广东紫菜Hsp70基因cDNA克隆与表达分析

The cDNA Cloning and Expression Analysis of the Hsp70 Gene from Pyropia guangdongensis

为探索广东紫菜(Pyropia guangdongensis)叶状体Hsp70基因在温度刺激下机体耐温相关分子机制,为广东紫菜的栽培生产提供技术参考,本研究利用RACE技术获得了广东紫菜Hsp70基因(PgHsp70)全长序列,并在此基础上采用实时荧光定量PCR技术,研究广东紫菜叶状体分别在不同温度(22℃、27℃和31℃)条件下处理0、1/6、1/2、1、6、12、24和36h后PgHsp70基因的差异表达。结果显示,PgHsp70基因序列长2004bp,包含一个1866bp的开放阅读框,可编码621个氨基酸,预测分子量为67.7kDa,理论等电点为4.87。基因表达水平定量分析表明,温度对PgHsp70基因表达水平有显著影响,PgHsp70基因在不同温度的表达水平变化趋势基本一致,均呈现先上调后下降的趋势,且均于1 h表达量到达最高水平,其中,在31℃ 1h表达量最高,为未经高温处理组的11倍,说明PgHsp70基因在应答高温胁迫中发挥着重要的作用。

In order to explore the molecular mechanisms of the Hsp70 gene in Pyropia guangdongensis, we have stimulated it with high temperature stresses. The aim was to provide technical references for the cultivation of P. guangdongensis. The full-length of PgHsp70 was obtained by rapid amplification of cDNA ends technical (RACE). On this basis, the expression of PgHsp70 with the different temperature (22℃, 27℃, and 31℃) stresses after 0, 1/6, 1/2, 1, 6, 12, 24, and 36 h were detected using quantitative real-time PCR. The results showed that the full-length cDNA of PgHsp70 was 2004 bp, including an open reading frame (ORF) of 1866 bp, encoding a polypeptide of 621 amino acids. The molecular mass of the deduced amino acid sequence was 67.7 kDa, with an estimated pI of 4.87. The expression of the PgHsp70, as measured by qRT-PCR, can be significantly induced by high-temperature stress. The three kinds of expression profiles for PgHsp70 were similar at different times, and all of them significantly increased first and then reached their maximum levels after one hour, and then dramatically decreased. Compared to the temperature stress of 22 ℃ and 27 ℃, the expression of the P. guangdongensis with the 31℃ temperature stress reached the highest level after being challenged for 1 h and was 11-fold higher than the normal. These results suggested that PgHsp70 plays an essential role in response to high-temperature stresses.