根据在香蕉果实抑制缩减文库中获得的1个14-3-3蛋白基因片段,采用PCR和RACE相结合的方法从香蕉(Musaacuminate L. AAA group cv. Brazilian)cDNA文库中筛选到其全长cDNA序列。序列测定和Blastx比对分析结果表明,该cDNA全长1 037 bp,含有1个完整的阅读框,其编码区编码261个氨基酸残基,具有植物14-3-3蛋白基因的保守结构域,并与其他植物来源的14-3-3蛋白具有很高的同源性,将其命名为Ma-14-3-3b(Musa acuminate 14-3-3)。采用遗传进化系统发育树的分析结果表明,Ma-14-3-3b与来源于单子叶植物的多数14-3-3蛋白基因序列在同一个进化枝上。采用RT-PCR对其在香蕉果实发育不同时期的表达进行分析结果表明,在香蕉果实发育的不同时期差异表达,推测其可能在果实的发育中起作用。
[Objective] The aim of the study is to clone and analyze the gene encoding 14-3-3 protein from banana. [Method] Combined with PCR amplification, RACE (rapid amplification of cDNA ends) technique was employed to clone 14-3-3 gene from banana; then the amplified sequence was sequenced and homologically analyzed. [Result] A new cDNA homologous with 14-3-3 protein genes were obtained by RT-PCR and RACE ( rapid amplification of cDNA ends ) approaches. The full length of this cDNA was 866 bp encoding 197 amino acids. Alignment of deduced amino acid sequence with those from other plants revealed that the cDNA shared high homology with 14-3-3 protein genes from other plants, and was designated as Musa acuminata 14-3-3 gene (Ma-14-3-3d). Phylogenetic analysis reveals that Ma-14-3-3d has closer genetic relationship with those from monocotyledon species than those from other species. [Conclusion] Ma-14-3-3d belongs to the same lineage of 14-3-3 from monocotyledon.
香蕉在进行遗传转化过程中,外植体容易褐化,从而降低了再生频率,影响到遗传转化的效率。为降低香蕉转化过程中外植体的褐化率,采用香蕉(Musa AAA group cv.Brazilian)未成熟雄花作为外植体,以农杆菌介导法进行遗传转化。结果表明,当改良的MS培养基中铵态氮与硝态氮(NH4+/NO3-)的摩尔比为20.6:67.6时,具有较强的抗褐化能力。当6-BA浓度为1.0mg/L时,外植体易于诱导出胚状体。
ROP(Rho-related GTPases from plants)蛋白是一类在植物信号转导方面有重要作用的蛋白质.将香蕉中的MaROP1基因连接在pET-30a载体上,并在大肠杆菌BL21(DE3)中诱导表达,获得了融合表达蛋白.将融合蛋白免疫新西兰大白兔制备了MaROP1的高效价、特异的多克隆抗体,为进一步深入研究MaROP1的生物学特性及其在香蕉果实成熟过程中的功能奠定了基础.
