CAJ | 학술논문

磷酸烯醇式丙酮酸羧化酶激酶(phosphoenolpyruvate carboxylase kinase, PPCK)是一种钙不依赖的丝氨酸/苏氨酸(Ser/Thr)类蛋白激酶,参与碳氮代谢等多个生物学过程,然而其在碱胁迫反应中的作用尚未见报道.本研究在前期野生大豆碱胁迫基因表达谱数据基础上,采用同源克隆的方法分离野生大豆(Glycine soja) PPCK1基因,该基因编码的氨基酸序列与大豆(Glycine max) PPCK1蛋白(AAQ83695.1)具有99%的相似性,被命名为GsPPCK1.在50 mmol L–1 NaHCO3胁迫处理3 h内,根和叶中GsPPCK1基因上调表达,属碱胁迫早期应答基因.通过农杆菌介导法对肇东苜蓿进行遗传转化,并对RT-PCR阳性的超表达转基因株系进行耐碱性分析,在100 mmol L–1 NaHCO3处理15 d后转基因株系生长状态良好,而非转基因对照株系明显萎蔫、失绿、甚至死亡;转基因株系的丙二醛含量和相对质膜透性显著低于非转基因株系(P<0.05),而叶绿素含量和根系活力显著高于非转基因对照(P<0.05),说明超量表达GsPPCK1基因增强了苜蓿的耐碱能力.以上结果表明, GsPPCK1参于植物耐碱胁迫反应过程,在碱胁迫基因工程研究领域具有良好的理论和实际应用意义.
린산희순식병동산최화매격매(phosphoenolpyruvate carboxylase kinase, PPCK)시일충개불의뢰적사안산/소안산(Ser/Thr)류단백격매,삼여탄담대사등다개생물학과정,연이기재감협박반응중적작용상미견보도.본연구재전기야생대두감협박기인표체보수거기출상,채용동원극륭적방법분리야생대두(Glycine soja) PPCK1기인,해기인편마적안기산서렬여대두(Glycine max) PPCK1단백(AAQ83695.1)구유99%적상사성,피명명위GsPPCK1.재50 mmol L–1 NaHCO3협박처리3 h내,근화협중GsPPCK1기인상조표체,속감협박조기응답기인.통과농간균개도법대조동목숙진행유전전화,병대RT-PCR양성적초표체전기인주계진행내감성분석,재100 mmol L–1 NaHCO3처리15 d후전기인주계생장상태량호,이비전기인대조주계명현위언、실록、심지사망;전기인주계적병이철함량화상대질막투성현저저우비전기인주계(P<0.05),이협록소함량화근계활력현저고우비전기인대조(P<0.05),설명초량표체GsPPCK1기인증강료목숙적내감능력.이상결과표명, GsPPCK1삼우식물내감협박반응과정,재감협박기인공정연구영역구유량호적이론화실제응용의의.
Environmental stresses, such as drought, high salty and alkali, adversely affect plant growth and productivity. Plants adapt to these environmental stresses by inducing numerous genes at the transcriptional level and by protein phosphorylation. Phosphoenolpyruvate carboxylase kinase (PPCK) is a Ca2+independent kinase in response to a range of signals in different plant tissues which plays a key role in the control of plant metabolism. As an important extension of our earlier studies summarized above on global transcriptome profiling of wild soybean under NaHCO3 treatment, an alkaline (NaHCO3) related gene GsPPCK1 was identified and subsequently cloned from Glycine soja, which has 99%similarity with PPCK1 of Glycine max (AAQ83695.1), named as GsPPCK1. Expression of GsPPCK1 mRNA was induced by NaHCO3 stress in roots and leaves. GsPPCK1 transcripts increased during 3 hour exposures to NaHCO3 stress. These results indicated that wild soybean PPCK1 was an early responded gene to alkaline stress. We transformed GsPPCK1 gene into alfalfa using a developed method, and transgenic alfalfa showed ob-servably enhanced tolerance to NaHCO3 stress compared with wild-type plants. Transgenic alfalfa grew well in the conditions of 100 mmol L–1 NaHCO3, while wild type plants exhibited discoloration and stunted growth, or even death. There were significantly changes in malondialdehyde content and relative membrane permeability caused by saline-alkaline stress in non-transgenic lines compared to transgenic lines (P<0.05). Moreover, compared with non-transgenic, transgenic alfalfa had higher levels of chloro-phyll content and root activity under alkali stress conditions. The result indicated that over-expression of GsPPCK1 in alfalfa could enhance alkaline tolerance. All results showed that GsPPCK1 gene could improve the tolerance of transgenic alfalfa to al-kali stress;therefore, the study on this field is of significance not only in theory but also in practice.