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马铃薯ACO和ACS基因过表达与RNAi载体的构建及遗传转化
Construction of Overexpression and RNAi Vectors of ACO and ACS Genes and Transformation of Potato
【作者】 刘英;
【导师】 司怀军;
【作者基本信息】 甘肃农业大学 , 植物学, 2014, 硕士
【摘要】 ACC合成酶(1-aminocyclopropane-1-carboxlic acid synthase,1-氨基环丙烷-1-羧酸合成酶,ACS)在乙烯合成过程中催化S-腺苷甲硫氨酸(S-adometsynthetase,SAMS)合成ACC,是乙烯合成过程中的限速酶。ACC氧化酶(1-aminocyclopropane-1-carboxlic acid oxidase,1-氨基环丙烷-1-羧酸氧化酶,ACO)催化ACC形成乙烯,因此又称为乙烯形成酶(ethylene-forming enzyme,EFE),两种酶在乙烯形成中都起关键作用。本论文以马铃薯栽培品种“甘农薯2号”试管苗作为材料,根据已报道的基因序列信息设计特异引物,利用RT-PCR克隆出马铃薯ACS和ACO基因序列,并进行了生物信息学分析。分别构建了过表达载体pBI121-ACO和pBI121-ACS,以及干扰表达载体pHellsgate8-ACO和pHellsgate8-ACO。利用农杆菌介导法,转化马铃薯栽培品种“甘农薯2号”,获得转基因植株,为研究ACO、ACS基因在马铃薯生长发育和逆境响应过程中的作用奠定基础。取得的结果如下:1.根据ACC合成酶(ACS)及ACC氧化酶(ACO)基因序列(http://solanaceae.plantbiology.msu.edu/,序列号分别为PGSC0003DMT400005971和PGSC0003DMT400014932)设计特异性引物,利用RT-PCR技术以马铃薯“甘农薯2号”试管苗克隆得到其基因特异性片段。将ACO、ACS基因全长序列连接到pMD18-T载体上,干扰片段经BP反应连接到pDONRTM201载体上,转化大肠杆菌(Escherichia coli)DH5α感受态细胞,进行测序。测序结果表明,ACS mRNA全长1832bp其中CDS区长1461bp,与茄科中的番茄同源性最高,达97%。ACO mRNA全长1614bp,CDS区906bp,与茄科中的番茄同源性最高,达96%。2.对马铃薯ACO、ACS基因进行生物信息学分析,结果表明,ACO共有301个氨基酸残基,分子量约34kDa,PI5.96。ACS共有486氨基酸残基,分子量约为55kDa,PI6.76。ACO具有9个alpha螺旋区域,11个beta片层区,13个转角和15个自由卷曲区。ACS具有12个alpha螺旋区域,22个beta片层区,32个转角和23个自由卷曲区。ACO有26个疏水区和26个亲水区。ACS具有39个疏水区和41个亲水区。马铃薯ACO、ACS基因编码的蛋白质和茄科中番茄同源蛋白在进化上属于同一个分支,分子进化最近,关系最为密切。3.将目的基因ACO与过表达载体pBI121定向克隆,得到过表达载体pBI121-ACO;利用Infusion技术将ACS基因与表达载体pBI121定向连接得到pBI121-ACS;通过LR反应,将ACO、ACS基因干扰片段与载体pHellsgate8定向连接,构建得到干扰表达载体pHellsgate8-ACO和pHellsgate8-ACS。4.将过表达载体pBI121-ACO和pBI121-ACS以及干扰表达载体pHellsgate8-ACO和pHellsgate8-ACS分别进行转入农杆菌(Agrobacteriumtumefaciens)菌株LBA4404中,制备农杆菌工程菌;利用农杆菌介导法,将载体分别转入马铃薯栽培品种“甘农薯2号”中,经过抗性筛选及PCR检测,分别获得4株(pBI121-ACO)和3株(pBI121-ACS)以及4株(pHellsgate8-ACO)和5株(pHellsgate8-ACS)转基因植株。
【Abstract】 ACC synthase (1-aminocyclopropane-1-carboxlic acid synthase, ACS) catalyzesthe synthesis of ethylene SAMS (S-adomet synthetase) synthetic ACC. ACS is arate-limiting enzyme in ethylene synthesis. ACC oxidase (1-aminocyclopropane-1-carboxlic acid oxidase, ACO) catalyzed ACC to ethylene. So ACO is also known EFE(ethylene-forming enzyme). Two enzymes in the synthesis of ethylene have played akey role. According to the gene sequences information which have been reported, thisstudy use potato cultivar "Gannongshu2" test-tube plantlet as material, RNA wasextracted from leaves of the potato-“Gannongshu2” to design gene special primers,the target genes ACS and ACO were cloned by RT-PCR and the target genes wereanalyzed by bioinformatics. This study built over expression vector pBI121-ACO andpBI121-ACS, and interference expression vector pHellsgate8-ACO andpHellsgate8-ACS. The vectors were introduced into Agrobacterium tumefaciens toprepare engineering bacteria, and obtained transgenic potato plants byagrobacterium-mediated method. This study lays the foundation for the study of ACOand ACS genes in potato growth and stress response process in the role, but also laidthe foundation for the use of genetic engineering ideal new potato varieties. Theresults are as following:1. With RT-PCR, design specific primers according to the gene sequence of ACCsynthase (ACS) and ACC oxidase (ACO)(http://solanaceae.plantbiology.msu.edu/,serial numbers are PGSC0003DMT400005971and PGSC0003DMT400014932) andits conservative fragments, obtain gene specific fragments after PCR amplificationand cloning. The full-length sequences of ACO and ACS will be attached to pMD18-Tcarrier. The interference fragments will be attached to carrier pDONRTM201by BPreaction. After transformation of Escherichia coli DH5α competent cells weresequenced. The results show that the full-length of ACS mRNA is1832bp in whichthe CDS district is1461bp, the homology is as high as97%compared with tomato.The full-length of ACO mRNA is1614bp in which the CDS district is906bp, thehomology is as high as96%compared with tomato.2. ACO was analyzed by bioinformation method, which indicated ACO encodeda protein of301amino acid, molecular weight almost34kDa, PI5.96containing9alpha helix,11beta pleated sheet,13beta turn and15nonregular coil.Bioinfornmation analysis showed ACS encoded a protein of486amino acid, molecular weight almost55kDa, PI6.76containing12alpha helix,22beta pleatedsheet,32beta turn and23nonregular coil. ACO contained26hydrophobic regionsand26hydrophilic regions. ACS contained39hydrophobic regions and41hydrophilic regions. Both ACS and ACO occupied the same evolutionary positionwith tomato.3. The plastid pBI121-ACO and pBI121-ACS was built by ACO and ACS genefragment, respectively, with Infusion method. The plastid pHellsgate8-ACO andpHellsgate8-ACS was built by ACO and ACS interference fragment, respectively, withLR reaction.4. The pBI121-ACO, pBI121-ACS, pHellsgate8-ACO and pHellsgate8-ACS wastransform to A.tumefaciens (LBA4404), respectively, then consequence infected thepotato “Gannongshu2”. Using PCR analysis,16transformed plants was detectedcontaining4pBI121-ACO infected plants,3pBI121-ACSinfected plants,4pHellsgate8-ACO infected plants and5pHellsgate8-ACS infected plants.
【Key words】 Potato; ACC synthetase; ACC oxidase; Bioinformatics analysis; RNAi; Genetic transformation;