马尾松PmSPX2基因的克隆、原核表达及表达模式分析

Cloning,prokaryotic expression and expression pattern analysis of Pinus massoniana PmSPX2 gene

SPX保守结构域(SPX domain)在植物营养胁迫中发挥着重要作用。SPX2为SPX家族中的SPX亚家族成员,已证明其在拟南芥、水稻中具有调控植物磷信号的功能。通过马尾松幼苗三代全长转录组数据和染色体步移法,克隆PmSPX2基因的全长序列及该基因的启动子序列;分析预测启动子序列上的顺式作用元件;利用IPTG诱导大肠杆菌表达PmSPX2蛋白,将原核表达产物进行质谱鉴定;分析PmSPX2的亚细胞定位;检测PmSPX2在低磷胁迫不同时期马尾松幼苗各组织部位的表达情况。结果表明:PmSPX2cDNA全长2019 bp,最长开放阅读框1059 bp,编码352个氨基酸,蛋白质大小约为39.966 kDa,等电点(pI)为5.91;克隆得到951 bp的PmSPX2启动子序列,预测到MYB转录因子结合位点、缺磷响应元件P1BS(PHR1 Binding Sequence)以及W-box元件等关键顺式作用元件;马尾松缺磷条件下,PmSPX2在不同组织部位均呈差异性表达;亚细胞定位结果显示该基因编码的蛋白定位在细胞核中。本研究为PmSPX2基因参与马尾松缺磷反应及其在磷信号调控机制的研究提供理论依据。

SPX conserved domain(SPX domain)plays an important role in plant nutrition stress.SPX2 is a member of the SPX subfamily of SPX family,and it has function of regulating plant phosphorus signals in Arabidopsis and Oryza sativa.The full cDNA and promoter sequence of PmSPX2 gene were obtained through three-generation full-length transcriptome data of Pinus massoniana and chromosome walking method.The cis-acting elements on the promoter sequence were analyzed and predicted.IPTG was used to induce expression of PmSPX2 protein in Escherichia coli,and the prokaryotic expression product was identified by mass spectrometry.Its subcellular localization was analyzed by employing transient expression system.The expression of PmSPX2 in different tissues of P.massoniana seedlings under low-phosphorus stress was analyzed by RT-qPCR.The results showed that PmSPX2 cDNA has a total length of 2019 bp,the longest open reading frame is 1059 bp,encoding 352 amino acids,the putative protein size is about 39.966 kDa,and the isoelectric point(pI)is 5.91.About 951 bp PmSPX2 promoter sequence was cloned,and key cis-acting elements such as MYB transcription factor binding site,phosphorus-deficiency response element P1 BS(PHR1 Binding Sequence)and W-box element were included in this fragment.The expression analysis showed that PmSPX2 was differentially expressed in different tissues under phosphorus deficiency condition.The results of subcellular localization showed that the protein encoded by this gene was localized in the nucleus.This study provides a theoretical basis for the study of PmSPX2 gene’s involvement in the phosphorus-deficiency response of P.massoniana and its mechanism of phosphorus signal regulation.