矮牵牛3个赤霉素2-氧化酶基因的克隆及初步功能分析

摘要

赤霉素(GA)是一类重要的植物内源性激素,在植物生长和发育过程中发挥重要的作用,其调控作用也极大地影响观赏植物的观赏性.矮牵牛(Petunia hybrida Vilm)是进行比较基因组学研究的优良材料,同时也是一个模式观赏植物.目前,在拟南芥(Arabidopsis thaliana)和水稻(Oryza sativa)中,大多数与赤霉素生物合成和降解相关的基因已经被克隆和研究,但在矮牵牛中鲜有相关的研究报道.本实验利用RACE技术从矮牵牛中获得了3个赤霉素2-氧化酶的全长cDNA序列,分别命名为PhGA20x1～3(登录号:GU059939、JQ323102和JQ323101).系统发生分析显示这3个基因都属于C19GA2氧化酶分支,与番茄(Solanum lycopersicum)的SlGA20x2,4和5,烟草(Nicotiana tabacum)的NtGA20x1～3最为接近.荧光定量PCR分析表明,PhGA20x1～3的表达谱相似,均在茎、叶、开花第一天的雄蕊和雌蕊、开始萎焉的花瓣中有较高的表达,在萼片、开花第一天的花瓣、授粉后7d的果实中表达量极低.不过,PhGA20x3 mRNA的丰度较PhGA20x1和2的低很多,当使用相同的模板和扩增效率相似的引物时,其Ct值较PhGA20x1和2的分别低4～5个循环(Ct PhGA2ox1～26,Ct PhGA2ox2～25,Ct PhGA2ox3～30).利用根癌农杆菌(Agrobacterium tumef aciens)介导法获得2×35S::PhGA20x1转基因矮牵牛植株表现出节间长度明显变短、叶色变深等缺乏赤霉素的表型,提示了PhGA20x1可能编码一个功能基因.%Gibberellins (GAs) are endogenous hormones that play important roles in the regulation of plant growth and development.Their regulation functions also have a great impact on the plant's ornamental performance.Petunia is a model system for comparative research,and a model plant for investigating the traits that influence omamental performance.Although most of the genes involved in the biosynthesis and catabolism of the bioactive GAs have been identified in Arabidopsis thaliana and rice(Oryza sativa),no GA metabolism genes in petunia (Petunia hybrida) had been cloned and analysed previously.In this study,three full-length GA 2-oxidase cDNAs were isolated from petunia using a RACE PCR method and we named them PhGA2ox1～3(GenBank accession numbers:GU059939,JQ323102 and JQ323101).Evolutionary analysis suggested that proteins encoded by the three cDNAs all belong to C19 CGA 2-oxidase clade,closing to SlGA2ox2,-4,and-5 of Solanum lycopersicum and NtGA2ox1～3 of Nicotiana tabacum,qRT-PCR analysis indicated that their expression profiles were similar.A relatively higher expression level was observed in stems,leaves,stamens and pistils on the first day of anthesis,and in wilting petals,compared to a very low expression level in sepals and petal on the first day of anthesis and in fruits seven days after pollination.However,PhGA2ox3 transcripts were much less abundant than those of PhGA2ox1 and-2 as the cycle threshold (Ct) values of PhGA2ox3 were four cycles lower than those of PhGA2ox1 and-2 for the same eDNA template (Ct phCA2ox1～26,Ct PhGA2ox2～25 and Ct PhGA2ox～30),using primers with similar efficiency.2 ×35S::PhGA2ox1 transgenic petunia plants were created by Agrobacterium tumefaciens-mediated transformation.The transgenic plants exhibited various degrees of GA-deficient phenotypes such as shortened internodes,smaller and dark-green leaves,which were reported and well described by other researchers,suggesting the PhGA2ox1 eDNA encodes a functional gene.