atnhx1和hvbadh1基因的克隆及对三种植物的转化研究
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摘要
土壤盐渍化是限制植物生长的主要原因之一。为了对抗盐渍化威胁，植物体在漫长的进化过程中形成了两条对抗盐胁迫的通用途径。一条是在遭受盐胁迫后重建细胞内遭到破坏的离子稳态，另一条是在胞质内积累组织相容性渗调剂解除高盐对植物体造成的渗透胁迫伤害。前者涉及细胞对Na~+、K~+和Cl~-的选择性吸收以及随后发生的将可能导致细胞毒害的过多离子(如Na~+、Cl~-等)区隔化到液泡中的过程。Na~+区隔化是一个植物主动将胞质中Na~+聚集到液泡中进行渗透调节的耗能过程，这个过程的实现离不开液泡膜Na~+／H~+逆向转运蛋白的作用。就第二条途径而言，目前常见的组织相容性溶剂包括甜菜碱(如甘氨酸甜菜碱)及其类似物、糖和多羟基醇(例如甘露醇、山梨醇和海藻糖等)以及氨基酸(如脯氨酸等)。尽管人们现在发现不同植物在遭遇到盐胁迫时积累的渗透调节物质不尽相同，但是在众多组织相容性渗透调节物质中，甘氨酸甜菜碱被普遍认为是植物应对包括盐胁迫在内的渗透胁迫时最重要的渗透调节者。高等植物中，甜菜碱合成过程是由胆碱经过连续两步不可逆氧化而实现的。首先，胆碱在胆碱单加氧酶(choline monooxigenase，CMO)的催化下被氧化合成甜菜碱醛；甜菜碱醛继而在甜菜碱醛脱氢酶(betaine aldehyde dehydrogenase，BADH)的作用下，被氧化成甘氨酸甜菜碱。
本文根据已报道的拟南芥液泡膜Na~+／H~+逆向转运蛋白AtNHX1编码基因序列(NCBI收录号：AF510074)设计了一对PCR引物，通过RT-PCR,直接从经过100 mmol／L NaCl．处理24h的拟南芥幼苗中克隆到编码液泡膜Na~+／H~+逆向转运蛋白，长度为1，617 bp的amhx1-cDNA片段，该cDNA片段含有编码一条含有538个氨基酸的多肽链的完整ORF。同源性比对结果显示，实验得到的atnhx1-cDNA序列与原报道的atnhx1基因具有99.7％的同源性。为了构建能够在植物中表达atnhx1-cDNA的植物表达载体，我们将CaMV35S启动子，TMV RNA 5’UTR中的Ω片段，atnhx1-cDNA和NOS polyA终止子串连在一起构成一个完整的表达盒，并使该表达盒整合到位于双元植物表达载体pNT质粒上T-DNA区的多克隆位点上，结果成功构建了含有atnhx1-cDNA的重组双元植物表达载体。用液氮冻融法将pNT-atnhx1质粒导入农杆菌LBA4404感受态细胞中，构建能够直接用于转化实验的农杆菌LBA4404(pNT-atnhx1)植物转化系统。
研究中对卡那霉素筛选浓度、外植体共培养时间、农杆菌菌液浓度、农杆菌侵染时间和乙酰丁香酮作用浓度等5个影响农杆菌对受体植物转化效率的因素进行了优化，进而建立了稳定高效的草木樨状黄芪和菊苣农杆菌LBA4404(pNT-atnhx1)高效遗传转化体系。应用这两个系统分别对．草木樨状黄芪和菊苣进行遗传转化，获得了3个草木樨状黄芪、2个菊苣转基因株系和大量的转基因愈伤组织。对所获得的卡那霉素抗性株系进行PCR、southern杂交和RT-PCR等分子生物学检测结果表明：筛选标记基因nptⅡ及目的基因atnhx1-cDNA已成功地整合到转基因株系的基因组中，并且可以在RNA水平上进行转录。
对草木樨状黄芪和菊苣转基因株系愈伤组织进行盐胁迫耐受试验，游离脯氨酸含量、K~+和Na~+含量测定的检测的结果表明：在相同胁迫条件下，转基因愈伤组织的相对生长率均明显高于未转化的野生型愈伤组织，两种植物的转基因株系愈伤组织内K~+／Na~+比值也始终显著高于野生型，而转基因株系愈伤组织的游离脯氨酸含量也始终高于野生型。可见，atnhx1基因的导入显著提高了草木樨状黄芪和菊苣细胞水平上的耐盐性。
草木樨状黄芪和菊苣转基因株系叶片K~+和Na~+含量测定和相对电导率测定结果显示，在相同胁迫条件下，两种植物的转基因株系叶片内K~+／Na~+比值始终显著高于野生型，而相对电导率则低于野生型植株叶片。可见，atnhx1基因的导入提高了草木樨状黄芪和菊苣植株水平上的生理耐盐性。
根据大麦，小麦，玉米、菠菜、高梁，拟南芥等6种不同植物已报道甜菜碱醛脱氢酶氨基酸序列进化保守区域的特点，设计一对兼并引物，运用RT-PCR技术从青稞中克隆得到728bp cDNA片段。根据该cDNA片段的序列测定结果，设计用于3’，5’RACE实验的引物。通过3’RACE获得538 bp cDNA片段，其中包含目的基因包括3’UTR在内的3’末端全部序列信息。根据RACE的结果，应用RT-PCR方法从青稞中成功获得长度为1，512bp编码BADH的全长编码ORF，通过氨基酸同源性比对发现该ORF的推定表达产物与大麦BADH同工酶BBD_2的同源性为98.4％，而与小麦、玉米和水稻等禾本科作物的BADH同源性分别为97％，84.7％和85.1％。这表明本研究克隆到的序列是青稞BADH的编码cDNA，我们将其命名为hvbadh1，并投递到GeneBank，获得注册号EF492983。hvbadh1在原核表达系统TB1-pMAL c-2-X中的表达研究显示，hvbadh1可以正常表达出分子量为54.2KD的蛋白质。
将克隆得到的hvbadh1的编码ORF，插入到添加了植物表达CaMV 35S启动子和Nos polyA终止子调控元件的植物表达载体pCAMBIA1301质粒相应的克隆位点中，构建了重组的双元植物表达载体pCAM-ba质粒，通过冻融法将其转入到根癌农杆菌LBA4404中，成功地构建了hvbadh1基因的农杆菌植物转化系统LBA4404(pCAM-ba)，用于后期对烟草的遗传转化。
采用农杆菌LBA4404(pCAM-ba)介导的叶圆盘法，将hvbadh1基因的编码ORF转移到受体烟草-秦烟95中。对所获得的潮霉素抗性烟草株系进行PCR和RT-PCR等分子生物学检测结果表明：在得到的2个烟草转化株系中，hptⅡ基因及hvbadh1基因已成功地整合到基因组中，并且可以在RNA水平上进行转录。
Salinity is a major constraint to plant productivity. In order to adapt to the salt stress, there are two common mechanisms found in plants, one of which is to rebuild the cellular ion homeostasis, the other is to accumulate the compatible solutes. Rebuilding the cellular ion homeostasis involves the intracellular regulation of absorbing Na~+, K~+ and Cl~- as well as subsequent vacuolar compartmentalization to prevent the accumulation of toxic ions in the cytosol. Vacuolar sequestration of Na~+is an important and cost-effective strategy for osmotic adjustment that also reduces the Na~+ concentration in the cytosol. Na~+ sequestration into vacuolar depends on the expression and activity of Na~+/H~+ antiportors. On the other hand, the compatible solutes, including betaines and related compounds, polyols and sugairs (such as mannitol, sorbitol, and trehalose), and amino acids (such as praline), are accumulated differently among plant species. Among them, betaines appear to be a critical determinant of stress tolerances.
In this study, the open reading frame(ORF) of atnhx1-cDNA, encoding the A. thaliana vacuolar Na~+/H~+ antiportor, was cloned by using one-step RT-PCR from the Arabidopsis thaliana seedlings after 24h treatment with 100 mmol/L NaCl. Two primers for PCR were designed following the atnhx1 sequence from NCBI (accession number: AF 510074). The atnhx1-cDNA we obtained is 1,617 bp, which just was an open reading frame encoding a predicted polypeptide of 538 amino acids, and was high homologue (99.7%) to the original atnhx1 sequence. In order to construct an expression vector which can express atnhx1-cDNA in plant, an expression cassette, including a CaMV35S promoter, aΩfragment of TMV RNA 5'UTR, atnhx1-cDNA and a NOS polyA sequence, was introduced into the T-DNA region of the binary vector pNT. The plant selectable marker gene of this recombined vector is nptII delivering kanamycin resistance to the transgenic plants. This recombined binary vector named as pNT-atnhx1 was transferred into Agrobacterium tumefaciens LBA4404 by freeze-thaw method, which could be used for plant transformation directly.
In the experiment, a highly-efficient and stable protocol for Agrobacterium mediated transformation was established and was adapted for the genetic transformation of two plant species, Astragalus melilotoides Pall and Cichoriuminty bus L. Five factors affecting the efficiency of Agrobacterium mediated transformation were optimized, such as the threshold concentration of kanamycin for selection, the pre-culture time of the explants, the suitable infection time of the Agrobacterium, the working concentration of the Agrobacterium and the best working concentration of acetosyringone (AS). Using this protocol, atnhx1-cDNA was transfermed into A. melilotoides Pall and C.bus L successfully. 3 T_0 generation transgenic strains of A melilotoides Pall and 2 transgenic strains of C.bus L and some of kanamycin-resistant calli were obtained. The PCR, Southern blotting and RT-PCR identification of the T_0 transgenic plants show that the atnhx1-cDNA has already integrated into the genome of the transgenic plants and the target gene can express at the level of RNA transcription.
Both the transgenic calli and the wild-type calli of A. melilotoides Pall and C.bus L were respectively cultured on the media containing different concentration of NaCl in order to test their resistance to NaCl. The results demonstrated that relative growth rates of the transgenic calli were higher than that of the wild-type calli.
After being cultured respectively on the media containing different concentration of NaCl, the accumulations of free proline ,K~+ and Na~+ in the cells of both the transgenic calli and the wild-type calli of A. melilotoides Pall and C.bus L were investigated. The results showed that both the concentration of free proline and K~+/Na~+ in the transgenic calli were higher than that in wild-type calli under the same stress condition. Therefore, the intergration of atnhxl gene enhanced the resistance to the salt stress of the transgenic calli.
In order to investigate whether the atnhxl gene transformation can enhance the salt tolerance of the T_0 transgenic plants, the K~+ and Na~+ concentration and relative conductivity of the transgenic plant leaves of A. melilotoides Pall and C.bus L were estimated when they were cultured under the stress triggered by various concentration of NaCl from 0 to 250mmol/L. The results indicated that the K~+/Na~+ rate of the transgenic plants was higher than that of wild-type plant under the same stress condition, but the situation of relative conductivity was on the opposite. Therefore, the transfer of atnhx1 gene can also enhance the salt tolerance of the target plant.
According to the multiply alignment of amino acid sequences of several plants Betaine Aldehyde Dehydrogenase(BADH) from NCBI, a pair of degenerate primers were designed against the conserved regions that was used for RT-PCR to amplify a 728bp cDNA segment from Hordeum vulgare L.var.nudum Hook.f. Based on the sequence of this cDNA segment, primers were designed for 3' RACE, 5'RACE and RT-PCR. Then a 538bp cDNA segment (the sequence of 3' end of badh) was acquired by 3' RACE. Finally by RT-PCR, a 1, 657bp cDNA fragment was cloned from Hordeum vulgare L.var.nudum Hook.f. This cDNA fragment contained a 1,512bp ORF coding the putative Betaine Aldehyde Dehydrogenase(BADH) and comprising 503 amino acid residues. Compared by blast, this putative Betaine Aldehyde Dehydrogenase was high homologue to other plants' BADH, and the highest homology to bbd2, a badh gene from barely, was 98.4%. The result indicates the gene cloned from Hordeum vulgare L.var.nudum Hook.f is a BADH gene named hvbadh1, and its Genbank accession number is EF492983. Expressing hvbadhl in the E.coli protein fusion and purification system, a new fusion protein about 96.3KD, composed by hvBADH1 (about 54.2KD) and MBP (about 42KD), was found by SDS-PAGE.
In order to construct an expression vector which can express hvbadhl in plants, an expression cassette, including a CaMV35S promoter, hvbadhl and a NOS polyA sequence, was introduced into the T-DNA region of the binary vector pCAMBIA1301. The plant selectable marker gene of the vector is hptII delivering hygromycin B resistance to the transgenic plants. This recombined binary vector named as pCAM-ba was transfermed into Agrobacterium tumefaciens LBA4404 by freeze-thaw method, which could be used for plant transformation directly.
2 regenerated T_0 transgenic plants of Nicotiana tabacum Qinyan95 with hygromycin B resistance were obtained via Agrobacterium tumefaciens-mediated transformation and characterized by PCR, and RT-PCR. The PCR and RT-PCR analysis of the T_0 generation transgenic plants show that the hvbadh1 gene has already integrated into the genome of the transgenic plants and the target gene can express at the level of RNA transcription.
本文根据已报道的拟南芥液泡膜Na~+／H~+逆向转运蛋白AtNHX1编码基因序列(NCBI收录号：AF510074)设计了一对PCR引物，通过RT-PCR,直接从经过100 mmol／L NaCl．处理24h的拟南芥幼苗中克隆到编码液泡膜Na~+／H~+逆向转运蛋白，长度为1，617 bp的amhx1-cDNA片段，该cDNA片段含有编码一条含有538个氨基酸的多肽链的完整ORF。同源性比对结果显示，实验得到的atnhx1-cDNA序列与原报道的atnhx1基因具有99.7％的同源性。为了构建能够在植物中表达atnhx1-cDNA的植物表达载体，我们将CaMV35S启动子，TMV RNA 5’UTR中的Ω片段，atnhx1-cDNA和NOS polyA终止子串连在一起构成一个完整的表达盒，并使该表达盒整合到位于双元植物表达载体pNT质粒上T-DNA区的多克隆位点上，结果成功构建了含有atnhx1-cDNA的重组双元植物表达载体。用液氮冻融法将pNT-atnhx1质粒导入农杆菌LBA4404感受态细胞中，构建能够直接用于转化实验的农杆菌LBA4404(pNT-atnhx1)植物转化系统。
研究中对卡那霉素筛选浓度、外植体共培养时间、农杆菌菌液浓度、农杆菌侵染时间和乙酰丁香酮作用浓度等5个影响农杆菌对受体植物转化效率的因素进行了优化，进而建立了稳定高效的草木樨状黄芪和菊苣农杆菌LBA4404(pNT-atnhx1)高效遗传转化体系。应用这两个系统分别对．草木樨状黄芪和菊苣进行遗传转化，获得了3个草木樨状黄芪、2个菊苣转基因株系和大量的转基因愈伤组织。对所获得的卡那霉素抗性株系进行PCR、southern杂交和RT-PCR等分子生物学检测结果表明：筛选标记基因nptⅡ及目的基因atnhx1-cDNA已成功地整合到转基因株系的基因组中，并且可以在RNA水平上进行转录。
对草木樨状黄芪和菊苣转基因株系愈伤组织进行盐胁迫耐受试验，游离脯氨酸含量、K~+和Na~+含量测定的检测的结果表明：在相同胁迫条件下，转基因愈伤组织的相对生长率均明显高于未转化的野生型愈伤组织，两种植物的转基因株系愈伤组织内K~+／Na~+比值也始终显著高于野生型，而转基因株系愈伤组织的游离脯氨酸含量也始终高于野生型。可见，atnhx1基因的导入显著提高了草木樨状黄芪和菊苣细胞水平上的耐盐性。
草木樨状黄芪和菊苣转基因株系叶片K~+和Na~+含量测定和相对电导率测定结果显示，在相同胁迫条件下，两种植物的转基因株系叶片内K~+／Na~+比值始终显著高于野生型，而相对电导率则低于野生型植株叶片。可见，atnhx1基因的导入提高了草木樨状黄芪和菊苣植株水平上的生理耐盐性。
根据大麦，小麦，玉米、菠菜、高梁，拟南芥等6种不同植物已报道甜菜碱醛脱氢酶氨基酸序列进化保守区域的特点，设计一对兼并引物，运用RT-PCR技术从青稞中克隆得到728bp cDNA片段。根据该cDNA片段的序列测定结果，设计用于3’，5’RACE实验的引物。通过3’RACE获得538 bp cDNA片段，其中包含目的基因包括3’UTR在内的3’末端全部序列信息。根据RACE的结果，应用RT-PCR方法从青稞中成功获得长度为1，512bp编码BADH的全长编码ORF，通过氨基酸同源性比对发现该ORF的推定表达产物与大麦BADH同工酶BBD_2的同源性为98.4％，而与小麦、玉米和水稻等禾本科作物的BADH同源性分别为97％，84.7％和85.1％。这表明本研究克隆到的序列是青稞BADH的编码cDNA，我们将其命名为hvbadh1，并投递到GeneBank，获得注册号EF492983。hvbadh1在原核表达系统TB1-pMAL c-2-X中的表达研究显示，hvbadh1可以正常表达出分子量为54.2KD的蛋白质。
将克隆得到的hvbadh1的编码ORF，插入到添加了植物表达CaMV 35S启动子和Nos polyA终止子调控元件的植物表达载体pCAMBIA1301质粒相应的克隆位点中，构建了重组的双元植物表达载体pCAM-ba质粒，通过冻融法将其转入到根癌农杆菌LBA4404中，成功地构建了hvbadh1基因的农杆菌植物转化系统LBA4404(pCAM-ba)，用于后期对烟草的遗传转化。
采用农杆菌LBA4404(pCAM-ba)介导的叶圆盘法，将hvbadh1基因的编码ORF转移到受体烟草-秦烟95中。对所获得的潮霉素抗性烟草株系进行PCR和RT-PCR等分子生物学检测结果表明：在得到的2个烟草转化株系中，hptⅡ基因及hvbadh1基因已成功地整合到基因组中，并且可以在RNA水平上进行转录。
Salinity is a major constraint to plant productivity. In order to adapt to the salt stress, there are two common mechanisms found in plants, one of which is to rebuild the cellular ion homeostasis, the other is to accumulate the compatible solutes. Rebuilding the cellular ion homeostasis involves the intracellular regulation of absorbing Na~+, K~+ and Cl~- as well as subsequent vacuolar compartmentalization to prevent the accumulation of toxic ions in the cytosol. Vacuolar sequestration of Na~+is an important and cost-effective strategy for osmotic adjustment that also reduces the Na~+ concentration in the cytosol. Na~+ sequestration into vacuolar depends on the expression and activity of Na~+/H~+ antiportors. On the other hand, the compatible solutes, including betaines and related compounds, polyols and sugairs (such as mannitol, sorbitol, and trehalose), and amino acids (such as praline), are accumulated differently among plant species. Among them, betaines appear to be a critical determinant of stress tolerances.
In this study, the open reading frame(ORF) of atnhx1-cDNA, encoding the A. thaliana vacuolar Na~+/H~+ antiportor, was cloned by using one-step RT-PCR from the Arabidopsis thaliana seedlings after 24h treatment with 100 mmol/L NaCl. Two primers for PCR were designed following the atnhx1 sequence from NCBI (accession number: AF 510074). The atnhx1-cDNA we obtained is 1,617 bp, which just was an open reading frame encoding a predicted polypeptide of 538 amino acids, and was high homologue (99.7%) to the original atnhx1 sequence. In order to construct an expression vector which can express atnhx1-cDNA in plant, an expression cassette, including a CaMV35S promoter, aΩfragment of TMV RNA 5'UTR, atnhx1-cDNA and a NOS polyA sequence, was introduced into the T-DNA region of the binary vector pNT. The plant selectable marker gene of this recombined vector is nptII delivering kanamycin resistance to the transgenic plants. This recombined binary vector named as pNT-atnhx1 was transferred into Agrobacterium tumefaciens LBA4404 by freeze-thaw method, which could be used for plant transformation directly.
In the experiment, a highly-efficient and stable protocol for Agrobacterium mediated transformation was established and was adapted for the genetic transformation of two plant species, Astragalus melilotoides Pall and Cichoriuminty bus L. Five factors affecting the efficiency of Agrobacterium mediated transformation were optimized, such as the threshold concentration of kanamycin for selection, the pre-culture time of the explants, the suitable infection time of the Agrobacterium, the working concentration of the Agrobacterium and the best working concentration of acetosyringone (AS). Using this protocol, atnhx1-cDNA was transfermed into A. melilotoides Pall and C.bus L successfully. 3 T_0 generation transgenic strains of A melilotoides Pall and 2 transgenic strains of C.bus L and some of kanamycin-resistant calli were obtained. The PCR, Southern blotting and RT-PCR identification of the T_0 transgenic plants show that the atnhx1-cDNA has already integrated into the genome of the transgenic plants and the target gene can express at the level of RNA transcription.
Both the transgenic calli and the wild-type calli of A. melilotoides Pall and C.bus L were respectively cultured on the media containing different concentration of NaCl in order to test their resistance to NaCl. The results demonstrated that relative growth rates of the transgenic calli were higher than that of the wild-type calli.
After being cultured respectively on the media containing different concentration of NaCl, the accumulations of free proline ,K~+ and Na~+ in the cells of both the transgenic calli and the wild-type calli of A. melilotoides Pall and C.bus L were investigated. The results showed that both the concentration of free proline and K~+/Na~+ in the transgenic calli were higher than that in wild-type calli under the same stress condition. Therefore, the intergration of atnhxl gene enhanced the resistance to the salt stress of the transgenic calli.
In order to investigate whether the atnhxl gene transformation can enhance the salt tolerance of the T_0 transgenic plants, the K~+ and Na~+ concentration and relative conductivity of the transgenic plant leaves of A. melilotoides Pall and C.bus L were estimated when they were cultured under the stress triggered by various concentration of NaCl from 0 to 250mmol/L. The results indicated that the K~+/Na~+ rate of the transgenic plants was higher than that of wild-type plant under the same stress condition, but the situation of relative conductivity was on the opposite. Therefore, the transfer of atnhx1 gene can also enhance the salt tolerance of the target plant.
According to the multiply alignment of amino acid sequences of several plants Betaine Aldehyde Dehydrogenase(BADH) from NCBI, a pair of degenerate primers were designed against the conserved regions that was used for RT-PCR to amplify a 728bp cDNA segment from Hordeum vulgare L.var.nudum Hook.f. Based on the sequence of this cDNA segment, primers were designed for 3' RACE, 5'RACE and RT-PCR. Then a 538bp cDNA segment (the sequence of 3' end of badh) was acquired by 3' RACE. Finally by RT-PCR, a 1, 657bp cDNA fragment was cloned from Hordeum vulgare L.var.nudum Hook.f. This cDNA fragment contained a 1,512bp ORF coding the putative Betaine Aldehyde Dehydrogenase(BADH) and comprising 503 amino acid residues. Compared by blast, this putative Betaine Aldehyde Dehydrogenase was high homologue to other plants' BADH, and the highest homology to bbd2, a badh gene from barely, was 98.4%. The result indicates the gene cloned from Hordeum vulgare L.var.nudum Hook.f is a BADH gene named hvbadh1, and its Genbank accession number is EF492983. Expressing hvbadhl in the E.coli protein fusion and purification system, a new fusion protein about 96.3KD, composed by hvBADH1 (about 54.2KD) and MBP (about 42KD), was found by SDS-PAGE.
In order to construct an expression vector which can express hvbadhl in plants, an expression cassette, including a CaMV35S promoter, hvbadhl and a NOS polyA sequence, was introduced into the T-DNA region of the binary vector pCAMBIA1301. The plant selectable marker gene of the vector is hptII delivering hygromycin B resistance to the transgenic plants. This recombined binary vector named as pCAM-ba was transfermed into Agrobacterium tumefaciens LBA4404 by freeze-thaw method, which could be used for plant transformation directly.
2 regenerated T_0 transgenic plants of Nicotiana tabacum Qinyan95 with hygromycin B resistance were obtained via Agrobacterium tumefaciens-mediated transformation and characterized by PCR, and RT-PCR. The PCR and RT-PCR analysis of the T_0 generation transgenic plants show that the hvbadh1 gene has already integrated into the genome of the transgenic plants and the target gene can express at the level of RNA transcription.
引文
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