蒙古冰草抗旱相关基因克隆、表达及RNAi载体构建
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摘要
蒙古冰草(Agropyron mongolicum Keng)是我国荒漠草原和典型草原优良的禾本科牧草,具有抗旱、抗寒、耐盐碱、耐瘠薄、抗病虫等优异特性,是牧草和农作物抗性改良的重要基因资源。同时,蒙古冰草是冰草属中珍贵的二倍体物种,不仅具有较高的饲用价值,还具有重要的生态价值和遗传育种价值。我国拥有丰富的蒙古冰草种质资源,开展蒙古冰草抗逆性基因资源的挖掘和利用,对于我国蒙古冰草种质资源的保护和种质创新具有重要意义。本研究以蒙古冰草为材料,开展了以抗旱基因为主的受逆境胁迫表达基因的克隆及其抗逆分子机制研究,主要结果如下:
1.采用PCR和RACE技术,从蒙古冰草中克隆了1个第四组LEA基因,命名为MwLEA。该基因cDNA全长961bp,编码171个氨基酸。具有LEA-4超基因家族N端特征结构域,C端序列不具备保守性,二级结构中α-螺旋占主导,该基因前140个氨基酸的亲水性较强,尾部序列亲水性减弱,受干旱胁迫诱导表达。
2.采用RT-PCR技术,从蒙古冰草中克隆了1个第三组LEA基因家族成员,命名为MwLEA3,基因全长1004bp,包含3个内含子区和3个外显子区,编码189个氨基酸。具有8个重复的11aa-基元序列,氨基酸组成中丙氨酸、赖氨酸和苏氨酸三种氨基酸约占总量的50%,不含半胱氨酸和色氨酸。二级结构中α-螺旋占主导,亲水性较强,经Southern杂交分析,认为该基因在基因组中以基因家族形式存在。
3.采用RT-PCR方法对MwLEA3基因的表达模式分析显示,该基因在根茎中的表达量高于叶片中的表达量;受干旱、高盐、外源激素ABA的诱导,在植物体内高水平表达。不同诱导条件下的基因表达模式存在差异。为深入研究MwLEA3基因的功能,以MwLEA3基因为靶标构建了RNAi表达载体pRNAiMwLEA3。
4.采用RT-PCR方法从蒙古冰草中克隆到肌动蛋白(Actin)基因,命名为MwACT。在诱导表达分析中,MwACT不受外界因素诱导表达量恒定,是组成型表达的管家基因。为研究其他基因在蒙古冰草中表达提供内标参照。
5.从蒙古冰草中克隆到谷胱甘肽-S-转移酶(GSTs)类基因,命名为MwGSTs,该基因具有GSTs超基因家族保守结构域(C-terminal alpha helical domain)。二级结构和亲疏水性分析表明,MwGSTs基因具有结合疏水底物的H位点和较强的疏水特性。
6.本研究克隆的4个基因均与小麦同类基因高度同源,从遗传进化方面阐明了蒙古冰草进化程度与小麦接近。从理论上支持了蒙古冰草是小麦野生近缘种这一观点,从而确定蒙古冰草为小麦抗性改良基因资源的理想物种。
Mongolian wheatgrass is an important genetic resource for improving forge grass and crops resistances which is excellent forage grass with many superior characteristics, such as Drought Resistance, Cold Resistance, Disease and Insect Resistance, Tolerance to Saline and Barren in Desert Grassland and Typical Grassland of China. Meanwhile, it is a precious diploid species with genetic and feeding value, ecological value and breeding value in Agropyron Gaertn. There are abundant germplasm resources of Mongolian wheatgrass in China. Discovering and cloning genes related to stress-tolerance are the foundation for improving the stress tolerance in crops with genetic modification, which also plays a crucial role in protecting, innovating germplasm resources and understanding the genetic mechanism of drought tolerance in Mongolian wheatgrass. In this research, Mongolian wheatgrass were used as experimental materials to clone genes related to drought resistance and study mechanism of stress tolerance. The main results are as follows:
1. A group 4 of LEA gene, named as MwLEA, was cloned from Mongolian wheatgrass by PCR and 3’and 5’RACE amplification and sequencing, the full length gene of LEA-4 superfamily N-terminal conserved domains was 961 bp, which encodes 171 amino acids. C-terminal of this gene is not conservative.α-helix structure plays a dominant role in the secondary structure ,its expression induced by drought stresses,and it has stronger hydrophilicity of pre-140 amino acids and tail-end decreased.
2. A group 3 of LEA gene, named as MwLEA3, was cloned from Mongolia wheatgrass by RT-PCR amplification and sequencing, the full length gene of 8 copies 11-amino acid sequence motif was 1004 bp, including of 3 introns and 3 exons regions ,which encodes 189 amino acids. Ala, Lys and Thr are about 50% of amino acids composition, and no Cys and Trp.α-helix structure plays a dominant role in the secondary structure and has stronger hydrophilicity. This gene is in the form of gene-family in genome by Southern analysis.
3. Analysis on expression pattern of MwLEA3 was by RT-PCR. The results revealed that its mRNA level was positively related to the drought tolerance. The gene was also induced by high salt and exogenous ABA treatments. Substantially, the expression of MwLEA3 was higher in roots and stem than that in leaves and their expression patterns were different under different stresses. RNAi expression vector harboring the partial MwLEA3 gene were constructed to study on function of the MwLEA3 gene.
4. A partial Actin gene of Mongolian wheatgrass, named as MwACT, was cloned by RT-PCR amplification. MwACT of constitutive expression is housekeeping gene without change with outside factors, which can be used as internal standard when study other genes’expression and regulation in Mongo1ian wheatgrass.
5. A partial GSTs gene of Mongolian wheatgrass, named as MwGSTs, was cloned by RT-PCR amplification. The partial gene of GSTs superfamily C-terminal conserved domains. Analysis on the secondary structure and hydrophilicity revealed that it had substrate binding pocket (H- site ) and stronger hydrophobicity.
6. 4 genes of Mongo1ian wheatgrass isolated in the research have high homology with wheat, and further illustrate evolution extent of Mongo1ia wheatgrass close to wheat. In theory, support to the View of Mongo1ian wheatgrass being wild relatives of wheat, and it is the optimal species for improving stress-tolerance in wheat was further confirmed.
1.采用PCR和RACE技术,从蒙古冰草中克隆了1个第四组LEA基因,命名为MwLEA。该基因cDNA全长961bp,编码171个氨基酸。具有LEA-4超基因家族N端特征结构域,C端序列不具备保守性,二级结构中α-螺旋占主导,该基因前140个氨基酸的亲水性较强,尾部序列亲水性减弱,受干旱胁迫诱导表达。
2.采用RT-PCR技术,从蒙古冰草中克隆了1个第三组LEA基因家族成员,命名为MwLEA3,基因全长1004bp,包含3个内含子区和3个外显子区,编码189个氨基酸。具有8个重复的11aa-基元序列,氨基酸组成中丙氨酸、赖氨酸和苏氨酸三种氨基酸约占总量的50%,不含半胱氨酸和色氨酸。二级结构中α-螺旋占主导,亲水性较强,经Southern杂交分析,认为该基因在基因组中以基因家族形式存在。
3.采用RT-PCR方法对MwLEA3基因的表达模式分析显示,该基因在根茎中的表达量高于叶片中的表达量;受干旱、高盐、外源激素ABA的诱导,在植物体内高水平表达。不同诱导条件下的基因表达模式存在差异。为深入研究MwLEA3基因的功能,以MwLEA3基因为靶标构建了RNAi表达载体pRNAiMwLEA3。
4.采用RT-PCR方法从蒙古冰草中克隆到肌动蛋白(Actin)基因,命名为MwACT。在诱导表达分析中,MwACT不受外界因素诱导表达量恒定,是组成型表达的管家基因。为研究其他基因在蒙古冰草中表达提供内标参照。
5.从蒙古冰草中克隆到谷胱甘肽-S-转移酶(GSTs)类基因,命名为MwGSTs,该基因具有GSTs超基因家族保守结构域(C-terminal alpha helical domain)。二级结构和亲疏水性分析表明,MwGSTs基因具有结合疏水底物的H位点和较强的疏水特性。
6.本研究克隆的4个基因均与小麦同类基因高度同源,从遗传进化方面阐明了蒙古冰草进化程度与小麦接近。从理论上支持了蒙古冰草是小麦野生近缘种这一观点,从而确定蒙古冰草为小麦抗性改良基因资源的理想物种。
Mongolian wheatgrass is an important genetic resource for improving forge grass and crops resistances which is excellent forage grass with many superior characteristics, such as Drought Resistance, Cold Resistance, Disease and Insect Resistance, Tolerance to Saline and Barren in Desert Grassland and Typical Grassland of China. Meanwhile, it is a precious diploid species with genetic and feeding value, ecological value and breeding value in Agropyron Gaertn. There are abundant germplasm resources of Mongolian wheatgrass in China. Discovering and cloning genes related to stress-tolerance are the foundation for improving the stress tolerance in crops with genetic modification, which also plays a crucial role in protecting, innovating germplasm resources and understanding the genetic mechanism of drought tolerance in Mongolian wheatgrass. In this research, Mongolian wheatgrass were used as experimental materials to clone genes related to drought resistance and study mechanism of stress tolerance. The main results are as follows:
1. A group 4 of LEA gene, named as MwLEA, was cloned from Mongolian wheatgrass by PCR and 3’and 5’RACE amplification and sequencing, the full length gene of LEA-4 superfamily N-terminal conserved domains was 961 bp, which encodes 171 amino acids. C-terminal of this gene is not conservative.α-helix structure plays a dominant role in the secondary structure ,its expression induced by drought stresses,and it has stronger hydrophilicity of pre-140 amino acids and tail-end decreased.
2. A group 3 of LEA gene, named as MwLEA3, was cloned from Mongolia wheatgrass by RT-PCR amplification and sequencing, the full length gene of 8 copies 11-amino acid sequence motif was 1004 bp, including of 3 introns and 3 exons regions ,which encodes 189 amino acids. Ala, Lys and Thr are about 50% of amino acids composition, and no Cys and Trp.α-helix structure plays a dominant role in the secondary structure and has stronger hydrophilicity. This gene is in the form of gene-family in genome by Southern analysis.
3. Analysis on expression pattern of MwLEA3 was by RT-PCR. The results revealed that its mRNA level was positively related to the drought tolerance. The gene was also induced by high salt and exogenous ABA treatments. Substantially, the expression of MwLEA3 was higher in roots and stem than that in leaves and their expression patterns were different under different stresses. RNAi expression vector harboring the partial MwLEA3 gene were constructed to study on function of the MwLEA3 gene.
4. A partial Actin gene of Mongolian wheatgrass, named as MwACT, was cloned by RT-PCR amplification. MwACT of constitutive expression is housekeeping gene without change with outside factors, which can be used as internal standard when study other genes’expression and regulation in Mongo1ian wheatgrass.
5. A partial GSTs gene of Mongolian wheatgrass, named as MwGSTs, was cloned by RT-PCR amplification. The partial gene of GSTs superfamily C-terminal conserved domains. Analysis on the secondary structure and hydrophilicity revealed that it had substrate binding pocket (H- site ) and stronger hydrophobicity.
6. 4 genes of Mongo1ian wheatgrass isolated in the research have high homology with wheat, and further illustrate evolution extent of Mongo1ia wheatgrass close to wheat. In theory, support to the View of Mongo1ian wheatgrass being wild relatives of wheat, and it is the optimal species for improving stress-tolerance in wheat was further confirmed.
引文
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