紫花苜蓿MsSNARE基因的克隆及表達(dá)分析
發(fā)布時(shí)間：2018-07-13 17:32
【摘要】：紫花苜蓿(Medicago sativa L.),是一種栽培范圍廣、適應(yīng)性強(qiáng)、品質(zhì)好、產(chǎn)量高的豆科牧草。雖然自身具有很強(qiáng)的非生物脅迫抗性,但全球氣候變化及極端天氣的發(fā)生,依然會(huì)給苜蓿生產(chǎn)造成嚴(yán)重?fù)p失。因此,培育具有優(yōu)良抗性的紫花苜蓿品種十分重要。近年來(lái),關(guān)于紫花苜蓿相關(guān)基因的克隆、功能驗(yàn)證及遺傳轉(zhuǎn)化的研究進(jìn)展較快。植物中的SNARE蛋白可以提高植物的抗病性,通過(guò)與其他通道蛋白的互作影響植物的生長(zhǎng)發(fā)育及對(duì)環(huán)境刺激的應(yīng)答。本研究利用RACE法克隆了紫花苜蓿MsSNARE基因,并利用生物信息學(xué)軟件對(duì)其序列進(jìn)行分析;構(gòu)建了瞬時(shí)表達(dá)載體PBI-MsSNARE;通過(guò)qRT-PCR技術(shù)對(duì)該基因的表達(dá)模式進(jìn)行分析。主要研究結(jié)果如下:1.利用RACE法,從紫花苜蓿中成功克隆出MsSNARE基因cDNA的全長(zhǎng),經(jīng)序列分析并發(fā)現(xiàn)MsSNARE cDNA全長(zhǎng)1396 bp,開放閱讀框長(zhǎng)(ORF)1257 bp,共編碼418個(gè)氨基酸殘基,其編碼的蛋白屬于SNARE家族。GeneBank登錄號(hào)為:JX467688。2.qRT-PCR分析發(fā)現(xiàn)MsSNARE基因在紫花苜蓿的根、莖、葉中都有表達(dá),在根、莖和葉中的表達(dá)量差異性不顯著。3.qRT-PCR分析了MsSNARE基因在受到干旱、鹽脅迫、ABA誘導(dǎo)時(shí)的轉(zhuǎn)錄水平變化,結(jié)果發(fā)現(xiàn)在受到外界脅迫時(shí)MsSNARE表達(dá)水平均上調(diào)。4.構(gòu)建了MsSNARE基因瞬時(shí)表達(dá)載體PBI-MsSNARE,洋蔥表皮細(xì)胞亞細(xì)胞定位研究表明該基因的產(chǎn)物定位于細(xì)胞膜上。
[Abstract]:Alfalfa (Medicago sativa L.) is a leguminous forage with wide cultivation range, strong adaptability, good quality and high yield. Although it has strong abiotic stress resistance, the global climate change and the occurrence of extreme weather will still cause serious losses to alfalfa production. Therefore, it is very important to cultivate alfalfa varieties with good resistance. In recent years, studies on cloning, functional verification and genetic transformation of alfalfa related genes have developed rapidly. The SNARE protein in plants can improve plant disease resistance and affect the growth and development of plants and their responses to environmental stimuli through interaction with other channel proteins. In this study, the MsSNARE gene of alfalfa was cloned by race method, and its sequence was analyzed by bioinformatics software. The transient expression vector PBI-MsSNAREwas constructed, and the expression pattern of the gene was analyzed by qRT-PCR. The main results are as follows: 1. The full length of MsSNARE gene cDNA was cloned from alfalfa by race method. By sequence analysis, the full length of MsSNARE cDNA was found to be 1396 BP, and the open reading frame length (ORF) was 1257 BP, encoding 418 amino acid residues. The encoding protein belongs to the Snare family. The accession number of GeneBank is: JX467688.2.qRT-PCR analysis shows that the MsSNARE gene is expressed in the roots, stems and leaves of alfalfa, but there is no significant difference in the expression levels in the roots, stems and leaves. 3.qRT-PCR analysis showed that the MsSNARE gene was subjected to drought. The results showed that the expression level of MsSNARE was up-regulated under the stress of salt stress, and the results showed that the expression level of MsSNARE was up-regulated by .4. The transient expression vector PBI-MsSNAREof MsSNARE gene was constructed. The subcellular localization of MsSNARE gene in epidermal cells of onion showed that the product of MsSNARE gene was located on the cell membrane.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S541.9
本文編號(hào)：2120186
[Abstract]:Alfalfa (Medicago sativa L.) is a leguminous forage with wide cultivation range, strong adaptability, good quality and high yield. Although it has strong abiotic stress resistance, the global climate change and the occurrence of extreme weather will still cause serious losses to alfalfa production. Therefore, it is very important to cultivate alfalfa varieties with good resistance. In recent years, studies on cloning, functional verification and genetic transformation of alfalfa related genes have developed rapidly. The SNARE protein in plants can improve plant disease resistance and affect the growth and development of plants and their responses to environmental stimuli through interaction with other channel proteins. In this study, the MsSNARE gene of alfalfa was cloned by race method, and its sequence was analyzed by bioinformatics software. The transient expression vector PBI-MsSNAREwas constructed, and the expression pattern of the gene was analyzed by qRT-PCR. The main results are as follows: 1. The full length of MsSNARE gene cDNA was cloned from alfalfa by race method. By sequence analysis, the full length of MsSNARE cDNA was found to be 1396 BP, and the open reading frame length (ORF) was 1257 BP, encoding 418 amino acid residues. The encoding protein belongs to the Snare family. The accession number of GeneBank is: JX467688.2.qRT-PCR analysis shows that the MsSNARE gene is expressed in the roots, stems and leaves of alfalfa, but there is no significant difference in the expression levels in the roots, stems and leaves. 3.qRT-PCR analysis showed that the MsSNARE gene was subjected to drought. The results showed that the expression level of MsSNARE was up-regulated under the stress of salt stress, and the results showed that the expression level of MsSNARE was up-regulated by .4. The transient expression vector PBI-MsSNAREof MsSNARE gene was constructed. The subcellular localization of MsSNARE gene in epidermal cells of onion showed that the product of MsSNARE gene was located on the cell membrane.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S541.9
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1 張通;紫花苜蓿MsSNARE基因的克隆及表達(dá)分析[D];西北農(nóng)林科技大學(xué);2016年
，本文編號(hào)：2120186
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