交换形成相关基因的RNAi载体构建及转化甘蓝型油菜研究
发布时间：2018-11-05 13:19
【摘要】：减数分裂中,同源染色体相互识别、配对后在非姊妹染色单体间发生片段交换,导致同源重组的发生。同源重组是生物多样性产生的重要来源之一。近年来,随着研究者对模式植物拟南芥、水稻减数分裂机制的深入研究,越来越多的参与交换形成的基因被发掘,使得人工调控交换的发生及其频率成为可能,在作物遗传育种中具有重要的潜在应用价值。甘蓝型油菜(Brassica napus,AACC)为白菜(B.rapa,AA)与甘蓝(B.oleracea,CC)杂交形成的异源四倍体,是我国重要的油料作物之一。由于缺少天然及人工突变体,对其减数分裂调控机制的研究鲜有报道。本研究的主要目的是利用RNAi技术降低甘蓝型油菜中相关基因(均为拟南芥减数分裂中参与交换形成的同源基因)的表达,分析甘蓝型油菜减数分裂中交换形成的分子机制,并为通过同源/部分同源重组引入野生近缘种优异基因建立材料基础。主要结果如下:1)利用RT-PCR扩增,从甘蓝型油菜Westar中获得FIGL1、MHF1、XRCC2、ASY1和MUS81目标干涉区段。以质粒pFGC-5941为骨架,构建了上述五个基因的RNAi载体,并通过酶切与测序验证。2)利用农杆菌介导的遗传转化技术,以下胚轴为外植体,将上述载体分别转入甘蓝型油菜Westar、J9709和JW76中,抗性筛选后共得到再生植株78株。3)对其中66株进行PCR检测,共鉴定到阳性植株48株,平均转化效率为72.73%。其中带有FIGL1载体阳性植株25株,MHF1阳性株8株,XRCC2阳性株15株。另外,获得MUS81转化再生苗12株,尚未鉴定;未获得ASY1转化植株。4)根据各基因保守序列设计引物,通过qRT-PCR技术分析转基因阳性植株花蕾中目标基因的表达情况。结果表明,在14株阳性植株中目标基因表达量不同程度低于野生型材料。5)与野生型相比,各阳性植株花粉母细胞减数分裂未发现明显异常。这与拟南芥中上述基因的突变体表型相一致,这些突变体均具有正常减数分裂进程,但同源染色体交换频率增加。
[Abstract]:In meiosis homologous chromosomes recognize each other and exchange fragments between non-sister chromatids after pairing leading to homologous recombination. Homologous recombination is one of the important sources of biodiversity. In recent years, with the research of model plant Arabidopsis thaliana and rice meiosis mechanism, more and more genes involved in exchange formation have been discovered. It has important potential application value in crop genetics and breeding. Brassica napus (Brassica napus,AACC) is one of the most important oil crops in China because it is an allotetraploid formed by crossing Chinese cabbage (B.rapa) with cabbage (B.oleracea CC). Due to the lack of natural and artificial mutants, there are few reports on the mechanism of meiosis regulation. The main purpose of this study was to reduce the expression of related genes in Brassica napus by RNAi, and to analyze the molecular mechanism of exchange formation in meiosis of Arabidopsis thaliana. And to establish the material foundation for introducing the excellent genes of wild relative species by homologous / partial homologous recombination. The main results are as follows: 1) the target interference regions of FIGL1,MHF1,XRCC2,ASY1 and MUS81 were obtained from Westar of Brassica napus by RT-PCR amplification. The RNAi vectors of the five genes mentioned above were constructed with plasmid pFGC-5941 as the skeleton, and verified by enzyme digestion and sequencing. 2) using Agrobacterium tumefaciens mediated genetic transformation technique, the following cotyls were used as explants to transfer the above vectors into Brassica napus Westar, In J9709 and JW76, 78 regenerated plants were obtained after screening. 66 of them were detected by PCR, 48 positive plants were identified, and the average transformation efficiency was 72.73. There were 25 FIGL1 vector positive plants, 8 MHF1 positive plants and 15 XRCC2 positive plants. In addition, 12 regenerated MUS81 plants were obtained, which were not identified, and no ASY1 transformed plants were obtained. 4) primers were designed according to the conserved sequences of each gene, and the expression of target genes in flower buds of transgenic positive plants was analyzed by qRT-PCR technique. The results showed that the expression of target gene in 14 positive plants was lower than that of wild type. 5) compared with wild type, the meiosis of pollen mother cell of each positive plant was not abnormal. This is consistent with the surface pattern of the above genes in Arabidopsis. These mutants all have normal meiosis process, but the frequency of homologous chromosome exchange is increased.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S565.4
本文编号：2312213
[Abstract]:In meiosis homologous chromosomes recognize each other and exchange fragments between non-sister chromatids after pairing leading to homologous recombination. Homologous recombination is one of the important sources of biodiversity. In recent years, with the research of model plant Arabidopsis thaliana and rice meiosis mechanism, more and more genes involved in exchange formation have been discovered. It has important potential application value in crop genetics and breeding. Brassica napus (Brassica napus,AACC) is one of the most important oil crops in China because it is an allotetraploid formed by crossing Chinese cabbage (B.rapa) with cabbage (B.oleracea CC). Due to the lack of natural and artificial mutants, there are few reports on the mechanism of meiosis regulation. The main purpose of this study was to reduce the expression of related genes in Brassica napus by RNAi, and to analyze the molecular mechanism of exchange formation in meiosis of Arabidopsis thaliana. And to establish the material foundation for introducing the excellent genes of wild relative species by homologous / partial homologous recombination. The main results are as follows: 1) the target interference regions of FIGL1,MHF1,XRCC2,ASY1 and MUS81 were obtained from Westar of Brassica napus by RT-PCR amplification. The RNAi vectors of the five genes mentioned above were constructed with plasmid pFGC-5941 as the skeleton, and verified by enzyme digestion and sequencing. 2) using Agrobacterium tumefaciens mediated genetic transformation technique, the following cotyls were used as explants to transfer the above vectors into Brassica napus Westar, In J9709 and JW76, 78 regenerated plants were obtained after screening. 66 of them were detected by PCR, 48 positive plants were identified, and the average transformation efficiency was 72.73. There were 25 FIGL1 vector positive plants, 8 MHF1 positive plants and 15 XRCC2 positive plants. In addition, 12 regenerated MUS81 plants were obtained, which were not identified, and no ASY1 transformed plants were obtained. 4) primers were designed according to the conserved sequences of each gene, and the expression of target genes in flower buds of transgenic positive plants was analyzed by qRT-PCR technique. The results showed that the expression of target gene in 14 positive plants was lower than that of wild type. 5) compared with wild type, the meiosis of pollen mother cell of each positive plant was not abnormal. This is consistent with the surface pattern of the above genes in Arabidopsis. These mutants all have normal meiosis process, but the frequency of homologous chromosome exchange is increased.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S565.4
【参考文献】
相关期刊论文 前1条
1 刘春霞;何群燕;金危危;;植物减数分裂中的染色体配对、联会和重组研究进展[J];遗传;2010年12期
，本文编号：2312213
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