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

【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.