马尾松PmPGK1和PmGPIC基因的克隆和表达分析

Cloning and Expression Analysis on PmPGK1 and PmGPIC Genes in Pinus massoniana

摘要:

为了解马尾松（Pinus massoniana）磷酸甘油酸激酶1（PGK1）与胞质溶胶葡萄糖磷酸异构酶（GPIC）的功能，采用RACE技术克隆了PmPGK1和PmGPIC基因，并进行了生物信息学分析与亚细胞定位，采用实时荧光定量PCR技术分析PmPGK1和PmGPIC的表达特性。结果表明，PmPGK1和PmGPIC全长为2 106和1 848 bp，分别编码507和566个氨基酸。PmPGK1和PmGPIC分别定位于叶绿体和胞质溶胶。PmPGK1表达量为新叶 > 老叶 > 新茎 > 根 > 花；而PmGPIC为老叶 > 花 > 新叶 > 新茎 > 根。低温胁迫24 h，PmPGK1和PmGPIC的表达量均随时间延长先降低后升高，且PmGPIC的表达量在处理2 h后即降至较低水平；高浓度CO₂胁迫24 h，PmPGK1的表达量随时间延长呈降低-升高-再降低的变化趋势，PmGPIC的表达下调但变化较不显著。因此，推测PmPGK1主要参与卡尔文循环及叶绿体/质体糖酵解，PmGPIC主要参与细胞质基质糖酵解；PmPGK1、PmGPIC活性在低温胁迫下均受抑制；PmPGK1活性在CO₂胁迫下受到显著抑制，而PmGPIC活性的影响不大。

Abstract:

To understand the functions of phosphoglycerate kinase 1 (PGK1) and cytosolic glucose phosphate isomerase (CPIC) of Pinus massoniana, the cDNA of PmPGK1 and PmGPIC were cloned by RACE, and the bioinformatic and subcellular localization of PmPGK1 and PmGPIC were analyzed, and then their expression patterns were performed by qRT-PCR. The results showed that the full-length cDNA of PmPGK1 and PmGPIC were 2 106 and 1 848 bp, encoding 507 and 566 amino acids, respectively. PmPGK1 and PmGPIC proteins were located in chloroplast and cytosol, respectively. The expression of PmPGK1 was in order of new leaf > old leaf > new stem > root > flower, while that of PmGPIC was old leaf > flower > new leaf > new stem > root. Under low temperature stress for 24 hours, the expression of PmPGK1 and PmGPIC decreased at first and then increased, and the expression of PmGPIC decreased to a low level after 2 hours. Under high CO₂ stress for 24 hours, the expression of PmPGK1 was significantly down-regulated, showing a trend of decrease-increase-decrease, while the down-regulation of PmGPIC was not obvious. Therefore, it was suggested that PmPGK1 mainly participated in the Calvin cycle and chloroplast/plast glycolysis, and PmGPIC was mainly involved in cytosolic glycolysis. The activities of PmPGK1 and PmGPIC were inhibited under low temperature stress, and PmPGK1 activity was significantly inhibited under high CO₂ stress, while PmGPIC activity was less affected.