【作者】 张俊芳；

【导师】 黄俊生；

【作者基本信息】 海南大学 ， 作物遗传育种， 2013， 博士

【摘要】 香蕉是世界重要的热带水果,因其多为三倍体,长期以来,香蕉育种多以筛选突变体为主,然而,人们对香蕉自发突变的分子机理知之甚少。研究突变机理,可以更好的了解突变的规律,为更好的利用突变进行有效的育种提供指导。香蕉果皮带毛突变体由“巴西”香蕉突变而来,其花梗、果实、雄花苞片等部位均表现有毛特征,花期提前。本研究以香蕉果皮带毛突变体为试材,构建突变体／野生型SSH库,筛选与突变有关的候选基因,同时,进一步优化香蕉遗传转化条件,为更好的验证香蕉基因功能奠定基础。主要研究结果如下：利用抑制性差减杂交(SSH)技术,分别以香蕉果皮带毛突变体和野生型为"tester"和‘’driver",构建正反双向两个差减cDNA文库,共获得591个基因片段,其中来自正向文库的325个,来自反向文库的266个。所得序列经BLAST比对分析结果显示：正向文库中206条序列与NCBI数据库中已知功能基因有较高同源性,7条序列与未知功能基因具有同源性,112条序列未能找到同源基因；反向文库中238条序列与NCBI数据库已知功能基因有较高同源性,9条序列与未知功能基因具有同源性,19条序列未能找到同源基因。通过gene ontology (GO)功能推测及归类,可将这些基因分为21亚类。根据前人关于植物表皮毛发育调控的研究结果和差减cDNA文库中筛选到的基因的功能推测,本研究选取4组22个可能与表皮毛发育相关的基因,采用半定量RT-PCR和实时荧光定量PCR的方法分析它们在野生型与突变体中的表达差异及在果实表皮毛发育过程中的表达变化。4组基因如下：(1)3个可能的关键转录因子：①MYB在表皮毛快速发育过程中,野生型中的表达量明显高于突变体中；②WD40在表皮毛发育的初始阶段在突变体中的表达量较高；③B-ZIP在表皮毛发育的成熟期及后期在突变体中的表达量稍高；(2)9个与激素信号相关的基因：①ARP(auxin repressed protein)在表皮毛快速生长期,突变体中表达量高于野生型；②ILR (IAA-amino acid hydrolase ILRl)在果实表皮毛发育过程中表现为折线状,虽然在两种表型中的趋势一致,但在表达量上还有一定差异：③2个ABAR(abscisic acid8’-hydroxylase)表达模式不同,编号M49在突变体和野生型中的表达差异不大,M286在表皮毛发育初期在野生型中的表达明显高于突变体；④4个ARF (auxin response factor)表达趋势各异,但总体表现为在表皮毛快速生长期,野生型中的表达高于突变体；⑤P2/ERF在野生型中的表达量始终高于突变体；(3)4个MAPK基因中,其中1个片段表现为表皮毛发育期在野生型中的表达量较高,另外3个在表皮毛发育期在两种表型差异不大；(4)与细胞分裂有关的6个基因,在两种表型中的变化趋势大体相同,基本上表现为随着果实及其表皮毛的发育表达量逐渐降低。可见,突变引起一系列基因的表达出现变化。MaERF基因在果实表皮毛发育过程中,在野生型中的表达量始终大于在突变体中的表达量。为了深入了解其功能,通过RACE技术获得其cDNA全长,对其编码的氨基酸序列进行生物信息学分析,认为该基因属于ERF亚家族的B2亚群,含有一个AP2/ERF保守结构域,多个亲水区和疏水区,但无跨膜结构,为非分泌蛋白：MaERF基因在基因组中低拷贝存在,含有一个内含子。RT-PCR分析表明,MaERF在香蕉各组织中均有表达,在生殖器官中的表达量较高。MaERF在拟南芥中的超表达研究显示,转基因植株表型没有明显变化,可能与表皮毛发育关系不大。MaMYB基因在突变体和野生型香蕉果皮中表达也有明显差异,通过RACE技术获得其cDNA全长,对其编码的氨基酸序列进行生物信息学分析,认为该基因属于R2R3MYB基因亚族,含有两个保守结构域,多个亲水区和疏水区,但无跨膜结构,为非分泌蛋白；MaMYB基因在基因组中低拷贝存在,含有一个内含子。RT-PCR分析表明,MaMYB在香蕉各组织中均有表达,其中在生殖器官中的表达量较高。MaMYB基因在拟南芥中的超表达研究显示,转基因植株叶片表皮毛减少,花器官表皮毛缺失,根系加长,果夹较野生型短,表明MaMYB可能参与了调控果皮表皮毛的发育,同时还可能与果实发育有关。为了更好的利用功能基因进行香蕉生物技术育种,对“巴西”香蕉胚性悬浮细胞遗传转化体系进行了优化,结果表明悬浮细胞对卡那霉素有一定抗性,适合作为筛选剂的抗生素为潮霉素和除草剂(Basta),浓度分别为10mg/L和5mg/L；悬浮细胞每2周继代一次,以继代5-6d的悬浮细胞为试材,以bar基因为筛选标记,悬浮细胞与菌液共培养6h后直接进行筛选培养,再生植株的转化率较高,可达到75%,阳性植株抗除草剂能力明显提高。更多还原

【Abstract】 Banana (Musa spp.) is very important fruits around the world. For a long time, the principal means for banana breeding were selected mutants because of its triploid. However, the molecular mechanism of banana mutations poorly understood. The law about mutation would be known better and provide more effective guidance for breeding through research mutation mechanism. The pilose fruit mutant was from Brazil banana(Musa spp."Cavendish" AAA), it is characterized with the rachis, fruit and brach were combined by hairy phenotypes, and early flowering. In this research, suppression subtractive hybridization library (SSH) had been built by the mutant and wild and some genes about mutant were selected. To lay the foundation for better validation banana gene function, conditions of embryogenic suspension cells transformation were further optimized. The main results were following:The pros and cons of two subtractive cDNA libraries were built with banana pilose fruit mutant and wild type as "tester" and "driver" by suppression subtractive hybridization (SSH) technology and a total of591gene fragments were obtained, including325from the forward library and266from the reverse library. The results of BLAST analysis showed that the forward library which have206fragments with high homology to known functional genes from NCB1database,7fragments with homology but unknown function,112fragments failed to find the homology gene; the forward library which have266fragments with high homology to known functional genes from NCBI database,9fragments with homology but unknown function,19fragments failed to find the homology gene. These genes can be divided into21categories by gene ontology (GO).According to the results of previous research on the regulation of plant trichome and genes function prediction from subtractive cDNA libraries,4groups including22genes that may be associated with trichome were detected by RT-PCR and fluorescence quantitative Real-time PCR about their differences expression in wild and mutant during fruit development. The results were as follows:(1)3key transcription factors:①In the process of trichomes fast development, MYB was significantly higher expression level in wild than the mutant;②WD40was higher expression level in mutant in the initial stage of the trichome development;③B-ZIP was slightly higher expression levels in mutant in the maturity and late stage of the trichome development;(2)9genes about hormone-related:①ARP (auxin repressed protein) was higher expression in mutant than wild in the process of trichomes fast development;②ILR was similar expression trend but some differences level in the two phenotypes of fruit development;③The two ABAR (abscisic acid8’-hydroxylase) genes had different expression patterns, the expression of No. M49was little difference in mutant and wild, the expression of No. M286was significantly higher in wild than mutant:④4ARF (auxin response factor) were different expression, they were all higher expression in wild than mutant in the process of trichomes fast development;⑤AP2/ERF was always higher expressionn wild than mutant;(3)1gene of4MAPK were high expression in the wild type, the expression of the other3genes were little difference;(4)6genes related to the cell division were similar expression in two phenotypes, the more development of fruit, the lower expression.So, a series of genes expression had been changed in mutation.MaaERF is always higher expression in the wild than the mutant during fruit development. Full-length cDNA of MaERFwas obtained by RACE technology. The analysis of the amino acid sequence of this gene showed it belongs ERF subfamily B2subsets contains one AP2/ERF conserve domain, some hydrophilic and hydrophobic regions, but no transmembrane domain structure; MaERF gene contains one intron and low-copys in the genome. RT-PCR analysis showed that MaERF was expressed in all organizations of banana, but the expression was higher in reproductive organs. Overexpression MaERF in Arabidopsis, there was no significantly diversification of phenotype, indicating that the gene were little effect in the process of trichome.One AYB gene named MaMYB was obtained by RACE technology for better understood its role in the banana peel trichome process. The analysis of the amino acid sequence of this gene showed it belongs R2R3MYB subgenotype family, contains two conserve domains, some hydrophilic and hydrophobic regions, but no transmembrane domain structure; MaMYB gene contains one intron and low-copys in the genome. RT-PCR analysis showed that MaMYB was expressed in all organizations of banana, but the expression was higher in reproductive organs. Overexpression MaMYB in Arabidopsis, the trichomes reduced in transgenic plants leaves and missing in their flower organs, roots were longer, the fruit clip shorter than the wild, so that MaMYB not only participated in the development of trichomes but also related to fruit development. In the process of transformation about banana embryogenic suspension cells, the suspension cells had some resistance to kanamycin, hygromycin and herbicide (Basta) were suitable as a selecting agent antibiotic, and the concentrations were10mg/L and5mg/L; Banana embryogenic suspension cells were subculture5-6days, and co-culture with Agrobacterium6h and then direct selected on selective medium, bar gene as a selection marker,75%regenerated plants were positive plants, and their herbicide-resistant capacity has significantly improved.更多还原