月季RhMAX2A基因的克隆与表达分析

Cloning and Expressions of RhMAX2A in Rosa hybrida

【目的】克隆月季(Rosa hybrida L.)RhMAX2A基因c DNA序列,分析其序列特征及其在不同组织中和去顶后的表达情况,为探究该基因在月季中的生物学功能及调控侧枝发生的转导机制提供理论支持。【方法】以月季品种滇红(Rosa hybrida‘Dianhong’)为材料,通过RT-PCR技术克隆RhMAX2基因的cDNA序列,利用生物信息学方法对其序列和所编码的蛋白质进行分析,利用烟草(Nicotiana tabacum)瞬时转化技术分析蛋白的亚细胞定位,同时采用实时荧光定量PCR(qRT-PCR)检测其在不同组织中及去顶后的表达情况。【结果】RhMAX2A基因(GeneBank登录号为OP055810)cDNA序列长1030 bp,编码246个氨基酸,该蛋白分子式为C_(2910)H_(4793)N_(1029)O_(1244)S_(210),相对分子质量为27.35 kD,总原子量为3909;该蛋白不稳定系数为53.07,脂肪系数为106.30,GRAVY值为0.049,是一类不稳定亲水性蛋白;RhMAX2A蛋白的二级结构主要由α-螺旋和无规则卷曲构成,且RhMAX2A为推定的F-box结构域,属于α/β水解酶家族。同源序列比对和系统进化树关系分析结果表明,RhMAX2A氨基酸序列(OP055810)与同属的古老月季品种月月粉(Rosa chinensis‘Old Blush’)氨基酸序列(XP_024283944.1)相似性最高,其次是同亚科的草莓(Fragria vesca subsp.vesca)(XP_004287076.1),三者亲缘关系较近。亚细胞定位结果显示,RhMAX2A编码蛋白位于细胞核。qRT-PCR检测结果显示,RhMAX2A基因在根、腋芽和节中表达,根中表达量最高,去顶处理显著上调RhMAX2A基因在根和腋芽中的表达。【结论】成功克隆了滇红RhMAX2A基因,其编码蛋白在细胞核上发挥作用,主要在根和腋芽中表达,且受去顶诱导上调表达。

【Objective】Biological functions and lateral branching transduction mechanism of RhMAX2A were investigated by cloning the cDNA and determining the after-decapitation expressions in tissues of Rosa hybrida.【Methods】The cDNA sequence of RhMAX2A was cloned from hybrid tea rose Dianhong by RT-PCR for a bioinformatic analysis.The subcellular location of the gene was determined by transient transformation of PC1300s-RhMAX2A-GFP in tobacco leaves.After decapitating the plant,expressions of the gene in different organs were determined by qRT-PCR.【Results】The cDNA of RhMAX2A(GeneBank accession number:OP055810)was 1030 bp in length encoding 246 amino acids with the chemical formula of C_(2910)H_(4793)N_(1029)O_(1244)S_(210),a molecular weight of 27.35 kD,and a total atomic weight of 3909.The instability coefficient of the unstable hydrophilic protein was 53.07,the fat coefficient,106.30,and the GRAVY value,0.049.The protein secondary structure was mainlyα-helix and random coil of a presumed F-box domain belonging to theα/βhydrolase family.RhMAX2A(OP055810)had the highest homology with that in R.chinensis Old Blush(XP_024283944.1)followed by that in Fragaria vesca subsp.vesca(XP_004287076.1)of the same closely related subfamily.The encoded protein was in the nucleus.The gene expressed most highly in the roots but also in the axillary buds,and nodes among tested organs.Decapitation on the plant significantly upregulated RhMAX2A in the roots and axillary buds.【Conclusion】RhMAX2A was successfully cloned from R.hybrida Dianhong,which functioned in the nucleus,expressed mainly in the roots and axillary buds,and could be upregulated by plant decapitation.