本文選題：蒺藜苜蓿 + U-box��； 參考：《中國農(nóng)業(yè)科學(xué)院》2016年博士論文

【摘要】：U-box蛋白在植物中廣泛存在說明該類蛋白在植物體內(nèi)發(fā)揮重要作用。很多U-box蛋白具有泛素連接酶E3活性以及在泛素化過程中特異地識別靶標(biāo)蛋白等生理生化功能。AtPUB4蛋白是在擬南芥中最新發(fā)現(xiàn)的具有E3連接酶活性的PUB蛋白,研究表明AtPUB4基因能影響花粉絨氈層細(xì)胞的生長發(fā)育,利用其特性有可能創(chuàng)制溫敏型雄性不育材料。蒺藜苜蓿(Medicago truncatula)是豆科苜蓿屬的一年生草本植物。二倍體,具有生育期短、基因組小、自花授粉、易于轉(zhuǎn)化、再生時間較短等特點,被作為豆科模式植物進行研究,2011年已完成全基因組測序。目前,有關(guān)蒺藜苜蓿U-box基因家族的研究尚未見報道。本試驗克隆兩個蒺藜苜蓿U-box基因MtPUB1和MtPUB2,通過生物信息學(xué)及生物技術(shù)手段分析了克隆基因結(jié)構(gòu)、亞細(xì)胞定位、原核表達(dá)及基因的表達(dá)特性,成功構(gòu)建了植物表達(dá)載體對擬南芥進行遺傳轉(zhuǎn)化。最后對MtPUB基因家族進化過程進行了分析。研究結(jié)果如下:1.利用同源克隆法獲得蒺藜苜蓿MtPUB1和MtPUB2基因,該基因cDNA全長2448和2400bp,分別編碼815和799個氨基酸,蛋白分子量為88.36 KDa和87.96 KDa,兩個蛋白都是疏水性蛋白,無信號肽和跨膜結(jié)構(gòu)。2.亞細(xì)胞定位預(yù)測MtPUB1基因定位在細(xì)胞質(zhì),MtPUB2基因定位在線粒體基質(zhì)。構(gòu)建原核表達(dá)載體,獲得MtPUB1蛋白。3.利用實時熒光定量PCR分析MtPUB1和MtPUB2基因在蒺藜苜蓿不同組織的表達(dá)特異性,結(jié)果表明MtPUB1基因在花中表達(dá)量最高,MtPUB2在莖中表達(dá)量最高,兩個基因在根部表達(dá)量最低。MtPUB1在NaCl、PEG、ABA誘導(dǎo)后,表達(dá)量上升,低溫誘導(dǎo)表達(dá)量變化不明顯。MtPUB2在NaCl誘導(dǎo)后表達(dá)量下降,在PEG、ABA及低溫誘導(dǎo)下表達(dá)量都呈現(xiàn)出上升趨勢。4.構(gòu)建植物表達(dá)載體pBI121-MtPUB1和pBI121-MtPUB2,采用農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化擬南芥Atpub4突變體,通過卡納抗性篩選和PCR、RT-PCR檢測后,獲得MtPUB1的T1代轉(zhuǎn)基因擬南芥種子。經(jīng)過對轉(zhuǎn)化擬南芥突變體和野生型擬南芥花粉掃描電鏡觀測,花粉改變不明顯。5.通過對MtPUB基因的功能歧化分析,U-box和ARM兩種功能結(jié)構(gòu)域的主要歧化位點位于3個保守的基序上,在此基礎(chǔ)上定位了9個歧化位點。6.對MtPUB基因序列選擇壓力的分析結(jié)果表明,MtPUB基因在強的負(fù)選擇壓力下經(jīng)過復(fù)制后保留發(fā)揮基因功能,其中114個負(fù)選擇位點所在序列與多序列聯(lián)配獲得保守序列相符,推測這一區(qū)域可使MtPUB蛋白與PREDEN發(fā)生特異性結(jié)合,因此產(chǎn)生不同功能。
[Abstract]:The widespread presence of U-box proteins in plants suggests that they play an important role in plants. Many U-box proteins have the activity of ubiquitin ligase E3 and the physiologic and biochemical functions such as target recognition protein. AtPUB4 protein is a newly discovered PUB protein with E3 ligase activity in Arabidopsis thaliana. The results showed that AtPUB4 gene could affect the growth and development of pollen tapetum cells, and the thermo-sensitive male sterile material could be created by using its characteristics. Medicago truncatula (Tribulus terrestris) is an annual herb of Alfalfa. Diploid, with the characteristics of short growth period, small genome, self-pollination, easy transformation and short regeneration time, has been studied as a legume model plant, and complete genome sequencing has been completed in 2011. At present, the study of U-box gene family of Tribulus terrestris has not been reported. In this study, two U-box genes of Tribulus terrestris, MtPUB1 and MtPUB2, were cloned. The structure, subcellular location, prokaryotic expression and expression characteristics of the cloned genes were analyzed by bioinformatics and biotechnology. Plant expression vector was successfully constructed for genetic transformation of Arabidopsis thaliana. Finally, the evolution process of MtPUB gene family was analyzed. The results are as follows: 1. The MtPUB1 and MtPUB2 genes of Tribulus terrestris were obtained by homologous cloning. The cDNA of the gene was 2448 and 2400bprespectively, encoding 815 amino acids and 799 amino acids, respectively. The molecular weight of the proteins was 88.36 KDa and 87.96 KDa. both proteins were hydrophobic proteins, no signal peptide and transmembrane structure. Subcellular localization predicted that the MtPUB1 gene was located in the cytoplasm of MtPUB2 gene in the mitochondrial matrix. Prokaryotic expression vector was constructed and MtPUB1 protein. 3. 3 was obtained. The expression specificity of MtPUB1 and MtPUB2 genes in different tissues of Medicago terrestris was analyzed by real-time fluorescence quantitative PCR. The results showed that the MtPUB1 gene expression was the highest in the flower and the lowest in the root. The expression of MtPUB2 decreased after induction of NaCl. The plant expression vectors pBI121-MtPUB1 and pBI121-MtPUB2 were constructed and transformed into Arabidopsis thaliana Atpub4 mutants by Agrobacterium tumefaciens. Transgenic Arabidopsis thaliana seeds of T1 generation of MtPUB1 were obtained by screening of Kana resistance and RT-PCR detection of pBI121-MtPUB2. The transformation of Arabidopsis mutants and wild type Arabidopsis pollen was observed by scanning electron microscope. Based on the analysis of the function of MtPUB gene, the main disambiguation sites of U-box and ARM were located on three conserved motifs, and the nine loci. 6 were located on the basis of this analysis. The results of sequence selection pressure of MtPUB gene showed that MtPUB gene remained functional after replication under strong negative selection pressure, and 114 negative selection sites were located at the same sequence as multiple sequences to obtain conserved sequences. It is speculated that this region can bind MtPUB protein to PREDEN specifically and thus produce different functions.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q943.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 魏臻武;蓋鈞鎰;;豆科模式植物——蒺藜苜蓿[J];草業(yè)學(xué)報;2008年01期

2 楊克珍;葉德;;植物雄配子體發(fā)生和發(fā)育的遺傳調(diào)控[J];植物學(xué)通報;2007年03期

3 孟祥紅,王建波,利容千;光敏胞質(zhì)不育小麥花藥發(fā)育過程中ATP酶的定位研究[J];作物學(xué)報;2000年06期

4 吳永敷,特木爾布和,許占有,羅慧寧;苜蓿雄性不育系雜交制種利用的研究[J];中國草地;1998年05期

5 鄧?yán)^新;劉文芳;肖翊華;;HPGMR花粉發(fā)育期花藥ATP含量及核酸與蛋白質(zhì)的合成研究[J];武漢大學(xué)學(xué)報(自然科學(xué)版);1990年03期

6 吳永敷;;國外對于苜蓿細(xì)胞質(zhì)雄性不育系的研究概況[J];國外畜牧學(xué).草原與牧草;1984年02期

7 吳永敷;;苜蓿雄性不育系的選育[J];中國草原;1980年02期

相關(guān)碩士學(xué)位論文 前1條

1 陳麗;紫花苜蓿雄性不育突變體的篩選及其細(xì)胞學(xué)觀察[D];蘭州大學(xué);2010年

，

本文編號：1884685