Cloning and Bioinformatics Analysis of Protein Phosphatase Genes MiSTPP1 and MiSTPP4 from Macadamia integrifolia
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摘要:
目的 通过澳洲坚果蛋白磷酸酶基因MiSTPP1和MiSTPP4克隆与生物信息学分析，以期为阐明澳洲坚果蛋白磷酸酶基因的功能及作用机制提供参考依据。 方法 基于澳洲坚果转录组数据和RT-PCR方法克隆澳洲坚果蛋白磷酸酶基因，利用生物信息学分析基因编码蛋白质的同源性、系统进化、理化性质、磷酸化位点、亚细胞定位、跨膜域和信号肽。 结果 获得2个新的澳洲坚果蛋白磷酸酶基因MiSTPP1和MiSTPP4，GenBank登录号分别为MT374548和MT374551。氨基酸同源性分析表明，MiSTPP1和MiSTPP4与其他植物来源的PP1蛋白具有极高的序列相似度，都包含典型结构域MPP_PP1_PPKL。进化树分析表明，MiSTPP1和MiSTPP4与PP1家族蛋白的亲缘关系最近。蛋白基本理化性质分析表明，MiSTPP1是一个不稳定的亲水性蛋白，而MiSTPP4蛋白是一个稳定的亲水性蛋白。磷酸化位点分析表明，MiSTPP1和MiSTPP4都以丝氨酸和苏氨酸磷酸化为主。亚细胞定位、跨膜域和信号肽预测表明，MiSTPP1和MiSTPP4极可能定位于细胞质，且都为非分泌蛋白和非跨膜蛋白。MiSTPP1和MiSTPP4的二级结构和三维结构主要含有α螺旋和无规则卷曲。 结论 MiSTPP1和MiSTPP4属于蛋白磷酸酶PP1家族基因，推测在响应逆境胁迫、信号转导等生理生化过程中发挥作用。 Abstract:Objective Two new protein phosphatase genes, MiSTPP1 and MiSTPP4, from macadamia (Macadamia integrifolia) were cloned for structure and function analyses by bioinformatics. Method The genes were cloned from M. integrifolia using transcriptome sequencing and RT-PCR technique. Homology, phylogenetic evolution, physicochemical properties, phosphorylation sites, subcellular localization, transmembrane domains, and signal peptides of the genes were analyzed. Result The cloned MiSTPP1 and MiSTPP4 from M. integrifolia were assigned with the GenBank accession numbers MT374548 and MT374551, respectively. The amino acid sequences of the genes had a similarity to other plant PP1 proteins containing the same typical structural domain MPP_PP1_PPKL. The evolutionary tree analysis showed that they closely related to PP1 family proteins. The basic physicochemical properties of MiSTPP1 indicated it be an unstable hydrophilic protein, and those of MiSTPP4 a stable hydrophilic protein. Phosphorylation sites on the genes were principally serine and threonine. The predicted subcellular localization, transmembrane domain, and signal peptide suggested that MiSTPP1 and MiSTPP4, as non-secretory and non-transmembrane proteins, were most likely located in the cytoplasm. The secondary and tertiary structures of the genes contained mainly α-helixes and random coils. Conclusion MiSTPP1 and MiSTPP4 from macadamia belonged to the protein phosphatase PP1 gene family and might play a role in response to stress and signal transduction as well as other physiological and biochemical processes of the plant. -
图 1 澳洲坚果MiSTPP1和MiSTPP4基因的克隆
注： M： Marker DL5000； 1： MiSTPP1扩增产物； 2： MiSTPP4扩增产物。
Figure 1. Isolations of MiSTPP1 and MiSTPP4 from M. integrifolia
Note: M: Marker DL5000; 1: amplification product of MiSTPP1; 2: amplification product of MiSTPP4.
图 2 MiSTPP1和MiSTPP4与其他植物PP1蛋白的同源性比对结果
注：结构域MPP_PP1_PPKL用下划线标出，丝氨酸/苏氨酸特异性蛋白磷酸酶特征序列用方框标出。
Figure 2. Homology between MiSTPP1, MiSTPP4, and other PP1 proteins
Note: Domain MPP_PP1_PPKL is underlined, serine/threonine specific protein phosphatases signature is boxed.
图 3 MiSTPP1和MiSTPP4与其他植物PP蛋白的系统进化关系
Figure 3. Relationship among phylogenetic trees of MiSTPP1, MiSTPP4, and other PP proteins
图 4 MiSTPP1蛋白（A）和MiSTPP4蛋白（B）的二级结构分析
Figure 4. Secondary structure of
MiSTPP1 (A) and MiSTPP4 (B) 
图 5 MiSTPP1蛋白（A）和MiSTPP4蛋白（B）的三维结构分析
Figure 5. 3D configurations of MiSTPP1 (A) and MiSTPP4 (B)
表 1 基因MiSTPP1和MiSTPP4克隆所用引物序列
Table 1. Primer sequences used in cloning MiSTPP1 and MiSTPP4
引物名称
Primer names引物序列（5′-3′）
Prime sequences退火温度
Tm/°CMiSTPP1-F TTGCCATTCTCATAATAGAGC 52.0 MiSTPP1-R TACCCAACACCTGTCATAAAC MiSTPP4-F CCCTGCTGTTCTCGATGACTT 55.2 MiSTPP4-R GCTTCTCCCTTTCTTTACACTTTT 
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