龙眼DlbZIP21基因克隆与表达分析

Cloning and Expression Analysis of DlbZIP21 in Longan

【目的】分析bZIP转录因子基因DlbZIP21在石硖和四季蜜两个龙眼品种不同组织和年周期表达水平的差异性,为揭示不同龙眼品种成花差异的分子调控机制提供理论依据。【方法】以石硖和四季蜜龙眼为试材,克隆其DlbZIP21基因,并对其编码蛋白理化性质特性、磷酸化位点和结构域进行生物信息学分析,利用实时荧光定量PCR分析该基因的时空表达特征。【结果】克隆获得石硖和四季蜜龙眼DlbZIP21基因分别命名为DlbZIP21-sx和DlbZIP21-sj,其开放阅读框(ORF)均为1353 bp,各编码450个氨基酸残基,预测蛋白质分子量分别为49.80和49.81 kD,理论等电点均为6.53。BLAST比对和系统发育进化树分析结果表明,DlbZIP21-sx与DlbZIP21-sj蛋白的同源性为99.78%,同时与大豆、水稻和菜豆的亲缘关系较近。DlbZIP21基因在花芽中的表达量表现为四季蜜龙眼比石硖龙眼高5倍;在年周期中,从1~12月DlbZIP21基因在四季蜜龙眼中的表达量均普遍高于石硖龙眼,其中在花芽分化期和开花期(12月—翌年4月)的表达量高于石硖龙眼6~8倍。【结论】DlbZIP21基因可能在四季蜜龙眼成花中发挥重要作用,且在诱导四季蜜龙眼一年多次成花过程中扮演重要角色。

【Objective】In order to reveal the molecular regulation mechanism and provide a theoretical basis of flower formation differences between longan cv.‘Shixia’and longan cv.Sijimi,the gene DlbZIP21 of the two varieties were cloned to analyze the differences in the expression levels of different tissues and annual cycles.【Method】The DlbZIP21 gene was cloned from‘Shixia’and Sijimi longan as template.Bioinformatics softwares were used to analyze the protein characteristics,phosphoric acid sites and structural domains encoded by the DlbZIP21 gene,spatiotemporal expression characteristics were analyzed by qRT-PCR.【Result】The cloned DlbZIP21 gene of‘Shixia’and‘Sijimi’was named DlbZIP21-sx and DlbZIP21-sj,respectively.The ORF length of the genes were both 1353 bp,both encoding 450 amino acids,and the predicted molecular weights of the protein were 49.80 and 49.81 kD,respectively.And the isoelectric point was 6.53.The result of Blast comparison indicated that DlbZIP21-sx and DlbZIP21-sj were close to soybean,rice and kidney bean.Phylogenetic tree analysis showed that the homology between DlbZIP21-sx and DlbZIP21-sj was 99.78%.It was also closely related to soybean,rice and kidney bean.Tissue expression levels analysis revealed that DlbZIP21 gene in flower bud of Sijimi was greatly higher than Shixia,which was 5 times higher.In the annual cycle,from January to December,the expression level of DlbZIP21 gene in Sijimi was generally higher than Shixia.The expression level of DlbZIP21 gene in‘Sijimi’was higher than Shixia at flower bud differentiation stage and flowering stage(December-April of the following year),which was 6-8 times higher.【Conclusion】DlbZIP21 gene plays a vital role in flower formation of longan and plays an important role in inducing longan to blossom many times a year.