胡杨PeAPY2基因的克隆及其转化细胞的耐盐性分析

Cloning of Apyrase Gene PeAPY2 from Populus euphratica and the Salt Tolerance of the Transformed Cells

从胡杨（Populus euphratica Oliv.）抗盐碱能力强于其它种类杨树。本实验室前期对胡杨的研究表明：盐胁迫下,胡杨腺苷三磷酸双磷酸水解酶基因（PeAPY）的转录水平上调,预示该基因可能在胡杨应对盐胁迫中发挥作用。本研究利用RT-PCR方法克隆了胡杨腺苷三磷酸双磷酸水解酶PeAPY2基因,将该基因在烟草BY-2细胞中过表达,分析PeAPY2基因与植物耐盐性的关系。结果证明,PeAPY2编码腺苷三磷酸双磷酸水解酶,其ORF区长为1404bp,编码467个氨基酸。对转PeAPY2基因的烟草BY-2细胞及野生型烟草BY-2细胞进行长期盐处理,结果表明,烟草BY-2细胞过表达PeAPY2基因后提高了耐盐性,但并没有明显改善由甘露醇（200~600mmol/L）引起的渗透胁迫。盐处理（50~150mmol/L,NaCl）2周后,野生型BY-2细胞鲜重和干重都低于转PeAPY2基因的烟草细胞。转基因细胞跟野生型细胞相比,Na＋的上升幅度较小,而K＋的下降幅度也较小。随着盐胁迫时间的延长,转基因细胞膜透性低于野生型细胞,在盐胁迫下细胞活力高于野生型细胞。说明烟草BY-2细胞过表达PeAPY2基因后改善了其对盐的耐受性,这对深入研究PeAPY2基因在胡杨耐盐机制中的作用具有重要的意义。

Populus euphratica has a higher capacity to tolerate salinity. Previous transcriptional profile showed that apyrase gene （PeAPY） was up-regulated under salinity conditions, indicating that apyrase of P. euphratica may play an important role in adapting to salt stress. To analyze the gene function, full length cDNA of PeAPY2 was cloned from P. euphratica by RT-PCR. Blast analysis shows that the cDNA encodes apyrase, which is a hydrophobic protein of 467 amino acids and has an ORF of 1 404 bp. Over-expression of PeAPY2 in tobacco BY-2 cells im-proved salt tolerance, but the ability to tolerate hyperosmotic stress that caused by mannitol （200~600 mmol/L） was not enhanced. After 2 weeks of NaCl stress （50~150 mmol/L）, wild-type cells had lower fresh and dry weights than PeAPY2-transgenic cells. Salt treatment caused a lower reduction of K＋and lower increase of Na＋in transgenic cells. Under salinity conditions, transgenic cells showed lower membrane permeabilization and higher cell viability compared to wild-type cells. This research reveals that overexpressed PeAPY2 gene could improve the ability to tol-erate salinity in tobacco cells. Our data provides useful information for the further study on the function of PeAPY2 in the salt tolerance of P. euphratica.