
 1.1  油料作物基因工程研究进展 1
 1.2  磷酸烯醇式丙酮酸羧化酶（PEPc）基因 2
 1.3  RNAi研究现状 2
 1.4  模式植物拟南芥中的PEPc基因 3
 1.5  本课题的研究目标及意义 4
2. 实验部分 5
 2.1  农杆菌介导的拟南芥转化 5
 2.2  转基因拟南芥的筛选与鉴定 7
 2.3  转基因拟南芥植株的性状分析 9
3. 结果与讨论 11
 3.1  农杆菌介导的拟南芥转化 11
 3.2  实验结果分析 11
 3.2  PEPi转基因拟南芥的筛选与鉴定 12
 3.3  转基因拟南芥植株的性状分析 14
4. 总结与展望 19
致谢 20
参考文献 21

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