CAJ | 학술논문

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。
호양（Populus euphratica Oliv）시우량적항역수충,유흔강적내염감성。전기호양전록조연구결과현시염협박가유도과당-1,6-이린산철최매기인（PeALD）전록상조,제시해기인가능대호양내염성방면유모충공헌。위분석PeALD기인재호양내염성중적작용,본연구이호양위재료,이용RT-PCR방법극륭료호양과당-1,6-이린산철최매기인적전장cDNA,통과기인전화법상시재연초중과량표체해기인,병대전기인주계적내염성진행초보분석。연구결과현시,극륭적cDNA편마과당-1,6-이린산철최매,ORF위1177bp,시유247개안기산편마적소수성단백,이론등전점위6.84,분자량위26.79kD。PCR여RT-PCR검측결과표명,외원적PeALD기인통과전화이경정합도연초적염색체중,병재4개주계중득도교강적표체。전화주계표형사선실험표명,재200mmol/L NaCl적MS배양기중배양15d후,야생형연초식주무명현고생장,협편전부황화병위축;이전기인연초고생장기본몰유수도억제,식주생장량호。설명재연초중과표체PeALD사전화식주적내염성현저제고。광합수거결과현시,ALD기인능구강저염협박대연초협편정광합속솔적영향,가능시PeALD과표체가속료잡이문순배적운전,제고료CO2적고정속솔,진이촉진료광합활성,통과광합작용촉진탄적적루,리우호흡작용화양화린산화산생ATP,위유지과막질자농도제도제공능량,종이유가능촉진질막상적Na＋/H＋역향전운,리우세포질막보지거염성。저사결과초보험증료PeALD기인적내염성공능,위진일보심입연구해기인재내염궤제중적작용전정료량호기출。
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.