辣椒水通道蛋白基因CaAQP的克隆与序列分析

 【目的】分析辣椒水通道蛋白基因CaAQP的序列特征,并研究其在抗寒品种P70中经4℃低温胁迫处理后的表达模式,为探讨辣椒的耐寒机理及辣椒品种耐寒性改良积累资料。【方法】利用RACE 技术进行cDNA全长克隆,采用生物信息学软件分析克隆基因的编码蛋白特性,并以4℃低温胁迫处理不同时间的抗寒品种P70为材料,利用Real time-PCR分析所克隆基因在辣椒低温胁迫前后的表达模式,以25℃处理为对照。【结果】在4℃低温胁迫处理后的耐寒辣椒品种P70叶片中分离到与水通道蛋白基因相关的差异表达片段,并利用RACE技术克隆得到该基因的全长cDNA,命名为CaAQP,登录号为GU116569。序列分析表明,该基因大小为1 032 bp,含5′非编码区为69 bp,3′非编码区为210 bp,CDS长753 bp,编码250个氨基酸, 蛋白分子量为25.7 kD。应用生物信息学软件对CaAQP分析表明,CaAQP含有6个跨膜区,有2个NPA单元,其氨基酸残基与MIP家族蛋白保守区序列完全一致。氨基酸序列比对发现,该序列与其它物种TIP类液泡膜水通道蛋白氨基酸序列有很高的同源性。聚类分析表明,CaAQP与番茄液泡膜水通道蛋白遗传距离最近,与禾本科的玉米和大麦遗传距离相对较远。Real time-PCR分析结果证实,该基因在低温胁迫下呈现下调表达的趋势。【结论】利用cDNA-AFLP并结合RACE技术首次在耐寒辣椒品种中克隆了水通道蛋白基因CaAQP,该基因属于MIP蛋白家族的一员,具有其典型的功能域,表达模式分析表明,该基因在低温胁迫过程中具有重要的调控作用,该研究结果为进一步探讨辣椒逆境胁迫过程中基因表达调控机制提供了重要信息。

Cloning and Sequence Analysis of the Aquaporins Gene CaAQP in Pepper

Abstract: 【Objective】 To provide an insight into mechanism of pepper’s cold resistance and cultivar development,the sequence characteristics of the aquaporin gene CaAQP in pepper were analyzed and the expression profiling of the chilling tolerant Capsicum annuum cv. P70 was studied after treatment at 4℃ cold stress. 【Method】 The rapid-amplification of cDNA ends (RACE) was used to amplify the full-length CaAQP gene, and the bioinformatics software was employed to analyze the structures and function of the coded protein. At the same time, the chilling tolerant pepper P70 treated at 4℃ was used as material and the real time-PCR was adoped to study the expression profile of gene CaAQP, the peper cv. treated at 25℃ was used as control. 【Result】 A gene encoding the aquaporin cDNA (designated as CaAQP, GenBank Accessin No. GU116569) was cloned from leaf of Capsicum annuum cv. P70 by RACE. The full-length CaAQP gene cDNA was 1 032 bp in length, which included a potential open reading frame of 753 bp, a 5′-untranslated region (5′-UTR) of 69 bp and a 3′-untranslated region (3′-UTR) of 210 bp, encoded a protein of 250 amino acids with a predicted molecular mass of 25.7 kD. Bioinformatics analysis demonstrated that CaAQP exhibited a typical structure with six membrane-spanning domains and an internal symmetry showing two highly conserved Asn-Pro-Ala (NPA) motifs, and possessing the MIP family signal consensus sequence. The CaAQP amino acids showed high identity with other 10 plant species TIP subfamily by NCBI homology comparison analysis. Phylogenetic analysis indicated that CaAQP was clustered with the SLAQP from Solanum lycopersicum. Real time-PCR analysis showed that the expression of CaAQP was down-regulated by low temperature stress. 【Conclusion】 An aquaporin gene CaAQP was firstly cloned by cDNA-AFLP and RACE from leaf of Capsicum annuum cv. P70. The CaAQP gene is a member of the MIP protein family, and has the typical function region. The expression profiling of this gene indicates that the CaAQP gene may play an important regulating role during low temperature stress. This study provides important information for the future research on the gene-expression regulation during cold stress.