澳洲坚果MiSTPP6基因克隆及低温胁迫下表达分析

Cloning and expression analysis of MiSTPP6 gene from Macadamia integrifolia under cold stress

  • 摘要: 【目的】克隆澳洲坚果丝氨酸/苏氨酸蛋白磷酸酶(PP1)家族基因(MiSTPP6),并分析其不同组织及低温胁迫下的表达模式,为澳洲坚果PP1家族基因的抗寒分子机制提供理论依据。【方法】基于澳洲坚果转录组测序结果,利用RT-PCR技术克隆MiSTPP6基因,对其序列进行生物信息学分析,并利用实时荧光定量PCR检测其在不同组织及低温胁迫下的表达情况。【结果】从澳洲坚果中克隆获得MiSTPP6基因(GenBank登录号为MT374553),其cDNA全长2129bp,最大的阅读框(ORF)长度为948bp,编码315个氨基酸残基,含有PP1蛋白家族的典型结构域(MPP_PP1_PPKL)和特征性序列(LRGNHE)。MiSTPP6蛋白为稳定的亲水性蛋白,以丝氨酸磷酸化为主,可能定位于细胞质,属于非分泌型蛋白或膜蛋白,与已知植物PP1蛋白的氨基酸序列具有很高的相似性。MiSTPP6蛋白的二级结构由α-螺旋(占40.00%)、无规则卷曲(占32.38%)、延伸链(占18.41%)和β-转角(占9.21%),其三级结构与模板蛋白4v0u.1.A的结构相似度为80.60%,GMQE值为0.89。MiSTPP6基因在根、茎、叶、刚开放的小花和谢花后30~45d的小果中均有表达,其中,MiSTPP6基因在刚开放的小花中的相对表达量最高。4 ℃低温胁迫后0.5~24.0h,MiSTPP6基因在澳洲坚果苗期叶片中的相对表达量较对照明显下调,整体上呈持续下降的趋势。【结论】MiSTPP6属于PP1家族基因,具有组织表达特异性,可能在澳洲坚果抗寒分子机制中发挥重要的调控作用。

     

    Abstract: 【Objective】To clone serine / thonine protein phosphatase(PP1)family gene MiSTPP6 from macadamia (Macadamia integrifolia)and analyze its expression patterns in different tissues and cold stress,so as to provide scientific basis for the molecular mechanism of PPI family gene in response to cold stress.【Method】Based on the transcriptome sequencing of macadamia,MiSTPP6 gene was cloned by RT-PCR and its sequences were analyzed by bioinformatics,and its expression in different tissues and cold stress was analyzed by real-time fluorescent quantitative PCR.【Result】A new protein phosphatase gene MiSTPP6 was cloned from M. integrifolia(GenBank accession number was MT374553). The full length cDNA and open reading frame(ORF)were 2129 bp and 948 bp,encoding 315 amino acidswith MPP_PP1_ PPKL domain and signature sequence(LRGNHE)belonged to the PP1 protein family. MiSTPP6 was a stable hydrophilic protein and mainly phosphorylated by serine,which might be located in the cytoplasm. MiSTPP6 was non-secreted transmembrane protein,which had high similarity with the amino acid sequence of known plant PP1 proteins. The secondary structure of MiSTPP6 consisted of α - helix(40.00%),random coil(32.38%),extended strand(18.41%)and β - turn (9.21%).The similarity of tertiary structure between MiSTPP6 and the template protein 4v0u.1.A was 80.60%,and the GMQE value was 0.89. MiSTPP6 was expressed in different tissues(roots,stems,leaves,small flowers and small fruits 30-45 d after blossom falling)of macadamia,with the highest expression in opened flower. After low temperature(4 ℃) stress for 0.5-24.0 h,the relative expression of MiSTPP6 in macadamia seedling leaves was significantly down-regulated compared with the control,and the overall trend continued to decrease.【Conclusion】MiSTPP6 belongs to PP1 family gene and has tissue expression specificity,it may play an important regulatory role in the molecular mechanism of response to cold stress in macadamia.

     

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