甜菜M14品系特异表达基因M14-341在模式植物中的表达研究

【作者】 汪立法；

【导师】 李海英；

【作者基本信息】 黑龙江大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 甜菜M14品系为郭德栋等人通过栽培甜菜(Beta vulgaris L.)和野生种白花甜菜(Beta corolliflora Zoss.)种间杂交及回交,获得的带有白花甜菜染色体的单体附加系(VV+1C,2n=19),附加染色体的传递率高达96.5%。甜菜M14品系是以二倍体孢子无融合生殖为主要繁殖方式,有性生殖为次要繁殖方式的兼性无融合生殖体。这种具有无融合生殖特性的甜菜M14品系的获得,为我们提供了一种良好的寻找无融合生殖相关基因的材料。本实验室前期采用抑制消减杂交技术(suppression subtractive hybridization,SSH)和mRNA差异显示技术(differential-display reverse transcription-PCR,DDRT-PCR)获得了298个甜菜M14品系特异表达的ESTs(expression sequence tags,ESTs),并挑选其中有研究价值的EST作为获得全长的侯选EST,通过快速扩增cDNA末端法(rapid amplification of cDNA ends,RACE)获得了甜菜M14品系特异表达的M14-341基因的cDNA全长。经生物信息学分析,M14-341基因编码的蛋白包括MADS-box结构域和K-结构域,并与菠菜(Spinacia oleracea)开花转录因子有88%的相似性,可以判定M14-341基因属于MADS-box基因。为进一步研究M14-341基因的功能以及M14-341基因与无融合生殖现象的关系,本实验首先利用DNAMAN软件将M14-341蛋白与拟南芥(Arabidopsis thaliana)中部分MIKC族蛋白及其它物种的MADS-box蛋白进行了同源比对,结果表明,M14-341蛋白与拟南芥中AG蛋白同源性达74%,可以将其归为AG基因(MADS-box基因家族“ABCDE”模型中的C类基因)的同源基因,其功能为控制花器官的发育。随后,构建了带有M14-341基因cDNA全长的植物表达载体pBI-M14-341,分别利用农杆菌介导的叶盘法和花头浸泡法将M14-341基因转入模式植物烟草和拟南芥中,以分析转基因植株的表型变化进而推测M14-341基因的功能。经过PCR检测、RT-PCR、Southern印迹、Northern印迹及GUS染色等分子生物学检测后,共获得24株转基因烟草和12株转基因拟南芥,进一步研究表明,M14-341基因在转基因植株花器官中表达量较高,在叶和茎中表达量微弱。对转基因植株的表型观测发现:M14-341基因在转基因植株中的组成型表达主要影响了烟草的花期及花器官的发育,转基因烟草出现花期延迟,花瓣颜色变浅,数量变少,花萼与花瓣转变为雄蕊、雌蕊类似结构,雄蕊、雌蕊变长,果实与种子的少育或不育等现象;转M14-341基因的拟南芥出现结实率降低,死亡率增高等变化。对转M14-341基因烟草花器官与作为对照的未转基因的野生型烟草花器官的总蛋白进行双向电泳,结果显示:转M14-341基因烟草植株与野生型烟草对照植株两者的蛋白质点分布情况非常相似,蛋白质点主要集中在分子量20~100 kD之间,而且大部分蛋白质的表达丰度都很低;共发现表达差异蛋白点8个,其中转M14-341基因烟草植株中表达消失的蛋白质点1个;新表达的蛋白质点1个;表达程度减弱的蛋白质点5个;表达程度增强的蛋白质点1个。实验结果表明:甜菜M14品系特异表达基因M14-341与AG类基因有相似的功能,在植物花各轮器官的分化发育及调控花期中起重要的作用。更多还原

【Abstract】 Apomictic monosomic addition line of Beta corolliflora Zoss., designated as M14, was obtained from the hybridization and intercross of Beta vulgaris L. and Beta corolliflora Zoss. in sugar beet. M14, which constituted of the normal 18 Beta vulgaris L. chromosomes with the No.9 chromosome of Beta corolliflora Zoss.(VV+1C,2n=19), was found having a chromosome transmission frequency of 96.5% by transmission analysis. M14 served as ideal materials for study of apomixes phenomenon and the apomixes genes, possessing facultative apomixis characteristics that diplospory apomictic reproduction was the main reproductive mode with secondary reproductive mode of sexual reproduction.298 differential ESTs were obtained by the method of SSH and DDRT-PCR, of which one research value EST was selected as candidate ESTs in this study. Full-length cDNA sequences of M14-341 gene was obtained by RACE. Bioinformatics analysis results of M14-341 gene indicated that M14-341 protein contained MADS-box structural domain and K- structural domain . M14-341 gene had similarity of 88% with Spinacia oleracea anthesis transcription factor, which was assessed to be from MIKC family of MADS-box genes.In this research, M14-341 protein was aligned with MIKC family protein of Arabidopsis thaliana by DNAMAN, which indicated that homology of M14-341 protein with AG protein was up to 74%. M14-341 gene was classified as AG genes, belonging to category C genes of“ABCDE”model of MADS-box genes. Eukaryotic expression vector pBI-M14-341 of full-length cDNA sequence of M14-341 gene was constructed. In order to analyze phenotype properties of transformed plants as well as function of M14-341 gene, M14-341 gene was transformed into tobacco and Arabidopsis thaliana respectively utilizing leaf disc transformation and floral dip transformation mediated by Agrobacterium tumefaciens. The result of analysis of PCR , Southern blot, RT-PCR, Northern blot and GUS reaction showed that M14-341 gene was transformed into model plant, and expressed in flowers, stems and leaves in transformed plants. 24 transgenic tobacco plants and 12 transgenic Arabidopsis thaliana plants were obtained. Quantity of M14-341 gene expression was higher in flower of transgenic plant, and lower in leaf and stem. Constitutive expression of M14-341 gene effected florescence and development of floral organ in transgenic tobacco plants, which emerged a series of abnormal phenotypes, such as delaying of florescence, shoaling of petal color, decreasing inflorescence and so on. Degrading of seed setting rate and increasing of fatality appeared in transgenic Arabidopsis thaliana plants.Two-dimensional gel electrophoresis of non-transgenic and transgenic tobacco floral organ total protein were performed. The results showed that expressed protein spots of transgenic tobacco plants are similar to that of non-transgenic tobacco plants. Expressed protein spots ranged from 20 to 100 kD . 8 remarkable differential expression protein spots were detected, including 1 disappeared spot, 1 newly expressed spot, 5 decreasingly expressed spots and 1 increasingly expressed spot.The results of this research indicated that specifically expressed M14-341 gene from M14 has a similar function with AG genes. M14-341 gene plays an important role in differentiation and development of floral organ as well as in regulating florescence.更多还原