条斑紫菜水通道蛋白PyAQP1基因的克隆及功能分析

Cloning and Expression Analysis of PyAQP1 in Pyropia yezoensis

本研究从条斑紫菜中克隆了一个水通道蛋白(Aquaporin,简称AQP)基因PyAQP,命名为PyAQP1(登录号为KT735045)。序列分析表明,PyAQP1基因cDNA全长为1 151bp,开放阅读框(ORF)为1 002bp,编码333个氨基酸,含有200bp的内含子。氨基酸序列比对分析表明,PyAQP1具有水通道蛋白的保守结构域NPA和6个跨膜区域。MEGA5.0构建的系统发生树表明,该基因属于Glps类。采用基因枪介导的洋葱瞬时表达定位结果显示,PyAQP1基因定位于细胞质膜上。运用实时荧光定量PCR技术对PyAQP1基因在干旱、盐度和温度处理条件下在转录水平进行分析。在干旱处理条件下,PyAQP1基因随着失水率的增加,表达量逐渐降低,失水率达到80%后复水处理,随着复水时间的延长,表达量增加。在山梨醇和盐度胁迫处理下,PyAQP1基因的表达趋势基本一致,随着盐度的增加,表达量降低。温度处理条件下,在-8℃时,基因的表达量显著增加,0℃时,基因的表达量略高于正常处理条件。当温度高于正常生长温度时,基因的表达量随着温度的升高,呈现逐渐升高的趋势。本研究为深入了解条斑紫菜逆境适应机制中水通道蛋白的作用奠定了基础,同时为后续条斑紫菜抗逆品系的选育提供了理论指导。

Aquaporins are ancient channel proteins localized in biological membranes and facilitating water transport through cell membranes rapidly. Aquaporin plays an important role in plant water homeostasis through the facilitation of water transport and solutes. The red macroalgae P. yezoensis, classified as Ban- giophyceae, is a suitable species for the elucidation of molecular mechanisms regulating environmental stresses in marine red algae. However, currently little is known about the AQP genes in P. yezoensis. A PyAQP1 gene （GI Number is KT735045） was isolated throught silico clone, based on the transcriptomic data in this research. The full length of PyAQP1 contained one intron with the size of 200 bp, and an open reading frame of 1 151 bp encoding 333 amino acids. The isoelectric points （PI） and molecular weights （MW） of the PyAQP1 amino acid sequence were 35.4 kD and 6. 43, respectively. The analysis of Py- AQP1 amino acid sequences showed that PyAQP1 contained the MIP family signature NPA motif as well as six transmembrane regions. Analysis of multiple sequence alignment and phylogenetic tree showed that Py- AQP1 shared very low similarity to any other plant MIP but a surprisingly high similarity to a subclass of bacterial Glps. The plasmid construct encoding PyAQP1-GFP under the control of the 35S promoter was transformed into onion epidermal cells by particle bombardment. The transient expression analysis in onion epidermal cells showed us that the PyAQP1 protein was localized in the cell plasma membrane, compared to the control 35S： ：GFP in the nucleus, cytoplasm and plasma membrane of the cells. To better understand the short-term expression patterns of PyAQP1 in response to abiotic stress, qRT-PCR was used to investigate the expression patterns of PyAQP1 in different treatment conditions （salt, drought and temperature）. Under drought stress, the expression level of Py- AQP1 decreased with the increment of the water loss rate. It was noting that the expression increased when rehy- dration after dehydration reached 80%. The expression patterns of PyAQP1 under sorbitol and Nacl treatment was similar and the expression level of PyAQP1 decreased with salinity increasing. Under temperature stress, the expression of PyAQP1 increased significantly at --8 ℃, and the expression at 0 ℃ was slightly higher than nor- nml treatment. However, the expression of PyAQP1 gradually increased with the temperature increasing, when growth temperature increases by 10 ℃. In this work, we reported the isolation and expression characterization of the PyAQP1 gene in the P. yenozesis, which provide the insights for understanding the mechanisms of PyAQP1 responding to abiotic stresses.