澳洲坚果MiSTPP3和MiSTPP7基因克隆与表达分析

以澳洲坚果品种‘O.C.’为试材,利用RT-PCR技术克隆获得2个丝氨酸/苏氨酸磷酸酶基因,分别命名为MiSTPP3和MiSTPP7,其编码序列长度分别为2095 bp和1700 bp。序列分析和系统进化分析表明,MiSTPP3含有MPP_PP5_C结构域,属于PP5亚家族;MiSTPP7含有MPP_PP7结构域,属于PP7亚家族。蛋白理化性质和亚细胞定位分析表明,MiSTPP3是一个稳定的亲水性蛋白,可能定位于质膜;MiSTPP7是一个不稳定的亲水性蛋白,可能定位于线粒体基质。生物信息学分析表明,MiSTPP3和MiSTPP7都是非分泌跨膜蛋白,其二级和三级结构的主要元件都是α螺旋、无规则卷曲和延伸链。RT-qPCR分析表明,MiSTPP3和MiSTPP7在澳洲坚果根、茎、叶、花和小果中都有不同的表达量;MiSTPP3受低温胁迫下调表达,而MiSTPP7上调表达,且二者都受干旱胁迫上调表达。因此,推测MiSTPP3和MiSTPP7可能参与澳洲坚果的生长发育及逆境胁迫反应。

Cloning and Expression Analysis of MiSTPP3 and MiSTPP7 from Macadamia integrifolia

Two serine / threonine phosphatase genes were named MiSTPP3 and MiSTPP7, with coding sequences 2095 bp and 1700 bp in length, respectively, which were cloned by RT-PCR from macadamia cultivar ‘O.C.’ (Macadamia integrifolia). Sequence analysis and evolution analysis indicated that MiSTPP3 with the MPP_PP5_C domain belonged to PP5 subfamily, and MiSTPP7 with the MPP_PP7 domain belonged to PP7 subfamily. Physicochemical properties of protein and subcellular localization analysis showed that MiSTPP3 as a stable hydrophilic protein may be located in the plasma membrane, and MiSTPP7 as an unstable hydrophilic protein may be located in the mitochondrial matrix. Bioinformatics analysis indicated that MiSTPP3 and MiSTPP7 were non-secreted transmembrane proteins, α-helix, random coil and extended strand were the backbone of the secondary and 3D protein structures. RT-qPCR analysis indicated that MiSTPP3 and MiSTPP7 expressed differently in roots, stems, leaves, flowers and small fruits of M. integrifolia; MiSTPP3 was down regulated by low temperature stress, while MiSTPP7 was up-regulated, and both of them were up-regulated by drought stress. The results suggested that MiSTPP3 and MiSTPP7 may be involved in the growth and development of M. integrifolia and the stress response.