摘要
【目的】克隆月季独脚金内酯(SLs)信号应答底物受体即α/β折叠水解蛋白基因Dwarf 14(D14),对其进行亚细胞定位及表达分析,为探究该基因的生物学功能及其在SLs调控月季侧枝发生中的转导机制提供理论支持。【方法】以月季品种滇红为材料,克隆RhD14基因并进行生物信息学分析,同时采用实时荧光定量PCR(qRT-PCR)检测其在不同组织中的表达情况,利用烟草瞬时转化系统对RhD14基因编码蛋白进行亚细胞定位。【结果】克隆RhD14基因(GenBank登录号OP009358)cDNA序列1094 bp,包含完整的开放阅读框(ORF),长度为1045 bp,编码347个氨基酸残基,蛋白分子式为C_(1738)H_(2703)N_(441)O_(510)S_(15),相对分子量为38.41 kD,理论等电点(pI)为5.71,二级结构以α-螺旋和无规则卷曲为主,且RhD14为无跨膜结构和信号肽的稳定蛋白,属于α/β水解酶家族,亚细胞定位于细胞质和细胞膜上。同源序列比对和系统发育进化树分析结果显示,RhD14蛋白的氨基酸序列与同属的古老月季品种月月粉RcD14(XP_024283944.1)的相似性最高为98.05%,其次是同亚科的草莓FvD14(XP_004287076.1),相似性为89.75%,三者亲缘关系较近。实时荧光定量PCR(q RT-PCR)结果显示,RhD14基因在根、茎、叶、腋芽、节和顶芽中均有表达,其中RhD14基因在茎和腋芽中的相对表达量最高,极显著高于根(P<0.01,下同)。去顶处理后RhD14基因在茎和腋芽中的表达模式相似,均较未去顶处理明显上调表达,且随着处理时间的延长均呈先上升后下降的趋势,去顶处理后12 h达峰值,尤其是腋芽中RhD14基因在6、12、24和48 h的相对表达量与未去顶处理差异显著(P<0.05)或极显著。【结论】RhD14基因在细胞质和细胞膜上发挥作用,具有组织特异性,且受去顶诱导高效表达,推测该基因参与腋芽萌发过程。
【Objective】Strigolactone(SLs)signal response substrate receptor,α/βfolded hydrolyzed protein gene Dwarf 14(D14),was cloned from rose for bioinformatics analysis and subcellular localization in order to provide theoretical support for exploring the biological function of D14 gene and its transduction mechanism of SLs in regulating the lateral branching of Rosa hybrida Dianhong.【Method】Rosa hybrida Dianhong was the material,RhD14 gene was cloned and bioinformatics analysis was conducted.The expression levels of RhD14 in different tissues was detected by real-time quantitative PCR(qRT-PCR),and the subcellular localization of the protein encoded by RhD14 gene was performed by a tobacco transient transformation system.【Result】The cDNA sequence of the RhD14 gene(GenBank accession number OP009358)was 1094 bp,with a 1045 bp open reading frame(ORF),encoding 347 amino acid residues.The molecular formula of the protein was C_(1738)H_(2703)N_(441)O_(510)S_(15),the relative molecular weight was 38.41 kD,and the theoretical isoelectric point(pI)was 5.71.α-helix and random coil were the main secondary structures of its amino acid sequence.RhD14 was a stable protein without transmembrane structure and signal peptide,which belonged to theα/βhydrolase family.The subcellular localization results showed that the encoded protein of RhD14 was located in the cytoplasmic and cytomembrane membrane.Homologous sequence alignment and phylogenetic tree analysis showed that the amino acid sequence of RhD14(OP009358)had the highest similarity of 98.05%with that of the old garden rose,Rosa chinensis Old Blush-RcD14(XP_024283944.1),followed by Fragaria vesca subsp.vesca(XP_004287076.1)of the same subfamily with the similarity for 89.75%,which were closely related.The analysis of qRT-PCR showed that RhD14 gene was expressed in all of the test tissues(roots,stems,leaves,axillary buds,nodes,and apical buds).Taking roots as the control,the expression levels of RhD14 in stems and axillary buds were the highest,and were extremely significantly higher than that in roots(P<0.01,the same below).The expression pattern of RhD14 gene in stems and axillary buds after topping treatment was similar,both of which were significantly up-regulated compared with that of non-topping treatment.With the extension of treatment time,the expression of RHD14 gene firstly increased and then decreased,and reached the peak at 12 h after topping treatment.In particular,the relative expression levels of RhD14 gene in axillary buds at 6,12,24 and 48 h were significantly different(P<0.05)or extremely significant different compared with those without topping treatment.【Conclusion】RhD14 gene plays a role in the cytoplasmic and cytomembrane membrane with tissue specificity,which can be highly expressed under topping induction.It is inferred that RhD14 gene participates in the process of axillary bud germination.
作者
杜高齐
李雪娇
许彬
关文灵
孟静
DU Gao-qi;LI Xue-jiao;XU Bin;GUANWen-ling;MENG Jing(College of Horticulture and Landscape,Yunnan Agricultural University,Kunming,Yunnan 650201,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2023年第1期34-45,共12页
Journal of Southern Agriculture
基金
云南省基础研究计划项目(202201AT070256)
云南省科技人才与平台计划(202005AF150035)
云南省教育厅科学研究基金项目(2021J0112)。
