杨树SnRK基因克隆及遗传转化研究
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摘要
蔗糖非发酵相关蛋白激酶SnRK(SNF1-related protein kinase )是一类丝氨酸/苏氨酸蛋白激酶,通过多种复杂的信号传递途径对干旱、高盐及低温胁迫作出响应,激发下游抗逆基因的表达,提高植物的抗逆性。本研究基于杨树全基因组序列信息,克隆杨树SnRK基因,并进行遗传转化研究。主要研究结果如下:
1.根据已知的拟南芥SRK2C基因序列,在杨树基因组上定位并克隆获得了3个SnRK基因,初步命名为PtSnRK-1、PtSnRK-2和PtSnRK-3。序列分析结果表明3个基因的开放阅读框(ORF)分别为1356bp、1329bp和1320bp,推测分别编码451个、442个和439个氨基酸(aa)残基的蛋白质,分子量分别约为50.22Kda、49.22Kda和48.89Kda。3个PtSnRK基因均具有保守的Protein kinase结构域和NAF结构域。
2.利用Gateway技术构建了3个克隆基因的过量表达载体pH35GS:: PtSnRK-1、pH35GS::PtSnRK-2和pH35GS::PtSnRK-3。经农杆菌介导将这3个基因转入欧美杂种山杨T89和南林895杨,通过最适遗传转化条件及潮霉素筛选获得了一批转PtSnRK-1、PtSnRK-2和PtSnRK-3基因欧美杂种山杨T89和南林895杨植株,并对转基因植株进行了分子生物学鉴定和抗逆生理指标检测。
3.PCR和实时定量RT-PCR检测结果表明目的基因已经整合进杨树基因组并超量表达。逆境(PEG6000和NaCl处理)下对叶绿素含量、丙二醛含量、超氧化物歧化酶活性和叶片相对含水量等测定结果显示,3个基因的导入均显著地提高了转基因植株的抗旱和耐盐能力。
SNF1-related protein kinase(SnRK) is a Serine / threonine protein kinase. SnRK genes are capable of mediating signals initiated during drought , salt and cold stress, play important roles in transducing stress signals,increasing protein synthesization and regulating signal transduction. Using the poplar genome database,we cloned three SnRK genes in poplar, and compeleted the transformation of poplar. It would be useful for the development of the gene engineering in genetic improvement of poplar to clone SnRK genes in poplar and develop transgenic polpar.
The main conclusions of this study were as following:
1 Three SnRK genes were cloned from Populus trichocarpa,designated as PtSnRK-1 ,PtSnRK-2 and PtSnRK-3 respectively. Bioinformatics analysis of PtSnRK genes showed that the ORF of PtSnRK-1 was 1356bp and encoded for a polypeptide of 451 amino acids, 50.22KD molecular weight. The ORF of PtSnRK-2 was 1329bp and encoded for a polypeptide of 442 amino acids, 49.22KD molecular weight. The ORF of PtSnRK-3 was 1320bp and encoded for a polypeptide of 439 amino acids, 48.89KD molecular weight. All of the three PtSnRK genes had conserved protein kinase domain and NAF domain.
2 Overexpression vectors pH35GS::PtSnRK-1、pH35GS::PtSnRK-2 and pH35GS: PtSnRK-3 were constructed using gateway cloning technology,and then transfered the three genes into Populus×euramericana cv.‘Nanlin895’and Populus tremula×P. tremuloides Michx-clone T89 respectively mediated by Agrobacterium tumefaciems. Transgenic poplars were selected to survive on the culture medium with antibiotics,which includes Populus×euramericana cv.‘Nanlin895’and Populus tremula×P. tremuloides Michx-clone T89 with single PtSnRK-1,PtSnRK-2 and PtSnRK-3 gene. The molecular biological tests and plant physiological analysis were carried out to verify these transgenic poplars.
3 The three genes had been integrated into poplar genome and could express excessively,which verified by PCR and real time RT-PCR. The result of physiological analysis such as chloroplast content,SOD activity MDA content and leaf relative water content indicated that the drought-resistance and salt-resistance ability of transgenic poplars with PtSnRK genes were extremely enhanced.
1.根据已知的拟南芥SRK2C基因序列,在杨树基因组上定位并克隆获得了3个SnRK基因,初步命名为PtSnRK-1、PtSnRK-2和PtSnRK-3。序列分析结果表明3个基因的开放阅读框(ORF)分别为1356bp、1329bp和1320bp,推测分别编码451个、442个和439个氨基酸(aa)残基的蛋白质,分子量分别约为50.22Kda、49.22Kda和48.89Kda。3个PtSnRK基因均具有保守的Protein kinase结构域和NAF结构域。
2.利用Gateway技术构建了3个克隆基因的过量表达载体pH35GS:: PtSnRK-1、pH35GS::PtSnRK-2和pH35GS::PtSnRK-3。经农杆菌介导将这3个基因转入欧美杂种山杨T89和南林895杨,通过最适遗传转化条件及潮霉素筛选获得了一批转PtSnRK-1、PtSnRK-2和PtSnRK-3基因欧美杂种山杨T89和南林895杨植株,并对转基因植株进行了分子生物学鉴定和抗逆生理指标检测。
3.PCR和实时定量RT-PCR检测结果表明目的基因已经整合进杨树基因组并超量表达。逆境(PEG6000和NaCl处理)下对叶绿素含量、丙二醛含量、超氧化物歧化酶活性和叶片相对含水量等测定结果显示,3个基因的导入均显著地提高了转基因植株的抗旱和耐盐能力。
SNF1-related protein kinase(SnRK) is a Serine / threonine protein kinase. SnRK genes are capable of mediating signals initiated during drought , salt and cold stress, play important roles in transducing stress signals,increasing protein synthesization and regulating signal transduction. Using the poplar genome database,we cloned three SnRK genes in poplar, and compeleted the transformation of poplar. It would be useful for the development of the gene engineering in genetic improvement of poplar to clone SnRK genes in poplar and develop transgenic polpar.
The main conclusions of this study were as following:
1 Three SnRK genes were cloned from Populus trichocarpa,designated as PtSnRK-1 ,PtSnRK-2 and PtSnRK-3 respectively. Bioinformatics analysis of PtSnRK genes showed that the ORF of PtSnRK-1 was 1356bp and encoded for a polypeptide of 451 amino acids, 50.22KD molecular weight. The ORF of PtSnRK-2 was 1329bp and encoded for a polypeptide of 442 amino acids, 49.22KD molecular weight. The ORF of PtSnRK-3 was 1320bp and encoded for a polypeptide of 439 amino acids, 48.89KD molecular weight. All of the three PtSnRK genes had conserved protein kinase domain and NAF domain.
2 Overexpression vectors pH35GS::PtSnRK-1、pH35GS::PtSnRK-2 and pH35GS: PtSnRK-3 were constructed using gateway cloning technology,and then transfered the three genes into Populus×euramericana cv.‘Nanlin895’and Populus tremula×P. tremuloides Michx-clone T89 respectively mediated by Agrobacterium tumefaciems. Transgenic poplars were selected to survive on the culture medium with antibiotics,which includes Populus×euramericana cv.‘Nanlin895’and Populus tremula×P. tremuloides Michx-clone T89 with single PtSnRK-1,PtSnRK-2 and PtSnRK-3 gene. The molecular biological tests and plant physiological analysis were carried out to verify these transgenic poplars.
3 The three genes had been integrated into poplar genome and could express excessively,which verified by PCR and real time RT-PCR. The result of physiological analysis such as chloroplast content,SOD activity MDA content and leaf relative water content indicated that the drought-resistance and salt-resistance ability of transgenic poplars with PtSnRK genes were extremely enhanced.
引文
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