马尾松蔗糖磷酸合酶基因的克隆和表达分析

Cloning and expression analysis of Pinus massoniana sucrose phosphate synthase gene

通过挖掘4个马尾松蔗糖磷酸合酶基因(PmSPS1~PmSPS4),探究SPS基因在马尾松中的表达特性及功能。利用马尾松转录组数据挖掘SPS基因,对基因进行克隆及生物信息学分析,用实时荧光定量PCR检测其在马尾松不同组织、高低产脂种质和低磷胁迫中的相对表达量。结果从马尾松针叶中克隆获得4个SPS基因,开放阅读框(open reading frame, ORF)长度为1 701~3 243 bp,编码566~1 080个氨基酸。进化分析表明,PmSPS1和PmSPS3同属A亚族,PmSPS2和PmSPS4同属B亚族。组织特异性分析发现,PmSPS2和PmSPS4在针叶中表达量最高,PmSPS1和PmSPS3在树皮中表达量最高;PmSPS1和PmSPS3在幼嫩组织和成熟组织表达差异不显著,PmSPS2和PmSPS4在成熟针叶中的表达量显著高于嫩叶(P<0.05),在嫩茎和成熟茎间差异不显著。PmSPS1和PmSPS3的表达量在高、低产脂种质中差异不显著,PmSPS2和PmSPS4在高产脂种质针叶中的表达水平高于低产脂,在高产脂木质部中低于低产脂。低磷胁迫处理后,4个PmSPS基因在针叶中的表达水平均为上调,其中PmSPS2基因表达量最高,达到对照组的3.18倍。综合以上分析结果,克隆得到的4个马尾松SPS基因分布于A、 B亚族中,不同亚族基因呈现出不同的表达特性,为后续深入研究SPS家族基因在马尾松中的功能提供了理论依据。

To determine function and expression patterns of the sucrose phosphate synthase(SPS)gene in Masson pine(Pinus massoniana Lamb.),four sucrose phosphate synthase genes(PmSPS1 to PmSPS4)were studied.The four SPS genes were identified using Masson pine transcriptome data and were cloned from Masson pine.Bioinformatic analysis was conducted and the relative expressions of the genes were detected by qRT-PCR in different tissues,different-yield oleoresin germplasm,and following stress caused by low levels of phosphorus.The open reading frames of the genes ranged from 1701 to 3243 bp,encoding 566 to 1080 amino acids.Phylogenetic tree analysis confirmed that PmSPS1 and PmSPS3 belonged to subfamily A,with PmSPS2 and PmSPS4 in subfamily B.Tissue-specific expression analysis revealed that PmSPS2 and PmSPS4 were most highly expressed in needles,with PmSPS1 and PmSPS3 obviously expressed in bark.The expression of PmSPS1 and PmSPS3 did not highly differ between young and mature tissues.The expressions of PmSPS2 and PmSPS4 were significantly higher(P<0.05)in mature needles than in young needles,while there were no significant changes between young and mature stems.In addition,the expression levels of PmSPS1 and PmSPS3 were not significantly different between high and low-yield oleoresin germplasm,while the expression levels of PmSPS 2 and PmSPS 4 in needle of high-yield oleoresin germplasm were higher than those of low-yield oleoresin germplasm and lower in high-yield oleoresin xylem than in low-yield.The expression levels of all four PmSPS genes were up-regulated in needles in the presence of low phosphorus.PmSPS 2 expression was the highest,being 3.18 times higher than that of the control.The collective results showed that the four SPS genes obtained by cloning were distributed in subfamilies A and B.The genes of different subfamilies showed different expression characteristics.The results provide a theoretical basis for the subsequent in-depth study of the functions of SPS family genes in Masson pine.