中国野生华东葡萄抗白粉病基因（VpUSP）克隆及表达分析
摘要
应激蛋白(Universal stress protein, USP)是一种普遍存在于植物中的抗性相关蛋白,一般存在于细胞质中,在植物受到外界胁迫时表达上调,增强植物的抗逆性。
葡萄白粉病[Uncinula necator (Schw.) Burr.]是严重危害世界葡萄生产的一种真菌性病害。在葡萄著名产区如美国加州,法国,意大利,澳大利亚,中国新疆等地区,葡萄白粉病为害比较严重,给葡萄生产造成巨大的经济损失。利用基因工程手段与传统育种技术相结合,发掘抗病葡萄种质的抗病基因,培育抗病新品种是解决此问题的有效途径之一。因此,开展中国野生华东葡萄的usp基因的分子生物学研究具有十分重要的意义。
根据从本课题组前期构建的中国野生葡萄华东葡萄株系白河-35-1在白粉病诱导下的cDNA文库中获得的1条与广泛逆境蛋白相关的EST序列,设计特异引物,结合RACE技术克隆了中国野生华东葡萄白河-35-1应激蛋白基因(Universal stress protein, USP)全长序列进行序列分析,通过半定量RT-PCR技术,研究了VpUSP在中国野生华东葡萄抗病、感病株系中的表达变化。主要研究结果如下:
(1)结合RACE技术扩增出中国野生华东葡萄白河-35-1usp相关基因(VpUSP),生物信息分析表明该基因全长836 bp,开放阅读框为528bp,编码175个氨基酸,GenBank登录号为GU946975。以中国野生华东葡萄白河-35-1 VpUSP基因推导的氨基酸序列为检索序列,通过NCBI的BLASTx程序检测与其同源的氨基酸序列,结果发现:拟南芥、蚕豆、水稻中有相似序列,同源性在59%-62%之间,表明所获得序列为usp基因家族的成员。
(2)该基因在白粉菌诱导下的表达模式表明:应激蛋白在中国野生华东葡萄感病株系中,接种白粉病前后均有表达,但表达量变化不明显;然而,抗病的中国野生华东葡萄株系在接种白粉病后VpUSP基因表达量呈明显的增加趋势,并且在48h时达到表达高峰随后降低,初步表明中国野生华东葡萄VpUSP基因在与白粉病菌互作过程中具有表达活性。
(3)以白河-35-1试管苗为材料,研究不同激素浓度组合对白河-35-1叶片和叶柄、茎段不定芽再生的影响。结果表明,诱导白河-35-1叶片和叶柄、茎段的最佳培养基MS+IBA0.2mg/L+TDZ4mg/L+蔗糖15g/L+琼脂0.6%,再生率分别为9%、10%、8%。
(4)以白河-35-1试管苗为材料,研究不同激素浓度组合对白河-35-1的继代的影响,结果表明,最佳继代培养基为1/2MS+IBA1.0mg/L+NAA0.1mg/L+蔗糖28g/L+琼脂0.6%。
Universal stress protein(USP)is a small cytoplasmic bacterial protein that is upregulated when the cell is exposed to stress agents.USP enhances when the rate of cell survival during prolonged exposure to such conditions, and it may provide a general“stress endurance”activity.
Grape powdery mildew is a fungal disease which seriously harms the world’s grape production. In the famous grape growing areas, such as California, France, Italy, Australia and Xinjiang in China, the grape powdery mildew has down great harm and caused huge economic losses for grape production. Combination of genetic engineering and traditional breeding technology, identification of resistant gene in resistant grape germplasm and cultivating of new resistant variety are effective ways to solve the problem.
Here,we described the isolation and nucleotide sequence analysis of the EST from the cDNA library constructed of powdery mildew-infected leaves of‘Baihe-35-1’and compared its amino acids USP from other species. The major results of this dissertation are as follows:
1.A USP(Universal stress protein) related EST sequence from the cDNA library constructed of powdery mildew-infected leaves of‘Baihe-35-1’is screened. Full length of this gene is obtained by RACE and was designated as VpUSP(GenBank accession number GU946975) . It is 836bp ,which encondes 175 amino acids.We blast the sequence of Arabidopsis thaliana, Brassica rapa from GenBank,result shwoede that they shared 62%- 59%.
2.Semi-quantitative RT-PCR was employed to study the expression of the gene. The results showed constitutive expression of the gene was observed in susceptible lines. However, the gene could be induced by grapevine powdery mildew in resistant lines, and reached the climax after 48h, It suggests that VpUSP has the expression activity during the powdery mildew infecting leaves.
3. Different combination of plant growth regulators were studied to the“BaiHe-35-1”plantlets subculture growth condition.Result shows that the most suitable medium for the“BaiHe-35-1”plantlets subculture was1/2 MS+IBA1.0 mg/L+NAA0.1 mg/L+source28 g/L+agar0.6%.
4. Leaf、petiole and stem of“BaiHe-35-1”were used in this study. Different combination of plant growth regulators were studied to induced the adventitious buds. Result shows that the best medium in inducing the adventitionous buds was MS+IBA0.2 mg/L+TDZ4 mg/L+source 15g/L+agar0.6%.And the regeneration efficiency was 9%、10%、8%。.
葡萄白粉病[Uncinula necator (Schw.) Burr.]是严重危害世界葡萄生产的一种真菌性病害。在葡萄著名产区如美国加州,法国,意大利,澳大利亚,中国新疆等地区,葡萄白粉病为害比较严重,给葡萄生产造成巨大的经济损失。利用基因工程手段与传统育种技术相结合,发掘抗病葡萄种质的抗病基因,培育抗病新品种是解决此问题的有效途径之一。因此,开展中国野生华东葡萄的usp基因的分子生物学研究具有十分重要的意义。
根据从本课题组前期构建的中国野生葡萄华东葡萄株系白河-35-1在白粉病诱导下的cDNA文库中获得的1条与广泛逆境蛋白相关的EST序列,设计特异引物,结合RACE技术克隆了中国野生华东葡萄白河-35-1应激蛋白基因(Universal stress protein, USP)全长序列进行序列分析,通过半定量RT-PCR技术,研究了VpUSP在中国野生华东葡萄抗病、感病株系中的表达变化。主要研究结果如下:
(1)结合RACE技术扩增出中国野生华东葡萄白河-35-1usp相关基因(VpUSP),生物信息分析表明该基因全长836 bp,开放阅读框为528bp,编码175个氨基酸,GenBank登录号为GU946975。以中国野生华东葡萄白河-35-1 VpUSP基因推导的氨基酸序列为检索序列,通过NCBI的BLASTx程序检测与其同源的氨基酸序列,结果发现:拟南芥、蚕豆、水稻中有相似序列,同源性在59%-62%之间,表明所获得序列为usp基因家族的成员。
(2)该基因在白粉菌诱导下的表达模式表明:应激蛋白在中国野生华东葡萄感病株系中,接种白粉病前后均有表达,但表达量变化不明显;然而,抗病的中国野生华东葡萄株系在接种白粉病后VpUSP基因表达量呈明显的增加趋势,并且在48h时达到表达高峰随后降低,初步表明中国野生华东葡萄VpUSP基因在与白粉病菌互作过程中具有表达活性。
(3)以白河-35-1试管苗为材料,研究不同激素浓度组合对白河-35-1叶片和叶柄、茎段不定芽再生的影响。结果表明,诱导白河-35-1叶片和叶柄、茎段的最佳培养基MS+IBA0.2mg/L+TDZ4mg/L+蔗糖15g/L+琼脂0.6%,再生率分别为9%、10%、8%。
(4)以白河-35-1试管苗为材料,研究不同激素浓度组合对白河-35-1的继代的影响,结果表明,最佳继代培养基为1/2MS+IBA1.0mg/L+NAA0.1mg/L+蔗糖28g/L+琼脂0.6%。
Universal stress protein(USP)is a small cytoplasmic bacterial protein that is upregulated when the cell is exposed to stress agents.USP enhances when the rate of cell survival during prolonged exposure to such conditions, and it may provide a general“stress endurance”activity.
Grape powdery mildew is a fungal disease which seriously harms the world’s grape production. In the famous grape growing areas, such as California, France, Italy, Australia and Xinjiang in China, the grape powdery mildew has down great harm and caused huge economic losses for grape production. Combination of genetic engineering and traditional breeding technology, identification of resistant gene in resistant grape germplasm and cultivating of new resistant variety are effective ways to solve the problem.
Here,we described the isolation and nucleotide sequence analysis of the EST from the cDNA library constructed of powdery mildew-infected leaves of‘Baihe-35-1’and compared its amino acids USP from other species. The major results of this dissertation are as follows:
1.A USP(Universal stress protein) related EST sequence from the cDNA library constructed of powdery mildew-infected leaves of‘Baihe-35-1’is screened. Full length of this gene is obtained by RACE and was designated as VpUSP(GenBank accession number GU946975) . It is 836bp ,which encondes 175 amino acids.We blast the sequence of Arabidopsis thaliana, Brassica rapa from GenBank,result shwoede that they shared 62%- 59%.
2.Semi-quantitative RT-PCR was employed to study the expression of the gene. The results showed constitutive expression of the gene was observed in susceptible lines. However, the gene could be induced by grapevine powdery mildew in resistant lines, and reached the climax after 48h, It suggests that VpUSP has the expression activity during the powdery mildew infecting leaves.
3. Different combination of plant growth regulators were studied to the“BaiHe-35-1”plantlets subculture growth condition.Result shows that the most suitable medium for the“BaiHe-35-1”plantlets subculture was1/2 MS+IBA1.0 mg/L+NAA0.1 mg/L+source28 g/L+agar0.6%.
4. Leaf、petiole and stem of“BaiHe-35-1”were used in this study. Different combination of plant growth regulators were studied to induced the adventitious buds. Result shows that the best medium in inducing the adventitionous buds was MS+IBA0.2 mg/L+TDZ4 mg/L+source 15g/L+agar0.6%.And the regeneration efficiency was 9%、10%、8%。.
引文
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