玉米蛋白激酶基因ZmPti1、ZmPti1-1和ZmCIPK2的抗旱功能分析
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摘要
玉米是世界主要农作物之一。全球水资源短缺,干旱已成为玉米生产发展的主要限制因素。发现新的抗旱功能基因,培育抗旱玉米新材料,对于玉米的抗旱遗传育种具有重要的理论和现实意义。蛋白激酶在植物的抗旱过程中起到了非常重要的作用。本研究以京178和京501自交系玉米为材料,利用花粉管通道法、基因枪法构建受干旱胁迫诱导的蛋白激酶基因ZmPtil、ZmPtil-1、ZmCIPK2的过表达或RNAi(?)朱系,进一步分析了这些转基因系的抗旱功能。主要结果如下：
为了分析ZmPtil的抗旱功能,构建了植物表达载体pBPC-ZmPtil-bar,利用花粉管通道法转化玉米,对转ZmPtil基因玉米植株的不同抗旱指标进行了分析。结果表明,与对照相比,转基因株系的抗旱能力得到了明显的提高。干旱胁迫下,与对照相比,转ZmPtil基因植株具有较少的卷叶,植株高度和干重分别比对照高6.2%和12.6%。转基因植株的穗粒数和千粒重分别比对照高21.9%和8.7%。在干旱胁迫下,转基因植株的相对电导率和MDA含量分别比对照低14.8%和18.1%,而相对含水量、可溶性糖和脯氨酸含量则分别比对照高7.3%、2.5%和22.7%。
为了分析ZmPtil-1的抗旱功能,构建了植物表达载体35S-ZmPtil-1-DHA-NOS-pGreen0029,利用浸花法转化拟南芥,对转ZmPtil-1基因拟南芥植株的不同抗旱指标进行了分析。结果表明,与对照相比,转基因株系的抗旱水平得到了明显的提高。与对照植株相比,在干旱胁迫下,3个转基因株系的存活率分别为70%,76%和87%,显著高于野生型29%的存活率。干旱胁迫下,与对照相比,转ZmPtil-1基因拟南芥植株具有较少的枯萎叶。转基因植株的种子重比对照高58.7%。在干旱胁迫下,转基因植株的相对电导率和MDA含量分别比对照低13.7%和17.7%,而相对含水量、可溶性糖和脯氨酸含量则分别比对照高7.1%、3.6%和22.5%。
为了进一步分析ZmPtil-1对玉米抗旱性的影响,构建了植物表达载体pGreen0229-ZmPtil-1,利用基因枪法转化玉米,对转ZmPtil-1基因玉米植株的不同抗旱指标进行了分析。结果表明,与对照相比,转基因株系的抗旱能力得到了明显的提高。干旱胁迫下,与对照相比,转ZmPtil-1基因植株具有较少的卷叶,植株高度和干重分别比对照高8.2%和14.4%。转基因植株的穗粒数和千粒重分别比对照高22.2%和8.8%。在干旱胁迫下,转基因植株的相对电导率和MDA含量分别比对照低20.4%和24.1%,而相对含水量、可溶性糖和脯氨酸含量则分别比对照高9.4%、9.0%和24.1%。
CIPK是参与渗透胁迫、盐、低温、脱落酸(abscisic acid, ABA)和糖等信号转导途径的、植物所特有的一类蛋白激酶。ZmCIPK2(GenBank接受号为EF158033)是一个新的玉米CIPK激酶基因。组成型表达ZmCIPK2基因的拟南芥种子发芽和发芽后幼苗的早期生长对干旱胁迫超敏感。为了分析ZmCIPK2的抗旱功能,构建了RNAi表达载体pGreen0229-ZmCIPK2Sense-GFP-ZmCIPK2Antisense,利用花粉管通道法转化玉米,ZmCIPK2基因在RNAi玉米中的表达水平下降直至完全沉默。结果表明,与对照相比,RNAi株系的抗旱性得到了明显的提高。干旱胁迫下,与对照相比,ZmCIPK2基因RNAi植株具有较少的卷叶。在干旱胁迫下,ZmCIPK2RNAi株系的相对电导率和MDA含量分别比对照低16.1%和17.5%,而相对含水量、可溶性糖和脯氨酸含量则分别比对照高8.2%、8.8%和24.2%。
Maize is one of the most important crops in the world. With the global water shortage, drought stress has been a major constraint for maize production. It is of vital theoretical and practical significance for maize genetic breeding in drought resistance to analyze gene function in drought resistance, discover new drought resistance genes and cultivate new drought resistance maize lines. Protein kinase plays an important role in plant drought resistance process. In this research, transgenic ZmPtil, ZmPtil-1and ZmCIPK2, which induced by drought stress from maize, overexpressed or RNAi lines of Jing178or Jing501inbred maize were constructed via pollen tube pathway method or microprojectile bombardment. The function in drought resistance of these transgenic lines was further analyzed. The results showed as follows:
In order to analyze the drought resistance function of ZmPtil, the plant expression vector pBPC-ZmPtil-bar was constructed and transformed into maize via pollen tube pathway method. The different drought resistance index of transgenic ZmPtil maize plants was analyzed. The results showed that the transgenic plants can enhance the drought resistance comparing to control plants. Under drought stress, compared to wild-type plants, transgenic ZmPtil plants showed a stronger growth recovery phenotype with less leaf rolling and better growth with higher plant height and dry weight, which was6.2%and12.6%higher than WT, respectively. The grain number per ear and1000-grain weight of transgenic plants was21.9%and8.7%higher than WT, respectively. After drought stress, the relative electric conductivity and MDA content of transgenic plants was14.8%,18.1%lower than that that of WT, respectively, whereas the relative water content, total soluble sugars, proline content of transgenic plants was7.3%,2.5%,22.7%higher than that that of WT, respectively.
In order to analyze the drought resistance function of ZmPtil-1, the plant expression vector35S-ZwPri1-1-DHA-NOS-pGreen0029was constructed and transformed into Arabidopsis via floral dip method. The different drought resistance index of transgenic ZmPtil-1Arabidopsis plants was analyzed. The results demonstrated that over-expression of ZmPtil-1gene improved drought resistance in transgenic Arabidopsis. Under drought stress, compared to wild type, survival rate of the three transgenic lines, which was70%,76%and87%respectively, was significantly higher than that of wild type which was29%. Under drought stress, compared to wild-type plants, transgenic ZmPtil-1Arabidopsis plants showed a stronger growth recovery phenotype with less wither leaves and better growth. The seed weight of transgenic plants was58.7%higher than WT. After drought stress, the relative electric conductivity and MDA content of transgenic plants was13.7%,17.7%lower than that that of WT, respectively, whereas the relative water content, total soluble sugars, proline content of transgenic plants was7.1%,3.6%,22.5%higher than that that of WT, respectively.
In order to further analyze the effect of ZmPtil-1on the drought tolerance of maize, the plant expression vector pGreenO229-ZmPtil-1was constructed and transformed into maize via microprojectile bombardment method. The different drought resistance index of transgenic ZmPtil-1maize plants was analyzed. The results demonstrated that over-expression of ZmPtil-1gene improved drought resistance in transgenic maize. Under drought stress, compared to wild-type plants, transgenic ZmPtil-1plants showed a stronger growth recovery phenotype with less leaf rolling and better growth with higher plant height and dry weight, which was8.2%and14.4%higher than WT, respectively. The grain number per ear and1000-grain weight of transgenic plants was22.2%and8.8%higher than WT, respectively. After drought stress, the relative electric conductivity and MDA content of transgenic plants was20.4%,24.1%lower than that that of WT, respectively, whereas the relative water content, total soluble sugars, proline content of transgenic plants was9.4%,9.0%,24.1%higher than that that of WT, respectively.
A family of specific protein kinase CIPK participates in regulation of osmotic stress, salt, cold, ABA and sugar signal transduction in plant. ZmCIPK2(GenBank accession no. EF158033) is a new maize CIPK kinase gene. Transgenic ZmCIPK2Arabidopsis lines are hypersensitive to drought stress when germination and early seedling growth. In order to analyze the drought resistance function of ZmCIPK2, the RNAi expression vector pGreenO229-ZmCIPK2Sense-GFP-ZmCIPK2Antisense was constructed and transformed into maize via pollen tube pathway method. The expression level of ZmCIPK2in RNAi maize lines was declined to some extent and even completely silenced. The results showed that the RNAi lines can enhance the drought resistance comparing to control plants. Under drought stress, compared to wild-type plants, ZmClPK2RNAi plants showed a stronger growth recovery phenotype with less leaf rolling and better growth. After drought stress, the relative electric conductivity and MDA content of transgenic plants was16.1%,17.5%lower than that that of WT, respectively, whereas the relative water content, total soluble sugars, proline content of transgenic plants was8.2%,8.8%,24.2%higher than that that of WT, respectively.
为了分析ZmPtil的抗旱功能,构建了植物表达载体pBPC-ZmPtil-bar,利用花粉管通道法转化玉米,对转ZmPtil基因玉米植株的不同抗旱指标进行了分析。结果表明,与对照相比,转基因株系的抗旱能力得到了明显的提高。干旱胁迫下,与对照相比,转ZmPtil基因植株具有较少的卷叶,植株高度和干重分别比对照高6.2%和12.6%。转基因植株的穗粒数和千粒重分别比对照高21.9%和8.7%。在干旱胁迫下,转基因植株的相对电导率和MDA含量分别比对照低14.8%和18.1%,而相对含水量、可溶性糖和脯氨酸含量则分别比对照高7.3%、2.5%和22.7%。
为了分析ZmPtil-1的抗旱功能,构建了植物表达载体35S-ZmPtil-1-DHA-NOS-pGreen0029,利用浸花法转化拟南芥,对转ZmPtil-1基因拟南芥植株的不同抗旱指标进行了分析。结果表明,与对照相比,转基因株系的抗旱水平得到了明显的提高。与对照植株相比,在干旱胁迫下,3个转基因株系的存活率分别为70%,76%和87%,显著高于野生型29%的存活率。干旱胁迫下,与对照相比,转ZmPtil-1基因拟南芥植株具有较少的枯萎叶。转基因植株的种子重比对照高58.7%。在干旱胁迫下,转基因植株的相对电导率和MDA含量分别比对照低13.7%和17.7%,而相对含水量、可溶性糖和脯氨酸含量则分别比对照高7.1%、3.6%和22.5%。
为了进一步分析ZmPtil-1对玉米抗旱性的影响,构建了植物表达载体pGreen0229-ZmPtil-1,利用基因枪法转化玉米,对转ZmPtil-1基因玉米植株的不同抗旱指标进行了分析。结果表明,与对照相比,转基因株系的抗旱能力得到了明显的提高。干旱胁迫下,与对照相比,转ZmPtil-1基因植株具有较少的卷叶,植株高度和干重分别比对照高8.2%和14.4%。转基因植株的穗粒数和千粒重分别比对照高22.2%和8.8%。在干旱胁迫下,转基因植株的相对电导率和MDA含量分别比对照低20.4%和24.1%,而相对含水量、可溶性糖和脯氨酸含量则分别比对照高9.4%、9.0%和24.1%。
CIPK是参与渗透胁迫、盐、低温、脱落酸(abscisic acid, ABA)和糖等信号转导途径的、植物所特有的一类蛋白激酶。ZmCIPK2(GenBank接受号为EF158033)是一个新的玉米CIPK激酶基因。组成型表达ZmCIPK2基因的拟南芥种子发芽和发芽后幼苗的早期生长对干旱胁迫超敏感。为了分析ZmCIPK2的抗旱功能,构建了RNAi表达载体pGreen0229-ZmCIPK2Sense-GFP-ZmCIPK2Antisense,利用花粉管通道法转化玉米,ZmCIPK2基因在RNAi玉米中的表达水平下降直至完全沉默。结果表明,与对照相比,RNAi株系的抗旱性得到了明显的提高。干旱胁迫下,与对照相比,ZmCIPK2基因RNAi植株具有较少的卷叶。在干旱胁迫下,ZmCIPK2RNAi株系的相对电导率和MDA含量分别比对照低16.1%和17.5%,而相对含水量、可溶性糖和脯氨酸含量则分别比对照高8.2%、8.8%和24.2%。
Maize is one of the most important crops in the world. With the global water shortage, drought stress has been a major constraint for maize production. It is of vital theoretical and practical significance for maize genetic breeding in drought resistance to analyze gene function in drought resistance, discover new drought resistance genes and cultivate new drought resistance maize lines. Protein kinase plays an important role in plant drought resistance process. In this research, transgenic ZmPtil, ZmPtil-1and ZmCIPK2, which induced by drought stress from maize, overexpressed or RNAi lines of Jing178or Jing501inbred maize were constructed via pollen tube pathway method or microprojectile bombardment. The function in drought resistance of these transgenic lines was further analyzed. The results showed as follows:
In order to analyze the drought resistance function of ZmPtil, the plant expression vector pBPC-ZmPtil-bar was constructed and transformed into maize via pollen tube pathway method. The different drought resistance index of transgenic ZmPtil maize plants was analyzed. The results showed that the transgenic plants can enhance the drought resistance comparing to control plants. Under drought stress, compared to wild-type plants, transgenic ZmPtil plants showed a stronger growth recovery phenotype with less leaf rolling and better growth with higher plant height and dry weight, which was6.2%and12.6%higher than WT, respectively. The grain number per ear and1000-grain weight of transgenic plants was21.9%and8.7%higher than WT, respectively. After drought stress, the relative electric conductivity and MDA content of transgenic plants was14.8%,18.1%lower than that that of WT, respectively, whereas the relative water content, total soluble sugars, proline content of transgenic plants was7.3%,2.5%,22.7%higher than that that of WT, respectively.
In order to analyze the drought resistance function of ZmPtil-1, the plant expression vector35S-ZwPri1-1-DHA-NOS-pGreen0029was constructed and transformed into Arabidopsis via floral dip method. The different drought resistance index of transgenic ZmPtil-1Arabidopsis plants was analyzed. The results demonstrated that over-expression of ZmPtil-1gene improved drought resistance in transgenic Arabidopsis. Under drought stress, compared to wild type, survival rate of the three transgenic lines, which was70%,76%and87%respectively, was significantly higher than that of wild type which was29%. Under drought stress, compared to wild-type plants, transgenic ZmPtil-1Arabidopsis plants showed a stronger growth recovery phenotype with less wither leaves and better growth. The seed weight of transgenic plants was58.7%higher than WT. After drought stress, the relative electric conductivity and MDA content of transgenic plants was13.7%,17.7%lower than that that of WT, respectively, whereas the relative water content, total soluble sugars, proline content of transgenic plants was7.1%,3.6%,22.5%higher than that that of WT, respectively.
In order to further analyze the effect of ZmPtil-1on the drought tolerance of maize, the plant expression vector pGreenO229-ZmPtil-1was constructed and transformed into maize via microprojectile bombardment method. The different drought resistance index of transgenic ZmPtil-1maize plants was analyzed. The results demonstrated that over-expression of ZmPtil-1gene improved drought resistance in transgenic maize. Under drought stress, compared to wild-type plants, transgenic ZmPtil-1plants showed a stronger growth recovery phenotype with less leaf rolling and better growth with higher plant height and dry weight, which was8.2%and14.4%higher than WT, respectively. The grain number per ear and1000-grain weight of transgenic plants was22.2%and8.8%higher than WT, respectively. After drought stress, the relative electric conductivity and MDA content of transgenic plants was20.4%,24.1%lower than that that of WT, respectively, whereas the relative water content, total soluble sugars, proline content of transgenic plants was9.4%,9.0%,24.1%higher than that that of WT, respectively.
A family of specific protein kinase CIPK participates in regulation of osmotic stress, salt, cold, ABA and sugar signal transduction in plant. ZmCIPK2(GenBank accession no. EF158033) is a new maize CIPK kinase gene. Transgenic ZmCIPK2Arabidopsis lines are hypersensitive to drought stress when germination and early seedling growth. In order to analyze the drought resistance function of ZmCIPK2, the RNAi expression vector pGreenO229-ZmCIPK2Sense-GFP-ZmCIPK2Antisense was constructed and transformed into maize via pollen tube pathway method. The expression level of ZmCIPK2in RNAi maize lines was declined to some extent and even completely silenced. The results showed that the RNAi lines can enhance the drought resistance comparing to control plants. Under drought stress, compared to wild-type plants, ZmClPK2RNAi plants showed a stronger growth recovery phenotype with less leaf rolling and better growth. After drought stress, the relative electric conductivity and MDA content of transgenic plants was16.1%,17.5%lower than that that of WT, respectively, whereas the relative water content, total soluble sugars, proline content of transgenic plants was8.2%,8.8%,24.2%higher than that that of WT, respectively.
引文
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