玉米MYB家族ZmMYB3R基因的克隆及抗逆功能研究
發(fā)布時(shí)間：2018-09-18 07:50
【摘要】：玉米作為重要的糧食作物和工業(yè)原料,在其生物進(jìn)化過(guò)程中為應(yīng)對(duì)各種逆境脅迫形成了復(fù)雜的調(diào)控網(wǎng)絡(luò)。其中,通過(guò)轉(zhuǎn)錄因子與靶基因的結(jié)合來(lái)調(diào)控各種逆境相關(guān)基因的表達(dá),可以提高植物對(duì)逆境的抗性。研究表明,MYB家族轉(zhuǎn)錄因子能夠積極地響應(yīng)植物逆境脅迫。其中R2R3亞家族是高度保守的、最為廣泛的一類(lèi)參與植物逆境脅迫的亞家族。而R1R2R3亞家族基因在植物逆境脅迫中的功能報(bào)道還較少,只有個(gè)別基因在擬南芥和水稻以及小麥中被發(fā)現(xiàn)與耐鹽、耐旱等一些抗逆功能相關(guān)。本研究通過(guò)生物信息學(xué)分析并篩選了玉米中R1R2R3亞家族基因,結(jié)合PCR方法從玉米中克隆了Zm MYB3R基因。利用亞細(xì)胞定位、熒光定量PCR以及過(guò)表達(dá)轉(zhuǎn)基因手段等一系列研究方法明確了該基因在玉米抗逆過(guò)程中的功能,并對(duì)其響應(yīng)逆境脅迫的機(jī)制做了初步分析。具體結(jié)果如下:1.序列分析發(fā)現(xiàn)Zm MYB3R基因全長(zhǎng)為1692bp,編碼563個(gè)氨基酸,分子質(zhì)量為61.66k D,等電點(diǎn)為8.33。在N端有三個(gè)MYB結(jié)構(gòu)域,每個(gè)結(jié)構(gòu)域中有三個(gè)色氨酸殘基。同時(shí)Zm MYB3R基因與近緣物種逆境相關(guān)的3R基因的進(jìn)化關(guān)系非常緊密。2.熒光定量PCR結(jié)果表明Zm MYB3R基因受外源ABA、干旱和高鹽脅迫條件的誘導(dǎo)表達(dá)。同時(shí)組織表達(dá)模式分析顯示Zm MYB3R在各個(gè)組織中均有不同程度的表達(dá),其中在莖和葉片中表達(dá)量較高。3.通過(guò)亞細(xì)胞定位分析發(fā)現(xiàn),Zm MYB3R轉(zhuǎn)錄因子是核定位蛋白。轉(zhuǎn)錄活性分析表明該基因具有轉(zhuǎn)錄活性,通過(guò)篩選得出活性區(qū)域?yàn)?17-563bp。4.利用PCR方法構(gòu)建過(guò)表達(dá)載體結(jié)合農(nóng)桿菌侵染法獲得轉(zhuǎn)基因擬南芥植株。在MS培養(yǎng)基上添加Na Cl和Mannitol模擬高鹽、干旱環(huán)境。除此之外在真實(shí)環(huán)境下高鹽和干旱處理植株,并測(cè)定傷害度。脅迫處理結(jié)果顯示:相較于野生型,轉(zhuǎn)基因植株的耐鹽和抗旱能力明顯提高。5.ABA敏感性實(shí)驗(yàn)表明,轉(zhuǎn)基因植株種子的萌發(fā)率明顯高于野生型,葉片氣孔孔徑顯著減小。用外源的ABA處理轉(zhuǎn)基因型和野生型的擬南芥,發(fā)現(xiàn)轉(zhuǎn)基因型的擬南芥的發(fā)芽率明顯高于野生型。而轉(zhuǎn)基因擬南芥的氣孔在ABA處理后孔徑相對(duì)變的更窄。6.熒光定量PCR分析顯示轉(zhuǎn)基因植株中的ABA合成及下游相關(guān)基因的表達(dá)較野生型明顯上調(diào),表明Zm MYB3R通過(guò)調(diào)控ABA信號(hào)途徑增強(qiáng)植物的抗逆性。說(shuō)明Zm MYB3R可以依賴于ABA誘導(dǎo)下游逆境相關(guān)基因表達(dá),從而提高植物的非生物脅迫的耐受力。
[Abstract]:Maize, as an important food crop and industrial raw material, has formed a complex regulatory network in the course of its biological evolution to cope with various stresses. Among them, the expression of various stress-related genes can be regulated by the combination of transcription factors and target genes, which can improve the resistance of plants to stress. The results showed that MYB family transcription factors could respond positively to plant stress. Among them, R2R3 subfamily is highly conserved and most widely involved in plant stress. However, the function of R1R2R3 subfamily genes in plant stress was less reported. Only a few genes were found in Arabidopsis, rice and wheat, which were related to salt tolerance, drought tolerance and so on. In this study, R1R2R3 subfamily genes in maize were analyzed and screened by bioinformatics, and Zm MYB3R gene was cloned from maize by PCR method. A series of research methods, such as subcellular localization, fluorescence quantitative PCR and over-expression transgenic methods, were used to clarify the function of the gene in maize stress resistance, and the mechanism of its response to stress was preliminarily analyzed. The results are as follows: 1. Sequence analysis showed that the Zm MYB3R gene was 1692 BP, encoding 563 amino acids, with molecular weight of 61.66 KD and isoelectric point of 8.33. There are three MYB domains at the N terminal and three tryptophan residues in each domain. At the same time, the evolutionary relationship between Zm MYB3R gene and the 3R gene of relative species is very close. The results of fluorescence quantitative PCR showed that the expression of Zm MYB3R gene was induced by exogenous ABA, drought and high salt stress. At the same time, tissue expression pattern analysis showed that Zm MYB3R was expressed in different degree in all tissues, especially in stem and leaf. Subcellular localization analysis showed that Zm MYB3R transcription factor was a nuclear localization protein. The transcriptional activity analysis showed that the gene had transcriptional activity, and the active region was 217-563bp.4. Transgenic Arabidopsis thaliana plants were obtained by using PCR method to construct overexpression vector combined with Agrobacterium tumefaciens infection. Na Cl and Mannitol were added to MS medium to simulate high salt and dry environment. In addition, the plants were treated with high salt and drought in real environment, and the degree of injury was measured. The results of stress treatment showed that the sensitivity of transgenic plants to salt tolerance and drought resistance was significantly higher than that of wild type. The results of ABA sensitivity test showed that the germination rate of transgenic plants was significantly higher than that of wild type, and the stomatal aperture of leaves was significantly decreased. Transgenic type and wild type Arabidopsis thaliana were treated with exogenous ABA. It was found that the germination rate of transgenic type Arabidopsis thaliana was significantly higher than that of wild type. The pore diameter of transgenic Arabidopsis thaliana was narrower than that of ABA treatment. Fluorescence quantitative PCR analysis showed that ABA synthesis and downstream gene expression in transgenic plants were significantly up-regulated than those in wild type, indicating that Zm MYB3R enhanced plant stress resistance by regulating ABA signaling pathway. It was suggested that Zm MYB3R could induce downstream stress-related gene expression by ABA, thus enhancing plant abiotic stress tolerance.
【學(xué)位授予單位】：安徽農(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)】：Q943.2;S513
本文編號(hào)：2247256
[Abstract]:Maize, as an important food crop and industrial raw material, has formed a complex regulatory network in the course of its biological evolution to cope with various stresses. Among them, the expression of various stress-related genes can be regulated by the combination of transcription factors and target genes, which can improve the resistance of plants to stress. The results showed that MYB family transcription factors could respond positively to plant stress. Among them, R2R3 subfamily is highly conserved and most widely involved in plant stress. However, the function of R1R2R3 subfamily genes in plant stress was less reported. Only a few genes were found in Arabidopsis, rice and wheat, which were related to salt tolerance, drought tolerance and so on. In this study, R1R2R3 subfamily genes in maize were analyzed and screened by bioinformatics, and Zm MYB3R gene was cloned from maize by PCR method. A series of research methods, such as subcellular localization, fluorescence quantitative PCR and over-expression transgenic methods, were used to clarify the function of the gene in maize stress resistance, and the mechanism of its response to stress was preliminarily analyzed. The results are as follows: 1. Sequence analysis showed that the Zm MYB3R gene was 1692 BP, encoding 563 amino acids, with molecular weight of 61.66 KD and isoelectric point of 8.33. There are three MYB domains at the N terminal and three tryptophan residues in each domain. At the same time, the evolutionary relationship between Zm MYB3R gene and the 3R gene of relative species is very close. The results of fluorescence quantitative PCR showed that the expression of Zm MYB3R gene was induced by exogenous ABA, drought and high salt stress. At the same time, tissue expression pattern analysis showed that Zm MYB3R was expressed in different degree in all tissues, especially in stem and leaf. Subcellular localization analysis showed that Zm MYB3R transcription factor was a nuclear localization protein. The transcriptional activity analysis showed that the gene had transcriptional activity, and the active region was 217-563bp.4. Transgenic Arabidopsis thaliana plants were obtained by using PCR method to construct overexpression vector combined with Agrobacterium tumefaciens infection. Na Cl and Mannitol were added to MS medium to simulate high salt and dry environment. In addition, the plants were treated with high salt and drought in real environment, and the degree of injury was measured. The results of stress treatment showed that the sensitivity of transgenic plants to salt tolerance and drought resistance was significantly higher than that of wild type. The results of ABA sensitivity test showed that the germination rate of transgenic plants was significantly higher than that of wild type, and the stomatal aperture of leaves was significantly decreased. Transgenic type and wild type Arabidopsis thaliana were treated with exogenous ABA. It was found that the germination rate of transgenic type Arabidopsis thaliana was significantly higher than that of wild type. The pore diameter of transgenic Arabidopsis thaliana was narrower than that of ABA treatment. Fluorescence quantitative PCR analysis showed that ABA synthesis and downstream gene expression in transgenic plants were significantly up-regulated than those in wild type, indicating that Zm MYB3R enhanced plant stress resistance by regulating ABA signaling pathway. It was suggested that Zm MYB3R could induce downstream stress-related gene expression by ABA, thus enhancing plant abiotic stress tolerance.
【學(xué)位授予單位】：安徽農(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)】：Q943.2;S513
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 毛學(xué)峰;劉冬梅;劉靖;;中國(guó)大規(guī)模糧食進(jìn)口的現(xiàn)狀與未來(lái)[J];中國(guó)軟科學(xué);2016年01期
2 王爽;丁建麗;王璐;牛增懿;;基于地表光譜建模的區(qū)域土壤鹽漬化遙感監(jiān)測(cè)研究[J];干旱區(qū)地理;2016年01期
3 樊錦濤;蔣琛茜;邢繼紅;董金皋;;擬南芥R2R3-MYB家族第22亞族的結(jié)構(gòu)與功能[J];遺傳;2014年10期
4 李紅霞;汪妤;張戰(zhàn)鳳;彭惠茹;倪中福;;植物轉(zhuǎn)錄因子與作物抗逆脅迫關(guān)系的研究進(jìn)展[J];麥類(lèi)作物學(xué)報(bào);2013年03期
5 ;Regulation of AtbZIP1 Gene Expression by T-DNA Insertion in Arabidopsis thaliana[J];Journal of Northeast Agricultural University(English Edition);2010年03期
6 劉蕾;杜海;唐曉鳳;吳燕民;黃玉碧;唐益雄;;MYB轉(zhuǎn)錄因子在植物抗逆脅迫中的作用及其分子機(jī)理[J];遺傳;2008年10期
7 吳耀榮;謝旗;;ABA與植物脅迫抗性[J];植物學(xué)通報(bào);2006年05期
8 陳俊,王宗陽(yáng);植物MYB類(lèi)轉(zhuǎn)錄因子研究進(jìn)展[J];植物生理與分子生物學(xué)學(xué)報(bào);2002年02期
9 沈元月,黃叢林,張秀海,曹鳴慶;植物抗旱的分子機(jī)制研究[J];中國(guó)生態(tài)農(nóng)業(yè)學(xué)報(bào);2002年01期
10 郭北海,張艷敏,李洪杰,杜立群,李銀心,張勁松,陳受宜,朱至清;甜菜堿醛脫氫酶(BADH)基因轉(zhuǎn)化小麥及其表達(dá)[J];植物學(xué)報(bào);2000年03期
，本文編號(hào)：2247256
本文鏈接：http://sikaile.net/kejilunwen/jiyingongcheng/2247256.html
最近更新
教材專(zhuān)著
