Cloning, subcellular localization and expression analysis of CiSPL2 gene in pecan

【Objective】This study conducted the gene cloning of the pecan SQUAMOSA promoter binding protein(SPL) transcription factor gene CiSPL2,subcellular localization and expression analysis were also conducted,in order to provide a theoretical basis for exploring the molecular mechanism of this gene during the female flower bud differentiation in pecan. 【Method】The coding region(CDS) sequence of CiSPL2 gene was cloned using RT-PCR based on the pecan genome data,and the bioinformatics software was used to analyze its sequence characteristics and protein physical and chemical properties. The pCAMBIA1300-CiSPL2-GFP fusion expression vector was constructed,and subcellular localization was determined by observing fluorescence signals after transient transformation of tobacco. The expression patterns of Ci SPL2 gene in different tissues and female flower bud differentiation stages were studied based on the transcriptome data and real-time fluorescence quantitative PCR. 【Result】The CDS length of the CiSPL2 gene in pecan was 1398 bp,encoding a total of 465 amino acids residues with a relative molecular weight of 51.78 k D. The theoretical isoelectric point(pI) was 8.28,the instability coefficient was 49.89,the fatty acid amino acid index was 64.62,and the grand average of hydropathicity(GRAVY) was-0.617,indicating the protein was an unstable hydrophilic protein. The CiSPL2 protein sequence contained an SBP domain between the 183^rd and 257^th amino acids. Subcellular localization showed that CiSPL2protein was located in the nucleus. The secondary structure of CiSPL protein was mainly composed of α-helix(17.42%),extended chain(13.55%), β-turn(1.72%) and random coil(67.31%). CiSPL2 protein in pecan had the highest similarity with JrSPL2 protein in walnut(95.05%),followed by Betula platyphylla BlSPL2 protein with a similarity of 70.83%.CiSPL2 protein had the closest genetic relationship with the walnut JrSPL2 protein. The CiSPL2 gene promoter contained plant hormone response,drought stress response,and meristem expression elements. The expression level of CiSPL2 gene in female flowers and fruits was significantly higher than that in other tissues(P<0.05). The CiSPL2 gene was expressed at all stages of female flower bud differentiation,with the highest expression level during the formation of female inflorescence. 【Conclusion】The CiSPL2 gene contains a typical SBP conserved domain of the SPL transcription factor family,which may be involved in regulating differentiation and development of female flower buds in pecan.