紫花苜蓿MsDUF基因的克隆及功能研究
摘要
干旱和盐碱化是目前人类面临的世界性问题,也是抑制作物和牧草产量的主要环境因子,特别是在我国西北干旱半干旱地区显得尤为严重。紫花苜蓿(Medicago sativa L.)素有“牧草之王”的美称,是世界上利用最早、栽培最广的一种优良的豆科牧草,在我国西北干旱半干旱地区的畜牧业发展和生态环境建设中发挥着极其重要的作用。紫花苜蓿属于中等耐盐植物,但并不适合在盐碱地上长期种植。因此,培育具有较强耐盐性又有较强抗旱性的紫花苜蓿品种是当前我国西北干旱半干旱地区紫花苜蓿人工草地实现高产的根本出路之一。随着分子生物学技术的发展,利用基因工程技术手段来提高苜蓿的抗寒、抗旱、耐盐碱能力具有重要意义。
本研究利用RACE法克隆了紫花苜蓿抗旱相关基因MsDUF,构建了植物过表达载体pCAMBIA1301-MsDUF,利用农杆菌介导法将其转入到烟草和紫花苜蓿中,得到了转基因烟草和紫花苜蓿植株。对转基因植株进行干旱(20%PEG)和NaCl (200mmol/L)胁迫处理,测定相关生理指标,验证MsDUF基因的耐逆性,以期获得耐逆性更强的烟草和苜蓿材料。
主要研究结果如下：
1.利用RACE法,从紫花苜蓿中扩增MsDUF基因的全长cDNA。测序和序列分析结果表明,该基因全长714bp,开放阅读框全长633bp,编码210个氨基酸,GeneBank登录号为JX183734。
2.实时定量PCR分析MsDUF基因在转录水平上的表达。结果表明,MsDUF基因在根中表达量最高,同时该基因的表达还受干旱和盐胁迫的诱导。
3.构建了PBI221-GFP瞬时表达载体,进行亚细胞定位研究。将质粒用基因枪法转入洋葱表皮细胞,激光共聚焦显微镜下观察发现：MsDUF基因产物定位于细胞质中。
4.构建了植物过表达载体pCAMBIA1301-MsDUF,利用农杆菌介导法将其转入到烟草和紫花苜蓿中,经抗生素筛选,分别均获得10株转基因烟草和紫花苜蓿,经过PCR,RT-PCR检测,分别得到8株和7株阳性烟草苗和苜蓿苗,初步证明目的基因已经整合进烟草和苜蓿基因组中。
5.对转MsDUF基因的烟草和紫花苜蓿进行干旱(20%PEG)和NaCl(200mmol/L)胁迫处理后,测量相对含水量、丙二醛(MDA)、可溶性糖和脯氨酸含量,以及SOD活性。结果发现,转基因植株的丙二醛含量均比对照植株低；而相对含水量、可溶性糖和脯氨酸含量与对照相比均有明显升高。表明紫花苜蓿MsDUF基因的导入提高了烟草和紫花苜蓿的抗逆性。
Salinity and drought are two major abiotic factors that limit crop production and forage yield, especially in the arid and semi-arid areas of northwest China. Alfalfa (Medicago sativa L.) is one of the most important legume forages and plays a very important role in the agriculture and animal husbandry, and the ecological construction in the arid and semi-arid areas of northwest China. Alfalfa is a medium salt-tolerant plant, but not for long-term cultivation in saline soil. Breeding enhanced salt and drought resistant alfalfa cultivars is one of necessary ways to increasing forage yield of alfalfa pastures, in the arid and semi-arid areas of northwest China. With the development of biotechnology, genetic engineering technique is playing a vital role in studies on enhancing drought, salt and cold tolerance of plants.
In this study, we cloned MsDUF by rapid-amplification of cDNA ends(RACE), and constructed overexpression vector pCAMBIA1301-MsDUF. Then the vector was transformed into tobacco and alfalfa by Agrobacterium mediated transformation. To study the salt tolerance of transgenic tobaccos and alfalfa, NaCl (240mM) and20%PEG stress treatment was performed. In order to obtain more stress-tolerant tobacco and alfalfa materials, relative physiological indicators were determined to verify the effectiveness of MsDUF gene in improving plant tolerance to stress.
The main results were as following:
1.The complete coding cDNA of MsDUF was amplified from Medicago sativa pods mRNA by rapid-amplification of cDNA ends(RACE). Sequencing analysis reveals full length of open reading frame of MsDUF was633bp and encoded a protein of210amino acids residues, and the Genbank accession number is JX183734.
2.Real-time PCR was performed to reveal that transcript level of MsDUF in different tissues and under different stresses. The results indicated that MsDUF transcription was abundant in roots, and the expression of MsDUF was induced by NaCl and PEG treatments.
3.We constructed the fusion protein expression vector PBI221-GFP, and transformed it into onion epidermal cells using a gene gun. Subcellular localization of transiently expressed MsDUF-GFP fusion protein was detected by a confocal laser scanning microscope. The result revealed that MsDUF was localized in cytoplasm.
4.The coding region of MsDUF was inserted into the expression vector pCAMBIA1301to constructed the recombined overexpression vector pCAMBIA1301-MsDUF. Then the vector was transformed into tobacco and alfalfa by Agrobacterium mediated transformation. Ten kanamycin-resistant tobaccos and alfalfa were obtained. PCR and RT-PCR analysis showed that8and7plants were detected target fragment in tobaccos and alfalfa.
5.To study the salt tolerance of transgenic tobaccos and alfalfa, NaCl (240mM) and20%PEG stress treatment was performed. The relative water content (RWC), malondialdehyde (MDA) content, soluble sugar and proline content were measured. Results showed that the MDA content was significantly lower in the transgenic tobaccos that in wild ones. However, the RWC, MDA and proline content accumulated much more in transgenic tobaccos than in wild plants. It demonstrates a role of the MsDUF protein in stress protection and suggests the potential of the MsDUF gene for improving the salt stress tolerance of tobacco and alfalfa.
本研究利用RACE法克隆了紫花苜蓿抗旱相关基因MsDUF,构建了植物过表达载体pCAMBIA1301-MsDUF,利用农杆菌介导法将其转入到烟草和紫花苜蓿中,得到了转基因烟草和紫花苜蓿植株。对转基因植株进行干旱(20%PEG)和NaCl (200mmol/L)胁迫处理,测定相关生理指标,验证MsDUF基因的耐逆性,以期获得耐逆性更强的烟草和苜蓿材料。
主要研究结果如下：
1.利用RACE法,从紫花苜蓿中扩增MsDUF基因的全长cDNA。测序和序列分析结果表明,该基因全长714bp,开放阅读框全长633bp,编码210个氨基酸,GeneBank登录号为JX183734。
2.实时定量PCR分析MsDUF基因在转录水平上的表达。结果表明,MsDUF基因在根中表达量最高,同时该基因的表达还受干旱和盐胁迫的诱导。
3.构建了PBI221-GFP瞬时表达载体,进行亚细胞定位研究。将质粒用基因枪法转入洋葱表皮细胞,激光共聚焦显微镜下观察发现：MsDUF基因产物定位于细胞质中。
4.构建了植物过表达载体pCAMBIA1301-MsDUF,利用农杆菌介导法将其转入到烟草和紫花苜蓿中,经抗生素筛选,分别均获得10株转基因烟草和紫花苜蓿,经过PCR,RT-PCR检测,分别得到8株和7株阳性烟草苗和苜蓿苗,初步证明目的基因已经整合进烟草和苜蓿基因组中。
5.对转MsDUF基因的烟草和紫花苜蓿进行干旱(20%PEG)和NaCl(200mmol/L)胁迫处理后,测量相对含水量、丙二醛(MDA)、可溶性糖和脯氨酸含量,以及SOD活性。结果发现,转基因植株的丙二醛含量均比对照植株低；而相对含水量、可溶性糖和脯氨酸含量与对照相比均有明显升高。表明紫花苜蓿MsDUF基因的导入提高了烟草和紫花苜蓿的抗逆性。
Salinity and drought are two major abiotic factors that limit crop production and forage yield, especially in the arid and semi-arid areas of northwest China. Alfalfa (Medicago sativa L.) is one of the most important legume forages and plays a very important role in the agriculture and animal husbandry, and the ecological construction in the arid and semi-arid areas of northwest China. Alfalfa is a medium salt-tolerant plant, but not for long-term cultivation in saline soil. Breeding enhanced salt and drought resistant alfalfa cultivars is one of necessary ways to increasing forage yield of alfalfa pastures, in the arid and semi-arid areas of northwest China. With the development of biotechnology, genetic engineering technique is playing a vital role in studies on enhancing drought, salt and cold tolerance of plants.
In this study, we cloned MsDUF by rapid-amplification of cDNA ends(RACE), and constructed overexpression vector pCAMBIA1301-MsDUF. Then the vector was transformed into tobacco and alfalfa by Agrobacterium mediated transformation. To study the salt tolerance of transgenic tobaccos and alfalfa, NaCl (240mM) and20%PEG stress treatment was performed. In order to obtain more stress-tolerant tobacco and alfalfa materials, relative physiological indicators were determined to verify the effectiveness of MsDUF gene in improving plant tolerance to stress.
The main results were as following:
1.The complete coding cDNA of MsDUF was amplified from Medicago sativa pods mRNA by rapid-amplification of cDNA ends(RACE). Sequencing analysis reveals full length of open reading frame of MsDUF was633bp and encoded a protein of210amino acids residues, and the Genbank accession number is JX183734.
2.Real-time PCR was performed to reveal that transcript level of MsDUF in different tissues and under different stresses. The results indicated that MsDUF transcription was abundant in roots, and the expression of MsDUF was induced by NaCl and PEG treatments.
3.We constructed the fusion protein expression vector PBI221-GFP, and transformed it into onion epidermal cells using a gene gun. Subcellular localization of transiently expressed MsDUF-GFP fusion protein was detected by a confocal laser scanning microscope. The result revealed that MsDUF was localized in cytoplasm.
4.The coding region of MsDUF was inserted into the expression vector pCAMBIA1301to constructed the recombined overexpression vector pCAMBIA1301-MsDUF. Then the vector was transformed into tobacco and alfalfa by Agrobacterium mediated transformation. Ten kanamycin-resistant tobaccos and alfalfa were obtained. PCR and RT-PCR analysis showed that8and7plants were detected target fragment in tobaccos and alfalfa.
5.To study the salt tolerance of transgenic tobaccos and alfalfa, NaCl (240mM) and20%PEG stress treatment was performed. The relative water content (RWC), malondialdehyde (MDA) content, soluble sugar and proline content were measured. Results showed that the MDA content was significantly lower in the transgenic tobaccos that in wild ones. However, the RWC, MDA and proline content accumulated much more in transgenic tobaccos than in wild plants. It demonstrates a role of the MsDUF protein in stress protection and suggests the potential of the MsDUF gene for improving the salt stress tolerance of tobacco and alfalfa.
引文
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