蓖麻RcbZIP44基因克隆及表达特性

Cloning and expression characteristics of RcbZIP44 gene in castor

【目的】克隆蓖麻bZIP转录因子基因RcbZIP44,并进行表达特性分析,为探究bZIP转录因子在蓖麻生长发育及非生物胁迫下的生物学功能提供理论参考。【方法】从蓖麻品种通蓖5号中克隆bZIP转录因子基因家族成员RcbZIP44,对其序列进行生物信息学分析,借助烟草瞬时表达系统对RcbZIP44蛋白进行亚细胞定位,通过实时荧光定量PCR检测RcbZIP44基因在不同组织及非生物胁迫下的表达模式。【结果】克隆获得的RcbZIP44基因cDNA全长612 bp,包含1个456 bp的开放阅读框(ORF),编码151个氨基酸残基,无跨膜区域和信号肽结构,为不稳定的亲水性蛋白,存在21个潜在的磷酸化位点,二级结构以α-螺旋为主,定位于细胞核中。RcbZIP44基因启动子区域含有激素响应元件(ABRE、GARE-motif、TATC-box、TGACG-motif)、胁迫响应元件(LTR、ARE、MRE)等,推测RcbZIP44基因在非生物胁迫中发挥重要作用。RcbZIP44基因在茎中的相对表达量最高,显著高于根、子叶和真叶(P<0.05),说明其具有明显的组织表达特异性。在脱落酸(ABA)、盐、干旱和低温胁迫下,RcbZIP44基因在真叶和根中表现出不同的表达模式。RcbZIP44基因对低温胁迫敏感,处理6 h时的相对表达量最高;RcbZIP44基因在ABA和干旱胁迫下呈先增后减的表达模式,在处理12 h时的相对表达量最高;在盐胁迫中RcbZIP44基因表达较为平缓。【结论】RcbZIP44基因编码的蛋白含有bZIP家族特有的bZIP-plant-GBF1结构域,其主要在茎中发挥调控作用,且该基因受ABA、盐、干旱和低温等非生物胁迫诱导表达,推测RcbZIP44基因在蓖麻生长发育及响应非生物胁迫中发挥重要调控作用。

【Objective】Cloned the castor bZIP transcription factor gene RcbZIP44 and analyzed its expression characteristics to provide reference for exploring the biological functions of bZIP transcription factors in castor growth and development and under abiotic stress.【Method】The bZIP transcription factor gene family member RcbZIP44 from the castor variety Tongbi No.5 was cloned,bioinformatics analysis on its sequence was performed,the tobacco transient expression system was used to conduct subcellular localization analysis on the RcbZIP44 protein.The expression pattern of RcbZIP44 gene in different tissues and under abiotic stress was detected by real-time fluorescence quantitative PCR.【Result】The cloned full-length cDNA of the RcbZIP44 gene was 612 bp,containing a 456 bp open reading frame(ORF),encoding 151 amino acid residues.There was no transmembrane region and signal peptide structure.It was an unstable hydrophilic protein with 21 potential phosphorylation sites.The secondary structure was mainly consisted ofα-helix and was located in the nucleus.The promoter region of the RcbZIP44 gene contained hormone response elements(ABRE,GARE-motif,TATC-box,TGACG-motif),stress response elements(LTR,ARE,MRE),etc.It was speculated that the RcbZIP44 gene played an important role in abiotic stress.The relative expression level of RcbZIP44 gene in stems was the highest,significantly higher than that in roots,cotyledons and true leaves(P<0.05),indicating that it had obvious tissue expression specificity.Under abscisic acid(ABA),salt,drought and low-temperature stresses,RcbZIP44 gene showed different expression patterns in leaves and roots.RcbZIP44 gene was sensitive to low-temperature stress,and the relative expression level was the highest when treated for 6 h.RcbZIP44 gene showed an expression pattern of increasing first and then decreasing under ABA and drought stress,and the relative expression level was the highest when treated for 12 h.In salt stress,the expression of the RcbZIP44 gene was relatively stable.【Conclusion】The protein encoded by RcbZIP44 gene contains bZIP-plant-GBF1 domain which is unique in bZIP family.It mainly plays a regulatory role in stems.RcbZIP44 gene is induced by abiotic stresses such as ABA,salt,drought and low temperature.It is speculated that RcbZIP44 gene plays an important regulation role in castor growth and development and in response to abiotic stress.