唐古特白刺NtP5CS和NtCIPK2基因的克隆及功能分析
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摘要
强旱生盐生植物唐古特白刺(MNitrariaria tangutorum Bobr.)为中国特有的多年生落叶灌木,具有极强的抗逆能力,沙埋后可通过克隆生长形成白刺包,是土壤荒漠化和盐碱化防治的先锋植物。然而,由于遗传信息的缺乏,该植物抗逆分子机制的研究进展十分缓慢。本研究从唐古特白刺中克隆脯氨酸合成途径的关键基因二氢吡咯-5-羧酸合成酶NtP5CS和钙信号转导通路重要组分钙调磷酸酶B类似蛋白互作激酶NtCIPK2,分析两基因的序列信息、系统进化、细胞定位、启动子结构及表达特性,探讨其在大肠杆菌和拟南芥中抵抗非生物胁迫过程中发挥的作用,并进行紫花苜蓿的遗传转化和初步验证。本研究的开展对于深入了解唐古特白刺的抗逆分子机制,开发利用唐古特白刺抗逆资源及培育抗逆牧草新品种具有重要的意义。主要研究结果如下：
1.从唐古特白刺中分离获得NtP5CS和NtCIPK2基因,生物信息学分析发现,NtP5CS和NtCIPK2基因编码蛋白具有与拟南芥、水稻等其他物种同源蛋白一致的典型特征、关键结构域和活性调控位点,据此推测这两个基因编码蛋白与同源蛋白具有相似的功能,即在唐古特白刺Pro的合成及钙信号传导机制中发挥着重要的作用。亚细胞定位结果显示,NtP5CS主要分布于细胞膜和细胞核中,而NtCIPK2在整个细胞中广泛存在。
2.利用SiteFinding-PCR技术获得NtP5CS和NtCIPK2基因的启动子序列,长度分别为2566bp和1612bp。通过启动子预测软件分析,发现两个基因的启动子中含有大量逆境胁迫响应元件,如W-box、MYB、MYC、HSE和LTRE元件等,这些逆境响应元件的存在,表明NtP5CS和NtCIPK2基因的表达可能受到干旱、高盐、高温和低温等非生物胁迫因素的诱导；半定量RT-PCR结果证实,在干旱、高盐、高温及低温4种胁迫条件下,两个基因表达量明显升高,在唐古特白刺生境适应过程中发挥积极作用。
3.构建NtP5CS和NtCIPK2基因的原核表达载体,并进行大肠杆菌转化实验,模拟盐、碱、干旱、渗透、高温和低温6种胁迫条件,发现重组菌株的生长状况明显优于对照菌株,两个基因的表达均可显著提高大肠杆菌的多重抗逆能力；以唐古特白刺和拟南芥作为抗逆和非抗逆植物的代表,比较分析NtP5CS与AtP5CS基因的表达对大肠杆菌的影响。结果表明,NtP5CS相较AtP5CS而言,可以更有效的提高大肠杆菌对于6种胁迫的抗性,证明基因来源的不同可能导致植物抗逆能力的差异。
4.将NtP5CS和NtCIPK2基因在拟南芥中过量表达,在模拟盐和干旱胁迫条件下,转基因拟南芥的萌发率、根长和生长状况等表型特征均优于野生型植株,转基因拟南芥可溶性糖和脯氨酸的含量显著高于野生型植株,丙二醛MDA上升幅度和叶绿素下降幅度显著低于野生型植株,说明NtP5CS和NtCIPK2基因的表达从积累渗透调节物质,减缓膜脂过氧化反应,增强光合作用等方面提高了拟南芥的抗旱性和耐盐性。
5.以下胚轴作为外植体材料,通过农杆菌介导法,将NtP5CS和NtCIPK2基因转化紫花苜蓿“中苜一号”,PCR和RT-PCR检测证实外源基因已整合到苜蓿基因组中并实现过量表达,最终获得转NtP5CS或NtCIPK2基因紫花苜蓿再生植株各3株,为培育苜蓿抗逆品种奠定基础。
Nitraria tangutorum is a typical and native desert halophyte in northwest China with superior ability to resist salinity, alkalinity and drought. In addition, it can efficiently fix moving sands and decrease wind speed by clonal growth, which makes it an ideal plant for preventing soil desertification and alleviating the degree of soil salinity-alkalinity. However, the molecular mechanism underlying stress tolerance of this halophyte is still far from clear owing to the absence of genetic data. At the present study, NtP5CS and NtCIPK2genes were isolated from N. tangutorum to elucidate their characteristics of phylogenetic analysis, subcellular localization, promoter prediction and expression pattern, and to investigate their function in stress tolerance of Escherichia coli and Arabidopsis. Finally, NtPSCS-and NtCIPK2-overexpression Medicago sativa were obtained and preliminary validated. This study would be of great help in understanding the molecular mechanism of N. tangutorum in stress tolerance, exploring and utilizing this plant and breeding the stess-tolerant herbage. The main results are as follow:
1. Two novel genes NtP5CS and NtCIPK2were isolated from N. tangutorum by rapid amplification of cDNA ends (RACE) cloning. Bioinformatics analysis demonstrated that NtP5CS and NtCIPK2shared similar function with their orthologs from other species due to the same typical characteristics, conserved domains and regulatory sites, they play the important role in Pro synthesis and Ca2+signal transduction of N. tangutorum. NtP5CS protein was limited to nuclei and at the plasmalemma, whereas NtCIPK2protein was distributed throughout the whole cells.
2. To identify putative cis regulatory elements,2566bp and1600bp of the upstream sequences from the start codon of NtP5CS and NtCIPK2were isolated and analyzed. Many stress-related cis regulatory elements, such as W-box, MYB, MYC, HSE and LTR lay in the promoters. In accordance with promoter analysis, expression patterns by semi-quantative PCR were proved that the two genes showed upregulation of transcript by drought, salinity, cold and heat stress. The results suggested that NtP5CS and NtCIPK2mediated stress tolerance with regard to the adaptability of N. tangutorum to hostile environment.
3. The two genes were inserted into the prokaryotic expression vector pGEX4T-1and transformed into E. coli, meanwhile; using the homologous gene AtP5CS of Arabidopsis as the representative of glycophyte, the difference in effect of NtP5CS and AtP5CS on E. coli was also assessed. The recombinant strains showed better growth than the control strains against salinity, drought, alkali, heat, cold, and high osmolyte stress, and also the NtP5CS-transformed strains grew better than the AtP5CS-transformed strains. The results indicated overexpression of NtP5CS and NtCIPK2improved the sress tolerance of E. coli and NtP5CS functioned more efficiently than AtP5CS.
4. Under stressed conditions, higher seed germination rate, longer root and better growth was observed and proline synthesis, chlorophyll content, soluble sugar accumulation and MDA was significantly improved in transgenic lines compared to the control, so overexpression of NtPSCS and NtCIPK2in Arabidopsis improved the transgenic plants tolerance to drought and salt stress by osmotic adjustment, membrane oxidation alleviation and photosynthesis enhancement.
5. Used the cotyledon as explants for induction, we obtained3NtP5CS-transfromed M. sativa lines and3NtCIPK2-transfromed lines, respectively. The two genes were integrated into the genome and expressed normally in the regenerated plants.
1.从唐古特白刺中分离获得NtP5CS和NtCIPK2基因,生物信息学分析发现,NtP5CS和NtCIPK2基因编码蛋白具有与拟南芥、水稻等其他物种同源蛋白一致的典型特征、关键结构域和活性调控位点,据此推测这两个基因编码蛋白与同源蛋白具有相似的功能,即在唐古特白刺Pro的合成及钙信号传导机制中发挥着重要的作用。亚细胞定位结果显示,NtP5CS主要分布于细胞膜和细胞核中,而NtCIPK2在整个细胞中广泛存在。
2.利用SiteFinding-PCR技术获得NtP5CS和NtCIPK2基因的启动子序列,长度分别为2566bp和1612bp。通过启动子预测软件分析,发现两个基因的启动子中含有大量逆境胁迫响应元件,如W-box、MYB、MYC、HSE和LTRE元件等,这些逆境响应元件的存在,表明NtP5CS和NtCIPK2基因的表达可能受到干旱、高盐、高温和低温等非生物胁迫因素的诱导；半定量RT-PCR结果证实,在干旱、高盐、高温及低温4种胁迫条件下,两个基因表达量明显升高,在唐古特白刺生境适应过程中发挥积极作用。
3.构建NtP5CS和NtCIPK2基因的原核表达载体,并进行大肠杆菌转化实验,模拟盐、碱、干旱、渗透、高温和低温6种胁迫条件,发现重组菌株的生长状况明显优于对照菌株,两个基因的表达均可显著提高大肠杆菌的多重抗逆能力；以唐古特白刺和拟南芥作为抗逆和非抗逆植物的代表,比较分析NtP5CS与AtP5CS基因的表达对大肠杆菌的影响。结果表明,NtP5CS相较AtP5CS而言,可以更有效的提高大肠杆菌对于6种胁迫的抗性,证明基因来源的不同可能导致植物抗逆能力的差异。
4.将NtP5CS和NtCIPK2基因在拟南芥中过量表达,在模拟盐和干旱胁迫条件下,转基因拟南芥的萌发率、根长和生长状况等表型特征均优于野生型植株,转基因拟南芥可溶性糖和脯氨酸的含量显著高于野生型植株,丙二醛MDA上升幅度和叶绿素下降幅度显著低于野生型植株,说明NtP5CS和NtCIPK2基因的表达从积累渗透调节物质,减缓膜脂过氧化反应,增强光合作用等方面提高了拟南芥的抗旱性和耐盐性。
5.以下胚轴作为外植体材料,通过农杆菌介导法,将NtP5CS和NtCIPK2基因转化紫花苜蓿“中苜一号”,PCR和RT-PCR检测证实外源基因已整合到苜蓿基因组中并实现过量表达,最终获得转NtP5CS或NtCIPK2基因紫花苜蓿再生植株各3株,为培育苜蓿抗逆品种奠定基础。
Nitraria tangutorum is a typical and native desert halophyte in northwest China with superior ability to resist salinity, alkalinity and drought. In addition, it can efficiently fix moving sands and decrease wind speed by clonal growth, which makes it an ideal plant for preventing soil desertification and alleviating the degree of soil salinity-alkalinity. However, the molecular mechanism underlying stress tolerance of this halophyte is still far from clear owing to the absence of genetic data. At the present study, NtP5CS and NtCIPK2genes were isolated from N. tangutorum to elucidate their characteristics of phylogenetic analysis, subcellular localization, promoter prediction and expression pattern, and to investigate their function in stress tolerance of Escherichia coli and Arabidopsis. Finally, NtPSCS-and NtCIPK2-overexpression Medicago sativa were obtained and preliminary validated. This study would be of great help in understanding the molecular mechanism of N. tangutorum in stress tolerance, exploring and utilizing this plant and breeding the stess-tolerant herbage. The main results are as follow:
1. Two novel genes NtP5CS and NtCIPK2were isolated from N. tangutorum by rapid amplification of cDNA ends (RACE) cloning. Bioinformatics analysis demonstrated that NtP5CS and NtCIPK2shared similar function with their orthologs from other species due to the same typical characteristics, conserved domains and regulatory sites, they play the important role in Pro synthesis and Ca2+signal transduction of N. tangutorum. NtP5CS protein was limited to nuclei and at the plasmalemma, whereas NtCIPK2protein was distributed throughout the whole cells.
2. To identify putative cis regulatory elements,2566bp and1600bp of the upstream sequences from the start codon of NtP5CS and NtCIPK2were isolated and analyzed. Many stress-related cis regulatory elements, such as W-box, MYB, MYC, HSE and LTR lay in the promoters. In accordance with promoter analysis, expression patterns by semi-quantative PCR were proved that the two genes showed upregulation of transcript by drought, salinity, cold and heat stress. The results suggested that NtP5CS and NtCIPK2mediated stress tolerance with regard to the adaptability of N. tangutorum to hostile environment.
3. The two genes were inserted into the prokaryotic expression vector pGEX4T-1and transformed into E. coli, meanwhile; using the homologous gene AtP5CS of Arabidopsis as the representative of glycophyte, the difference in effect of NtP5CS and AtP5CS on E. coli was also assessed. The recombinant strains showed better growth than the control strains against salinity, drought, alkali, heat, cold, and high osmolyte stress, and also the NtP5CS-transformed strains grew better than the AtP5CS-transformed strains. The results indicated overexpression of NtP5CS and NtCIPK2improved the sress tolerance of E. coli and NtP5CS functioned more efficiently than AtP5CS.
4. Under stressed conditions, higher seed germination rate, longer root and better growth was observed and proline synthesis, chlorophyll content, soluble sugar accumulation and MDA was significantly improved in transgenic lines compared to the control, so overexpression of NtPSCS and NtCIPK2in Arabidopsis improved the transgenic plants tolerance to drought and salt stress by osmotic adjustment, membrane oxidation alleviation and photosynthesis enhancement.
5. Used the cotyledon as explants for induction, we obtained3NtP5CS-transfromed M. sativa lines and3NtCIPK2-transfromed lines, respectively. The two genes were integrated into the genome and expressed normally in the regenerated plants.
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