交換形成相關(guān)基因的RNAi載體構(gòu)建及轉(zhuǎn)化甘藍(lán)型油菜研究
發(fā)布時(shí)間：2018-11-05 13:19
【摘要】：減數(shù)分裂中,同源染色體相互識(shí)別、配對(duì)后在非姊妹染色單體間發(fā)生片段交換,導(dǎo)致同源重組的發(fā)生。同源重組是生物多樣性產(chǎn)生的重要來(lái)源之一。近年來(lái),隨著研究者對(duì)模式植物擬南芥、水稻減數(shù)分裂機(jī)制的深入研究,越來(lái)越多的參與交換形成的基因被發(fā)掘,使得人工調(diào)控交換的發(fā)生及其頻率成為可能,在作物遺傳育種中具有重要的潛在應(yīng)用價(jià)值。甘藍(lán)型油菜(Brassica napus,AACC)為白菜(B.rapa,AA)與甘藍(lán)(B.oleracea,CC)雜交形成的異源四倍體,是我國(guó)重要的油料作物之一。由于缺少天然及人工突變體,對(duì)其減數(shù)分裂調(diào)控機(jī)制的研究鮮有報(bào)道。本研究的主要目的是利用RNAi技術(shù)降低甘藍(lán)型油菜中相關(guān)基因(均為擬南芥減數(shù)分裂中參與交換形成的同源基因)的表達(dá),分析甘藍(lán)型油菜減數(shù)分裂中交換形成的分子機(jī)制,并為通過(guò)同源/部分同源重組引入野生近緣種優(yōu)異基因建立材料基礎(chǔ)。主要結(jié)果如下:1)利用RT-PCR擴(kuò)增,從甘藍(lán)型油菜Westar中獲得FIGL1、MHF1、XRCC2、ASY1和MUS81目標(biāo)干涉區(qū)段。以質(zhì)粒pFGC-5941為骨架,構(gòu)建了上述五個(gè)基因的RNAi載體,并通過(guò)酶切與測(cè)序驗(yàn)證。2)利用農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化技術(shù),以下胚軸為外植體,將上述載體分別轉(zhuǎn)入甘藍(lán)型油菜Westar、J9709和JW76中,抗性篩選后共得到再生植株78株。3)對(duì)其中66株進(jìn)行PCR檢測(cè),共鑒定到陽(yáng)性植株48株,平均轉(zhuǎn)化效率為72.73%。其中帶有FIGL1載體陽(yáng)性植株25株,MHF1陽(yáng)性株8株,XRCC2陽(yáng)性株15株。另外,獲得MUS81轉(zhuǎn)化再生苗12株,尚未鑒定;未獲得ASY1轉(zhuǎn)化植株。4)根據(jù)各基因保守序列設(shè)計(jì)引物,通過(guò)qRT-PCR技術(shù)分析轉(zhuǎn)基因陽(yáng)性植株花蕾中目標(biāo)基因的表達(dá)情況。結(jié)果表明,在14株陽(yáng)性植株中目標(biāo)基因表達(dá)量不同程度低于野生型材料。5)與野生型相比,各陽(yáng)性植株花粉母細(xì)胞減數(shù)分裂未發(fā)現(xiàn)明顯異常。這與擬南芥中上述基因的突變體表型相一致,這些突變體均具有正常減數(shù)分裂進(jìn)程,但同源染色體交換頻率增加。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S565.4
本文編號(hào)：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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S565.4
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 劉春霞;何群燕;金危危;;植物減數(shù)分裂中的染色體配對(duì)、聯(lián)會(huì)和重組研究進(jìn)展[J];遺傳;2010年12期
，本文編號(hào)：2312213
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