栽培大豆非生物胁迫诱导的蛋白激酶基因GmGSK的克隆及功能分析
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摘要
高盐、干旱、低温等非生物胁迫是影响作物产量的重要环境因素。植物通过长期进化逐渐形成特定的机制来抵御外界环境造成的胁迫,减轻对自身的伤害。其中蛋白激酶可通过对底物可逆的磷酸化来参与胁迫信号的传导,从而进一步调节外界胁迫对作物造成的伤害。因此,蛋白激酶在植物的抗逆途径中起到了非常重要的作用。
我们以小麦的TaGSK1基因序列为探针,通过电子克隆在栽培大豆中得到一条类似糖原合成酶激酶的cDNA序列,并通过RT-PCR的方法将其克隆。序列分析表明,此cDNA包含一条1230bp的完整开放阅读框,编码410个氨基酸,估计的分子量为46.5kD,等电点PI为8.64。蛋白序列分析表明,其编码一个与GSK-3同源的蛋白,含有丝氨酸／苏氨酸蛋白激酶活性位点,命名为GmGSK (Genbank登录号：FJ460228)。
Southern杂交表明,GmGSK基因在栽培大豆基因组中至少有两个拷贝。Northern杂交显示,在栽培大豆的根、茎、叶、叶柄及子叶中均能表达GmGSK,但不同的组织器官中GmGSK基因的表达量不同,在根中分布较多。半定量RT-PCR分析结果显示,GmGSK基因的表达可以被NaCl、NaHCO3、干旱、ABA和低温所诱导,这表明GmGSK可能参与了大豆非生物胁迫的信号传导过程或对非生物胁迫的耐受。
利用pCAMBIA1302构建GmGSK:GFP融合载体,通过农杆菌渗入法在烟草中叶片细胞中瞬时表达GmGSK:GFP融合蛋白。对GmGSK进行亚细胞定位发现,其分布在细胞膜与细胞质中。为了进一步研究其功能我们分别将其转入大肠杆菌(BL21)和酿酒酵母(INVScl)中,原核表达显示其蛋白大小确为46.5kD左右；在转GmGSK基因的酿酒酵母中,其对NaCl、NaHCO3和干旱等非生物胁迫的耐受能力明显强于非转基因酿酒酵母。
最后,通过构建pCAMBIA3300-GmGSK植物双元表达载体,将GmGSK转入烟草,过量表达GmGSK基因,验证其在植物中的生物学功能。结果表明,在高盐胁迫下转基因植株的生长状态、相对生物量、根系发达程度与对照相比均得到了明显的提高。对转基因烟草和非转基因烟草植株共同浇灌适当浓度NaCl,结果显示转基因烟草能明显提高对高盐的耐受性,而非转基因烟草则在不久后死亡。对其生理指标进行测定显示,在盐胁迫下转基因烟草的可溶性糖含量增长幅度明显快于非转基因对照,而外渗相对电导率增长速率则明显慢于非转基因对照,这些有助于提高转基因烟草的耐盐性。
以上结果表明,栽培大豆GmGSK基因在生物抵御外界非生物胁迫过程中有重要作用,过量表达GmGSK基因,能显著提高生物的抗逆性,这也为将来利用耐盐基因改善栽培大豆耐盐性方面,提供了一条新途径。
High salt, drought, low temperature and other abiotic stresses are important environmental factors to affect crop yield. Plants reduce their own damage through long-term evolution to resist the external environment stress resistance. Protein kinase may be reversible through the phosphorylation to participate in stress signal transduction, thereby further respond to external stresses on plants. Protein kinase played a very important role in the stress-tolerant pathway in plants.
We used wheat TaGSKl gene sequence as a query. A full-length cDNA of the GSK-3-like gene was obtained by in silico cloning from Soybean database of GenBank and was further isolated from G. max by RT-PCR. The complete cDNA open reading frame sequence is 1230 bp in length and encoded 410 amino acid residues with a theoretical molecular weight of 46.5 kD and an isoelectric point of 8.64. Protein sequence analysis showed that it encoded a protein homologous with GSK, containing serine/threonine protein kinase active site, named GmGSK (Genbank Access number:FJ460228).
Southern blot analysis revealed that it had at least two copies in G. max genome. Northern blot analysis indicated that GmGSK expressed in all tested tissues, with highest expression in root. Semi-quantitative RT-PCR analysis revealed that GmGSK gene could be induced by NaCl, NaHCO3, drought, ABA and cold, which suggested that GmGSK might be involved in soybean abiotic stress signal transduction process or tolerance to abiotic stress.
We used pCAMBIA1302 to construct GmGSK:GFP vector and GmGSK:GFP fusion protein was introduced into tobacco by Agroinfiltration. The subcellular localization of GmGSK indicated that it was distributed in the cell membrane and cytoplasmic. GmGSK was transformed into Escherichia coli. (BL21) and successfully expressed with a 46.5kD detected. And Saccharomyces cerevisiae (INVSc1) transformed with GmGSK increased their tolerance obviously to NaCl, NaHCO3 and drought stresses than wild type.
Finally, in order to further validate GmGSK biological functions in plants, a recombinant vector pCAMBIA3300-GmGSK was construted and transferred into tobacco for over-expression analysis. The results showed that the growth, relative biomass and rooting rate of transgenic tobaccos were significantly improved than wild type. Transgenic tobacco was treated with NaCl solution and results showed that transgenic tobacco could increase their salt tolerance significantly compared with wild type tobacco. Physiological analysis showed that transgenic tobacco increase the soluble sugar content significantly faster than wild type tobacco and extravasation relative conductivity growth rate was significantly slower than wild type tobacco under salt stress, which help the transgenic tobacco to improve salt tolerance.
These results suggested that soybean GmGSK gene played an important role to resist abiotic stress. Over-expressed GmGSK gene could significantly improve the salt tolerance of yeast and tobacco, which indicated that GmGSK gene could be a promising gene in stress tolerance improvement in soybean and other plants in the future.
我们以小麦的TaGSK1基因序列为探针,通过电子克隆在栽培大豆中得到一条类似糖原合成酶激酶的cDNA序列,并通过RT-PCR的方法将其克隆。序列分析表明,此cDNA包含一条1230bp的完整开放阅读框,编码410个氨基酸,估计的分子量为46.5kD,等电点PI为8.64。蛋白序列分析表明,其编码一个与GSK-3同源的蛋白,含有丝氨酸／苏氨酸蛋白激酶活性位点,命名为GmGSK (Genbank登录号：FJ460228)。
Southern杂交表明,GmGSK基因在栽培大豆基因组中至少有两个拷贝。Northern杂交显示,在栽培大豆的根、茎、叶、叶柄及子叶中均能表达GmGSK,但不同的组织器官中GmGSK基因的表达量不同,在根中分布较多。半定量RT-PCR分析结果显示,GmGSK基因的表达可以被NaCl、NaHCO3、干旱、ABA和低温所诱导,这表明GmGSK可能参与了大豆非生物胁迫的信号传导过程或对非生物胁迫的耐受。
利用pCAMBIA1302构建GmGSK:GFP融合载体,通过农杆菌渗入法在烟草中叶片细胞中瞬时表达GmGSK:GFP融合蛋白。对GmGSK进行亚细胞定位发现,其分布在细胞膜与细胞质中。为了进一步研究其功能我们分别将其转入大肠杆菌(BL21)和酿酒酵母(INVScl)中,原核表达显示其蛋白大小确为46.5kD左右；在转GmGSK基因的酿酒酵母中,其对NaCl、NaHCO3和干旱等非生物胁迫的耐受能力明显强于非转基因酿酒酵母。
最后,通过构建pCAMBIA3300-GmGSK植物双元表达载体,将GmGSK转入烟草,过量表达GmGSK基因,验证其在植物中的生物学功能。结果表明,在高盐胁迫下转基因植株的生长状态、相对生物量、根系发达程度与对照相比均得到了明显的提高。对转基因烟草和非转基因烟草植株共同浇灌适当浓度NaCl,结果显示转基因烟草能明显提高对高盐的耐受性,而非转基因烟草则在不久后死亡。对其生理指标进行测定显示,在盐胁迫下转基因烟草的可溶性糖含量增长幅度明显快于非转基因对照,而外渗相对电导率增长速率则明显慢于非转基因对照,这些有助于提高转基因烟草的耐盐性。
以上结果表明,栽培大豆GmGSK基因在生物抵御外界非生物胁迫过程中有重要作用,过量表达GmGSK基因,能显著提高生物的抗逆性,这也为将来利用耐盐基因改善栽培大豆耐盐性方面,提供了一条新途径。
High salt, drought, low temperature and other abiotic stresses are important environmental factors to affect crop yield. Plants reduce their own damage through long-term evolution to resist the external environment stress resistance. Protein kinase may be reversible through the phosphorylation to participate in stress signal transduction, thereby further respond to external stresses on plants. Protein kinase played a very important role in the stress-tolerant pathway in plants.
We used wheat TaGSKl gene sequence as a query. A full-length cDNA of the GSK-3-like gene was obtained by in silico cloning from Soybean database of GenBank and was further isolated from G. max by RT-PCR. The complete cDNA open reading frame sequence is 1230 bp in length and encoded 410 amino acid residues with a theoretical molecular weight of 46.5 kD and an isoelectric point of 8.64. Protein sequence analysis showed that it encoded a protein homologous with GSK, containing serine/threonine protein kinase active site, named GmGSK (Genbank Access number:FJ460228).
Southern blot analysis revealed that it had at least two copies in G. max genome. Northern blot analysis indicated that GmGSK expressed in all tested tissues, with highest expression in root. Semi-quantitative RT-PCR analysis revealed that GmGSK gene could be induced by NaCl, NaHCO3, drought, ABA and cold, which suggested that GmGSK might be involved in soybean abiotic stress signal transduction process or tolerance to abiotic stress.
We used pCAMBIA1302 to construct GmGSK:GFP vector and GmGSK:GFP fusion protein was introduced into tobacco by Agroinfiltration. The subcellular localization of GmGSK indicated that it was distributed in the cell membrane and cytoplasmic. GmGSK was transformed into Escherichia coli. (BL21) and successfully expressed with a 46.5kD detected. And Saccharomyces cerevisiae (INVSc1) transformed with GmGSK increased their tolerance obviously to NaCl, NaHCO3 and drought stresses than wild type.
Finally, in order to further validate GmGSK biological functions in plants, a recombinant vector pCAMBIA3300-GmGSK was construted and transferred into tobacco for over-expression analysis. The results showed that the growth, relative biomass and rooting rate of transgenic tobaccos were significantly improved than wild type. Transgenic tobacco was treated with NaCl solution and results showed that transgenic tobacco could increase their salt tolerance significantly compared with wild type tobacco. Physiological analysis showed that transgenic tobacco increase the soluble sugar content significantly faster than wild type tobacco and extravasation relative conductivity growth rate was significantly slower than wild type tobacco under salt stress, which help the transgenic tobacco to improve salt tolerance.
These results suggested that soybean GmGSK gene played an important role to resist abiotic stress. Over-expressed GmGSK gene could significantly improve the salt tolerance of yeast and tobacco, which indicated that GmGSK gene could be a promising gene in stress tolerance improvement in soybean and other plants in the future.
引文
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