洛旱2号小麦中4个干旱诱导基因的克隆和表达
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摘要
本文在分析干旱胁迫条件下小麦品种洛旱2号根系基因表达谱的基础上,通过RT-PCR技术克隆了4个干旱胁迫反应相关基因,分析了克隆基因在干旱、高盐胁迫和ABA处理条件下的表达特性;构建了其中一个基因TaLEA4的正义和反义植物表达载体,并通过基因枪法、花粉管通道法和农杆菌介导法进行了小麦和水稻的遗传转化,获得了经PCR鉴定呈阳性的转基因植株。主要结论为:
1.利用RT-PCR方法克隆了小麦干旱胁迫反应相关基因4个:克隆基因TaLEA4的cDNA片段为764bp,编码区为510 bp,5’-非编码区有94bp,3’-非编码区有160 bp,推测编码蛋白的分子量为17.53 kD,由169个氨基酸组成,进一步研究发现克隆基因TaLEA4在中国春基因组中包含一个100bp的内含子。TaRAB12基因cDNA片段长度为1013bp,其中编码区696bp,推测编码蛋白由231个氨基酸组成,分子量为23.14 kD;TaRAB56基因的cDNA片段长度为464bp,编码区为363bp,推测可编码120个氨基酸的多肽;TaRAB910基因的cDNA片段为950bp,其中编码区为549bp,可编码182个氨基酸的多肽,进一步分析发现该基因为受水分胁迫调控的膜蛋白基因。
2.分析了克隆基因在干旱和高盐(NaCl)胁迫及ABA处理条件下的表达特性。结果表明,在小麦幼苗根系中,在PEG6000胁迫诱导前期,TaLEA4基因的表达量逐渐上升,24h达到最强,48h时迅速回落;而在小麦幼苗的叶片中,该基因的在水分胁迫0.5h的表达量较强,其它时间点的表达水平都较低;在ABA处理胁迫条件下,该基因也表现出差异表达的特性。PEG胁迫条件下,小麦洛旱2号叶片中TaRAB56基因的表达呈现出逐渐增强后减弱的趋势,根系中TaRAB56基因在处理6h时有稍强于对照的表达;中国春叶片和根系中TaRAB56基因在胁迫不同时期的表达量与对照相比有较大差异。NaCl处理条件下,该基因在小麦洛旱2号中仅在0.5h时有稍强于对照的表达,而在中国春叶片和根系中该基因表达趋势不明显;在ABA胁迫过程中,TaRAB910基因在小麦幼苗叶片中出现了差异表达;但在NaCl胁迫过程中,该基因在根系中的表达量高于在叶片中的表达量。
3.构建了TaLEA4基因的正义和反义植物表达的载体,并通过基因枪、花粉管通道和农杆菌介导法分别进行了小麦和水稻的遗传转化研究。转基因植株的PCR检测结果表明:基因枪法获得转TaLEA4正义基因的转基因小麦阳性植株2株,转TaLEA4反义基因的转基因小麦阳性植株6株;花粉管通道法获得转反义TaLEA4基因小麦植株223株(其中以郑麦9023为受体的185株,以豫麦34为受体的38株),转正义TaLEA4基因小麦植株26株(其中以郑麦9023为受体的5株,以豫麦34为受体的21株);农杆菌介导法获得转基因水稻植株20株,其中转正义和反义TaLEA4基因的植株分别为4株和16株。
Based on the data obtained from the gene expression profiling under the drought-stress in the roots of wheat cultivar‘Luo Han No. 2’, four drought-induced genes were cloned by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and their expression patterns under the drought- and salt- stresses, as well as the ABA treatment, were studied; The sense and antisense expression vectors of one of the cloned genes were constructed and were used in the genetic transformation of wheat and rice plants by the particlebombardment, Agrobacterium-mediated and pollen tube mediated transformation methods, and some transgenic plantlets were obtained. The main results showed as follows:
1 Four drought-stress correlate genes were cloned by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The cDNA of TaLEA4 includes a sequence of 764bp in length and a coding region 510bp,which contains 94bp in 5' UTR and 160bp in 3' UTR . The deduced amino acids sequence (170 aa) of this gene is about 17.53KD and the pI is 6.05. The genemic cDNA of TaLEA4 in Chinese Spring was studied and a 100bp intron was found in the coding region of the gene. The TaRAB12 includes a sequence of 1013bp in length and a coding region 726bp,and the deduced amino acids sequence (241 aa) of this gene is about 23.98KD. The TaRAB56 is a gene which including a sequence of 464bp in length and a coding region 363bp,and the deduced amino acids was composed by 120aa.The TaRAB910 gene included a sequence of 950bp in length and a coding region 549bp,which the deduced amino acids were composed by 182aa, and the further analysis showed that this gene can code a water-stress regulated membrane protein.
2 Expression patterns of cloned genes under the drought-, NaCl- and ABA- stresses were studied via SQ-RT-PCR , then the results showed: in roots, the expression level of TaLEA4 was gradually increased with the water-stress accumulation, and it reached the highest level at the time point of 24 hours after PEG treatment, however, the expression level declined at the time point of 48 hours after PEG treatment; in leaves, the expression of the gene is strong at the time point of 0.5 hours after PEG treatment, but that is relatively weak at other time points detected; Undering the stress of ABA,the TaLEA4 have the Characteristics of differential expression . Expression patterns of the TaRAB56gene under the drought- and salt- stresses showed that: under the PEG mediated water-stress, the expression of TaRAB56 was increased gradually and then decreased in the leaves of LuohanNo. 2, and in root, the expression of the gene is relatively higher than that of control at time 6h of stress treatment. In Chinese Spring, the expression of the gene is greatly changed in the leaves and roots. Under the NaCl mediated salt-stress treatment, The expression of TaRAB56 was slightly stranger than that of control at the time point of 0.5 h in Luohan 2; while in Chinese Spring, the expression of the gene was not obviously changed during the salt-stress treatment in the leaves and roots tissues.The expression level of TaRAB910 under the ABA -stress in leave were significant differently comparing with it's expression in root, but the expression level of TaRAB910 in leaves were lower comparing with it's expression in roots when under NaCl stress.
3 The sense and antisense plant expression vectors of TaLEA4 gene were constructed, and they were used in the transformation of rice and wheat plant via microprojectile bombardment, Agrobacterium-mediated and pollen tube pathway mediated transformation. The identification of transgenic plants showed that 2 transgenic wheat plants with the sense TaLEA4 gene and 6 transgenic wheat plants with antisense TaLEA4 gene were obtained via the transformation of microprojectile bombardment. By the method of pollen-tube-pathway, 223 transgenic wheat plants with antisense TaLEA4 gene were obtained, and among them, 185 plants were the wheat variety "ZhengMai9023" and 38 of them were the wheat variety "YuMai34"; From the 26 positive plants with sense TaLEA4 gene, 5 transgenic plants were the wheat varietiy "ZhengMai9023" and 21 were the wheat varietiy "YuMai34". The PCR identification of rice putative transgenic plants transformed by Agrobacterium-mediated methods showed that 4 positive transgenic rice plants with the sense TaLEA4 gene and 16 transgenic plants with anti-sence TaLEA4 gene were obtained.
1.利用RT-PCR方法克隆了小麦干旱胁迫反应相关基因4个:克隆基因TaLEA4的cDNA片段为764bp,编码区为510 bp,5’-非编码区有94bp,3’-非编码区有160 bp,推测编码蛋白的分子量为17.53 kD,由169个氨基酸组成,进一步研究发现克隆基因TaLEA4在中国春基因组中包含一个100bp的内含子。TaRAB12基因cDNA片段长度为1013bp,其中编码区696bp,推测编码蛋白由231个氨基酸组成,分子量为23.14 kD;TaRAB56基因的cDNA片段长度为464bp,编码区为363bp,推测可编码120个氨基酸的多肽;TaRAB910基因的cDNA片段为950bp,其中编码区为549bp,可编码182个氨基酸的多肽,进一步分析发现该基因为受水分胁迫调控的膜蛋白基因。
2.分析了克隆基因在干旱和高盐(NaCl)胁迫及ABA处理条件下的表达特性。结果表明,在小麦幼苗根系中,在PEG6000胁迫诱导前期,TaLEA4基因的表达量逐渐上升,24h达到最强,48h时迅速回落;而在小麦幼苗的叶片中,该基因的在水分胁迫0.5h的表达量较强,其它时间点的表达水平都较低;在ABA处理胁迫条件下,该基因也表现出差异表达的特性。PEG胁迫条件下,小麦洛旱2号叶片中TaRAB56基因的表达呈现出逐渐增强后减弱的趋势,根系中TaRAB56基因在处理6h时有稍强于对照的表达;中国春叶片和根系中TaRAB56基因在胁迫不同时期的表达量与对照相比有较大差异。NaCl处理条件下,该基因在小麦洛旱2号中仅在0.5h时有稍强于对照的表达,而在中国春叶片和根系中该基因表达趋势不明显;在ABA胁迫过程中,TaRAB910基因在小麦幼苗叶片中出现了差异表达;但在NaCl胁迫过程中,该基因在根系中的表达量高于在叶片中的表达量。
3.构建了TaLEA4基因的正义和反义植物表达的载体,并通过基因枪、花粉管通道和农杆菌介导法分别进行了小麦和水稻的遗传转化研究。转基因植株的PCR检测结果表明:基因枪法获得转TaLEA4正义基因的转基因小麦阳性植株2株,转TaLEA4反义基因的转基因小麦阳性植株6株;花粉管通道法获得转反义TaLEA4基因小麦植株223株(其中以郑麦9023为受体的185株,以豫麦34为受体的38株),转正义TaLEA4基因小麦植株26株(其中以郑麦9023为受体的5株,以豫麦34为受体的21株);农杆菌介导法获得转基因水稻植株20株,其中转正义和反义TaLEA4基因的植株分别为4株和16株。
Based on the data obtained from the gene expression profiling under the drought-stress in the roots of wheat cultivar‘Luo Han No. 2’, four drought-induced genes were cloned by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and their expression patterns under the drought- and salt- stresses, as well as the ABA treatment, were studied; The sense and antisense expression vectors of one of the cloned genes were constructed and were used in the genetic transformation of wheat and rice plants by the particlebombardment, Agrobacterium-mediated and pollen tube mediated transformation methods, and some transgenic plantlets were obtained. The main results showed as follows:
1 Four drought-stress correlate genes were cloned by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The cDNA of TaLEA4 includes a sequence of 764bp in length and a coding region 510bp,which contains 94bp in 5' UTR and 160bp in 3' UTR . The deduced amino acids sequence (170 aa) of this gene is about 17.53KD and the pI is 6.05. The genemic cDNA of TaLEA4 in Chinese Spring was studied and a 100bp intron was found in the coding region of the gene. The TaRAB12 includes a sequence of 1013bp in length and a coding region 726bp,and the deduced amino acids sequence (241 aa) of this gene is about 23.98KD. The TaRAB56 is a gene which including a sequence of 464bp in length and a coding region 363bp,and the deduced amino acids was composed by 120aa.The TaRAB910 gene included a sequence of 950bp in length and a coding region 549bp,which the deduced amino acids were composed by 182aa, and the further analysis showed that this gene can code a water-stress regulated membrane protein.
2 Expression patterns of cloned genes under the drought-, NaCl- and ABA- stresses were studied via SQ-RT-PCR , then the results showed: in roots, the expression level of TaLEA4 was gradually increased with the water-stress accumulation, and it reached the highest level at the time point of 24 hours after PEG treatment, however, the expression level declined at the time point of 48 hours after PEG treatment; in leaves, the expression of the gene is strong at the time point of 0.5 hours after PEG treatment, but that is relatively weak at other time points detected; Undering the stress of ABA,the TaLEA4 have the Characteristics of differential expression . Expression patterns of the TaRAB56gene under the drought- and salt- stresses showed that: under the PEG mediated water-stress, the expression of TaRAB56 was increased gradually and then decreased in the leaves of LuohanNo. 2, and in root, the expression of the gene is relatively higher than that of control at time 6h of stress treatment. In Chinese Spring, the expression of the gene is greatly changed in the leaves and roots. Under the NaCl mediated salt-stress treatment, The expression of TaRAB56 was slightly stranger than that of control at the time point of 0.5 h in Luohan 2; while in Chinese Spring, the expression of the gene was not obviously changed during the salt-stress treatment in the leaves and roots tissues.The expression level of TaRAB910 under the ABA -stress in leave were significant differently comparing with it's expression in root, but the expression level of TaRAB910 in leaves were lower comparing with it's expression in roots when under NaCl stress.
3 The sense and antisense plant expression vectors of TaLEA4 gene were constructed, and they were used in the transformation of rice and wheat plant via microprojectile bombardment, Agrobacterium-mediated and pollen tube pathway mediated transformation. The identification of transgenic plants showed that 2 transgenic wheat plants with the sense TaLEA4 gene and 6 transgenic wheat plants with antisense TaLEA4 gene were obtained via the transformation of microprojectile bombardment. By the method of pollen-tube-pathway, 223 transgenic wheat plants with antisense TaLEA4 gene were obtained, and among them, 185 plants were the wheat variety "ZhengMai9023" and 38 of them were the wheat variety "YuMai34"; From the 26 positive plants with sense TaLEA4 gene, 5 transgenic plants were the wheat varietiy "ZhengMai9023" and 21 were the wheat varietiy "YuMai34". The PCR identification of rice putative transgenic plants transformed by Agrobacterium-mediated methods showed that 4 positive transgenic rice plants with the sense TaLEA4 gene and 16 transgenic plants with anti-sence TaLEA4 gene were obtained.
引文
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