胡杨果糖-1,6-二磷酸醛羧酶基因PeALD的克隆及耐盐性分析  

Cloning and Salt-tolerant Analysis of Fructose-1,6-Bisphosphate Aldolase Gene PeALD of Populus euphratica Oliv

作　　者：张帆[1] 常李伟[1] 丁明全[1] 侯佩臣[1] 沈昕[1] 陈少良[1] 

机构地区：[1]北京林业大学生物科学与技术学院,北京100083

出　　处：《基因组学与应用生物学》2011年第3期257-267,共11页Genomics and Applied Biology

基　　金：国家自然科学基金项目(30872005);北京市优秀博士学位论文指导教师专项基金(YB20081002201);北京市自然科学基金项目(6112017);中央高校基本科研业务费专项资金(JC2011-2)共同资助

摘　　要：胡杨(Populus euphratica Oliv)是优良的抗逆树种,有很强的耐盐碱性。前期胡杨转录组研究结果显示盐胁迫可诱导果糖-1,6-二磷酸醛羧酶基因(PeALD)转录上调,提示该基因可能对胡杨耐盐性方面有某种贡献。为分析PeALD基因在胡杨耐盐性中的作用,本研究以胡杨为材料,利用RT-PCR方法克隆了胡杨果糖-1,6-二磷酸醛羧酶基因的全长cDNA,通过基因转化法尝试在烟草中过量表达该基因,并对转基因株系的耐盐性进行初步分析。研究结果显示,克隆的cDNA编码果糖-1,6-二磷酸醛羧酶,ORF为1177bp,是由247个氨基酸编码的疏水性蛋白,理论等电点为6.84,分子量为26.79kD。PCR与RT-PCR检测结果表明,外源的PeALD基因通过转化已经整合到烟草的染色体中,并在4个株系中得到较强的表达。转化株系表型筛选实验表明,在200mmol/L NaCl的MS培养基中培养15d后,野生型烟草植株无明显高生长,叶片全部黄化并萎缩;而转基因烟草高生长基本没有受到抑制,植株生长良好。说明在烟草中过表达PeALD使转化植株的耐盐性显著提高。光合数据结果显示,ALD基因能够降低盐胁迫对烟草叶片净光合速率的影响,可能是PeALD过表达加速了卡尔文循环的运转,提高了CO2的固定速率,进而促进了光合活性,通过光合作用促进碳的积累,利于呼吸作用和氧化磷酸化产生ATP,为维持跨膜质子浓度梯度提供能量,从而有可能促进质膜上的Na+/H+逆向转运,利于细胞质膜保持拒盐性。这些结果初步验证了PeALD基因的耐盐性功能,为进一步深入研究该基因在耐盐机制中的作用奠定了良好基础。Being a stress-resistant tree species,Populus euphratica exhibits a typically high capacity to tolerate salinity.Transcriptomic studies of P.euphratica have shown that fructose-1,6-bisphosphate aldolase（PeALD） gene was up-regulated by NaCl stress,indicated that this enzyme may have some contributions to salt tolerance of P.euphratica.To analyze the role of PeALD in salt tolerance,full length cDNA of ALD was cloned from P.euphr-atica by RT-PCR.Meanwhile,over expression of PeALD in tobacoo was conducted by transferring of a PeALD construct into tobacoo genome and we also analyzed the salt-resistant characters of the transgenic lines.The result of our study indicated that the cloned cDNA was the ALD ortholog of P.euphratica based on BLAST analysis,with an 1 177 bp open reading frame,encoding a hydrophobic protein of 247 amino acids with a theoretical isoelectric point of 6.84 and a molecular mass of 26.79 kD.The data of PCR and RT-PCR indicated that the PeALD was integrated into tebacoo genome and over-expressed in four transgenic lines.Salt-tolerance test showed that transgenic plantlets survived in MS medium with 200 mmol/L NaCl after 15 d culture and plant growth was not inhibited.In contrast,the leaves of wild type plantlet got etiolated and shrinked after 15 d culture at the same saline condition.The data of photosynthetic study showed that over-expression of PeALD attenuated the NaCl-induced reduction of net photosynthetic rate in transgenic plants,presumably resulting from an enhanced turnover rate of the calvin cycle.The stable carbon assimilation was favorable for respiration maintenance,leading to a constant ATP supply by oxidative phosphorylation,which benefits H＋-ATPase to energise salt transport through the plasma and vacuolar membranes.In conclusion,our study demonstrated that ALD gene cloned from P.euphratica was able to enhance the capacity for salt tolerance of model plants,and should be provide the foundation for further study of PeALD in salt stress signaling.

关 键 词：胡杨 果糖-1 6-二磷酸醛羧酶基因 转基因烟草 碳流向 抗盐性 

分 类 号：Q943.2[生物学—植物学] S792.119[农业科学—林木遗传育种]