黑穗病菌胁迫下甘蔗SSH文库构建及差异表达基因的克隆与分析
发布时间：2018-09-04 11:15
【摘要】：甘蔗黑穗病是由黑粉菌(Sporisorium scitamineum)引起的一种气传真菌性病害,已经成为世界各植蔗区的主要病害之一。甘蔗黑穗病能引起蔗茎产量降低和蔗糖分的减少,给甘蔗产业造成较为严重的损失。杂交育种是甘蔗抗黑穗病育种最主要也是最重要的途径,但是,由于甘蔗抗黑穗病基因大多来源于野生种,而野生种中高产高糖基因与一些不利基因存在紧密连锁的关系,使得甘蔗传统杂交育种在导入抗病基因的同时,也导入了一些不良的非目标性状基因。因此,如果能在解析甘蔗与黑穗病菌互作的分子机理的基础上,挖掘抗病关键基因,进而通过转基因手段将此类基因转导入目标甘蔗品种中,达到定向改良品种抗性的目的,既可以避免不良性状基因的导入,又能保持目标甘蔗品种原有的优良性状和品质特性,同时兼具抗黑穗病的特性,是解决甘蔗抗黑穗病育种瓶颈的一种有效途径。本研究以甘蔗品种新台糖22号(ROC22)为实验材料,首先,使用抑制消减杂交(suppression subtractive hybridization, SSH)技术构建cDNA文库；其次,利用反向Northern杂交技术,筛选差异表达基因；再次,通过电子克隆和RT-PCR相结合的技术,克隆目的基因；最后,在对所克隆基因进行生物信息学分析的基础上,利用实时荧光定量PCR技术,检测这些基因的组织特异性表达情况以及在黑穗病菌、SA (salicylic acid)、MeJA (methyl-jasmonate)和ABA (abscisic acid)胁迫下的表达特性,以期揭示这些候选基因在甘蔗对黑穗病抗性中的作用机制,为后续利用甘蔗基因工程手段定向改良甘蔗品种对黑穗病的抗性,提供具有自主知识产权的基因资源,课题研究具有较为重要的理论和实践意义。主要实验结果如下： 1、以接种甘蔗黑穗病菌48h和接种无菌水48h时间点的蔗芽为材料,利用抑制消减杂交技术,构建黑穗病菌胁迫下的甘蔗正、反向SSH文库。随后从文库中随机挑取24个克隆进行PCR鉴定,结果显示插入片段长度主要分布于150～750bp之间,说明本研究构建的SSH文库质量良好。 2、从SSH文库中,筛选768个阳性克隆,进行反向Northern杂交,经过分析杂交信号强度,筛选获得190个差异表达基因。 3、对经反向Northern杂交验证到的190个阳性克隆进行测序及生物信息分析,去除重复、污染及低质量(100bp)序列后,获得了174个差异表达基因,这些基因主要存在于高分子配体(macromolecular complex)或细胞器(organelle)中,具有催化活性(catalytic activity)和结构分子活性(structural molecule activity),参与糖酵解(Glycolysis/Gluconeogenesis)、光合作物固碳(Carbon fixation in photosynthetic organisms)、半胱氨酸和蛋氨酸代谢(Cysteine and methionine metabolism)、二羧酸代谢(Glyoxylate and dicarboxylate metabolism)、氨基糖和核苷酸糖代谢(Amino sugar and nucleotide sugar metabolism)、氧化磷酸化(Oxidative phosphorylation)等代谢途径。 4、在差异表达基因测序及生物信息学分析的基础上,开展如下工作： 首先,从SSH文库中挑选6个差异表达EST序列,经电子克隆和RT-PCR扩增验证获得6个差异表达基因的全长cDNA序列,分别命名为细胞色素b5还原酶基因(Scb5R,GenBank登录号：KJ577591)、14-3-3蛋白基因(Sc14-3-3, GenBank登录号：KJ577592)、乙醇脱氢酶基因(ScADH, GenBank登录号：KJ577593)、泛素结合酶基因(ScUBc E2, GenBank登录号：KJ577594)、真核翻译起始因子5A基因(SceIF5A, GenBank登录号：KJ577595)、S-腺苷甲硫氨酸合成酶基因(ScSAM, GenBank登录号：KJ577596)； 其次,对这6个基因进行生物信息学分析：甘蔗Scb5R基因全长1257bp,ORF长840bp,编码氨基酸297个,编码蛋白是一种无信号肽,定位于内质网的稳定、亲水、碱性、非分泌蛋白,蛋白质二级结构主要为无规则卷曲,主要功能为转运结合；甘蔗Sc14-3-3基因全长1048bp, ORF长771bp,编码氨基酸256个,编码蛋白是一种无信号肽,定位于细胞质的稳定、亲水、酸性、非分泌蛋白,蛋白质二级结构主要为α-螺旋,主要功能为翻译和能量代谢；甘蔗ScADH基因全长1644bp, ORF长1140bp,编码氨基酸379个,编码蛋白是一种无信号肽,定位于叶绿体基质的稳定、亲水、酸性、非分泌蛋白,蛋白质二级结构主要为无规则卷曲,主要功能为能量代谢和翻译；甘蔗ScUBc E2基因全长997bp, ORF长447bp,编码氨基酸148个,编码一种无信号肽,定位于细胞质的不稳定、亲水、碱性、非分泌蛋白,蛋白质二级结构主要为无规则卷曲；甘蔗SceIF5A基因全长1174bp, ORF长483bp,编码氨基酸160个,编码蛋白是一种无信号肽,定位于细胞质的稳定、亲水、酸性、非分泌蛋白,蛋白质二级结构主要为无规则卷曲,主要功能为翻译和能量代谢；甘蔗ScSAM基因全长1788bp, ORF长1196bp,编码氨基酸396个,是一种无信号肽,定位于细胞质的稳定、亲水、酸性、非分泌蛋白,蛋白质二级结构主要为无规则卷曲,主要功能为能量代谢和翻译。 最后,通过实时荧光定量PCR技术,对这6个基因在叶片、侧芽、蔗髓和根等不同组织中的表达情况及其在黑穗病菌、SA、MeJA和ABA胁迫下的表达特征进行分析,结果显示：这6个基因的转录本,除甘蔗Sc14-3-3基因、ScADH基因外,其余均在侧芽中检测到最高的转录水平。在品种YC05-179中,甘蔗Scb5R基因在受黑穗病菌胁迫及SA、MeJA和ABA诱导后表达均呈“扬-抑-扬”的趋势；甘蔗Sc14-3-3基因的表达受到黑穗病菌、SA、MeJA和ABA强烈诱导表达；甘蔗ScADH基因受ABA诱导表达,同时受黑穗病菌先抑制后诱导表达,SA和MeJA先诱导后抑制表达；甘蔗ScUBc E2基因受SA和MeJA诱导表达。甘蔗SceIF5A基因受黑穗病菌、SA、MeJA和ABA诱导表达；甘蔗ScSAM基因在接种黑穗病菌后呈“扬-抑-扬”表达趋势,受SA诱导后,呈“扬-抑”趋势。
[Abstract]:Sugarcane smut is an airborne fungal disease caused by Sporisorium scitamineum. It has become one of the major diseases in sugarcane planting areas all over the world. Sugarcane smut can reduce the yield and sugar content of sugarcane and cause serious losses to sugarcane industry. It is also the most important way, however, because the resistance genes of sugarcane to Smut mostly come from wild species, and the high-yield and high-sugar genes in wild species are closely linked with some unfavorable genes, so the traditional sugarcane hybrid breeding has introduced some unfavorable non-target genes as well as disease-resistant genes. Based on the analysis of the molecular mechanism of interaction between sugarcane and smut fungus, the key genes for disease resistance were excavated, and then these genes were transfected into the target sugarcane varieties by transgenic means to achieve the goal of directional improvement of sugarcane resistance. In this study, sugarcane variety ROC22 was used as experimental material. First, suppression subtractive hybridization (SSH) was used to construct a cDNA library. Second, reverse Northern hybridization was used to construct the library. Finally, on the basis of bioinformatics analysis of the cloned genes, tissue-specific expression of these genes and SA (salicylic acid) were detected by real-time fluorescence quantitative PCR. MeJA (methyl-jasmonate) and ABA (abscisic acid) stress expression characteristics, in order to reveal the role of these candidate genes in the resistance of sugarcane to Smut mechanism, for the follow-up use of sugarcane genetic engineering means to improve the directional resistance of sugarcane varieties to smut, to provide gene resources with independent intellectual property rights, the research has more. This is an important theoretical and practical significance. The main results are as follows:
1. Sugarcane SSH libraries were constructed by suppression subtractive hybridization (SSH) using sugarcane buds inoculated with smut fungus for 48 h and sterile water for 48 h. 24 clones were randomly selected from the library for PCR identification. The results showed that the inserted fragments were mainly distributed between 150 and 750 bp, indicating that the SSH libraries of Sugarcane under smut stress were constructed. The constructed SSH library is of good quality.
2. 768 positive clones were screened from SSH library, and 190 differentially expressed genes were obtained by reverse Northern hybridization.
3. Sequencing and bioinformatics analysis of 190 positive clones verified by reverse Northern hybridization showed that 174 differentially expressed genes were obtained after removing repetitive, contaminated and low-quality (100 bp) sequences. These genes were mainly found in macromolecular complexes or organelles with catalytic activity. Activity and structural molecule activity are involved in Glycolysis / Gluconeogenesis, Carbon fixation in photosynthetic organisms, Cysteine and methionine metabolism, Glyoxylate and dicarboxylate metabolism, and aminoglycose metabolism. And nucleotide metabolism, oxidative phosphorylation and other metabolic pathways.
4, based on the sequencing of differentially expressed genes and bioinformatics analysis, the following work is carried out:
Firstly, six differentially expressed EST sequences were selected from SSH library, and the full-length cDNA sequences of six differentially expressed genes were obtained by electronic cloning and RT-PCR amplification. They were named as cytochrome b5 reductase gene (Scb5R, GenBank login number: KJ577591), 14-3-3 protein gene (Sc14-3-3, GenBank login number: KJ577592), ethanol dehydrogenase gene (S CADH, GenBank login number: KJ577593, ubiquitin-binding enzyme gene (ScUBc E2, GenBank login number: KJ577594), Eukaryotic translation initiation factor 5A gene (SceIF5A, GenBank login number: KJ577595), S-adenosylmethionine synthase gene (ScSAM, GenBank login number: KJ577596);
Secondly, bioinformatics analysis of the six genes was carried out: the sugarcane Scb5R gene was 1257 BP in length, the ORF was 840 BP in length and encoded 297 amino acids. The encoded protein was a non-signaling peptide, located in the stable, hydrophilic, alkaline and non-secretory endoplasmic reticulum. The secondary structure of the protein was mainly irregular curl, and the main function was transport binding. The total length of the gene is 1048 bp, the ORF is 771 bp, encoding 256 amino acids. The encoded protein is a non-signaling peptide, located in the cytoplasm of stable, hydrophilic, acidic, non-secretory protein. The secondary structure of the protein is mainly alpha-helix, and the main function is translation and energy metabolism. Protein is a non-signaling peptide, located in the stability of chloroplast matrix, hydrophilic, acidic, non-secretory protein, protein secondary structure is mainly irregular curl, the main function is energy metabolism and translation; sugarcane ScUBc E2 gene 997 bp, ORF 447 bp, coding amino acids 148, coding a non-signaling peptide, located in the cytoplasm of the instability SceIF5A gene of sugarcane is 1174 BP long, ORF is 483 BP long, and encodes 160 amino acids. The encoded protein is a non-signaling peptide, located in the cytoplasm of stable, hydrophilic, acidic, non-secreting proteins. The secondary structure of protein is mainly irregular curl, and the main function is as follows: Translations and energy metabolism; sugarcane ScSAM gene is 1788 BP long, ORF 1196 BP long, encoding 396 amino acids, is a signal-free peptide, located in the cytoplasm of stable, hydrophilic, acidic, non-secretory protein, protein secondary structure is mainly irregular curl, the main function of energy metabolism and translation.
Finally, the expression of these six genes in leaf, lateral bud, sugarcane pulp and root tissues and their expression characteristics under SA, MeJA and ABA stress were analyzed by real-time fluorescence quantitative PCR. The results showed that the transcripts of these six genes, except for Sc14-3-3 gene and ScADH gene, were detected in lateral buds of sugarcane. In YC05-179, the expression of Scb5R gene in sugarcane showed a trend of "rising-inhibiting-rising" under the stress of Smut Fungus and SA, MeJA and ABA, the expression of Sc14-3-3 gene in sugarcane was strongly induced by Smut Fungus, SA, MeJA and ABA, and the expression of ScADH gene in sugarcane was induced by ABA and inhibited by Smut Fungus at the same time. Sugarcane ScUBc E2 gene was induced by SA and MeJA, sugarcane SceIF5A gene was induced by Smut Fungus, SA, MeJA and ABA, and sugarcane ScSAM gene was expressed in a trend of "rising-inhibiting-raising" after inoculation with Smut fungus.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：S435.661;Q943.2
本文编号：2221920
[Abstract]:Sugarcane smut is an airborne fungal disease caused by Sporisorium scitamineum. It has become one of the major diseases in sugarcane planting areas all over the world. Sugarcane smut can reduce the yield and sugar content of sugarcane and cause serious losses to sugarcane industry. It is also the most important way, however, because the resistance genes of sugarcane to Smut mostly come from wild species, and the high-yield and high-sugar genes in wild species are closely linked with some unfavorable genes, so the traditional sugarcane hybrid breeding has introduced some unfavorable non-target genes as well as disease-resistant genes. Based on the analysis of the molecular mechanism of interaction between sugarcane and smut fungus, the key genes for disease resistance were excavated, and then these genes were transfected into the target sugarcane varieties by transgenic means to achieve the goal of directional improvement of sugarcane resistance. In this study, sugarcane variety ROC22 was used as experimental material. First, suppression subtractive hybridization (SSH) was used to construct a cDNA library. Second, reverse Northern hybridization was used to construct the library. Finally, on the basis of bioinformatics analysis of the cloned genes, tissue-specific expression of these genes and SA (salicylic acid) were detected by real-time fluorescence quantitative PCR. MeJA (methyl-jasmonate) and ABA (abscisic acid) stress expression characteristics, in order to reveal the role of these candidate genes in the resistance of sugarcane to Smut mechanism, for the follow-up use of sugarcane genetic engineering means to improve the directional resistance of sugarcane varieties to smut, to provide gene resources with independent intellectual property rights, the research has more. This is an important theoretical and practical significance. The main results are as follows:
1. Sugarcane SSH libraries were constructed by suppression subtractive hybridization (SSH) using sugarcane buds inoculated with smut fungus for 48 h and sterile water for 48 h. 24 clones were randomly selected from the library for PCR identification. The results showed that the inserted fragments were mainly distributed between 150 and 750 bp, indicating that the SSH libraries of Sugarcane under smut stress were constructed. The constructed SSH library is of good quality.
2. 768 positive clones were screened from SSH library, and 190 differentially expressed genes were obtained by reverse Northern hybridization.
3. Sequencing and bioinformatics analysis of 190 positive clones verified by reverse Northern hybridization showed that 174 differentially expressed genes were obtained after removing repetitive, contaminated and low-quality (100 bp) sequences. These genes were mainly found in macromolecular complexes or organelles with catalytic activity. Activity and structural molecule activity are involved in Glycolysis / Gluconeogenesis, Carbon fixation in photosynthetic organisms, Cysteine and methionine metabolism, Glyoxylate and dicarboxylate metabolism, and aminoglycose metabolism. And nucleotide metabolism, oxidative phosphorylation and other metabolic pathways.
4, based on the sequencing of differentially expressed genes and bioinformatics analysis, the following work is carried out:
Firstly, six differentially expressed EST sequences were selected from SSH library, and the full-length cDNA sequences of six differentially expressed genes were obtained by electronic cloning and RT-PCR amplification. They were named as cytochrome b5 reductase gene (Scb5R, GenBank login number: KJ577591), 14-3-3 protein gene (Sc14-3-3, GenBank login number: KJ577592), ethanol dehydrogenase gene (S CADH, GenBank login number: KJ577593, ubiquitin-binding enzyme gene (ScUBc E2, GenBank login number: KJ577594), Eukaryotic translation initiation factor 5A gene (SceIF5A, GenBank login number: KJ577595), S-adenosylmethionine synthase gene (ScSAM, GenBank login number: KJ577596);
Secondly, bioinformatics analysis of the six genes was carried out: the sugarcane Scb5R gene was 1257 BP in length, the ORF was 840 BP in length and encoded 297 amino acids. The encoded protein was a non-signaling peptide, located in the stable, hydrophilic, alkaline and non-secretory endoplasmic reticulum. The secondary structure of the protein was mainly irregular curl, and the main function was transport binding. The total length of the gene is 1048 bp, the ORF is 771 bp, encoding 256 amino acids. The encoded protein is a non-signaling peptide, located in the cytoplasm of stable, hydrophilic, acidic, non-secretory protein. The secondary structure of the protein is mainly alpha-helix, and the main function is translation and energy metabolism. Protein is a non-signaling peptide, located in the stability of chloroplast matrix, hydrophilic, acidic, non-secretory protein, protein secondary structure is mainly irregular curl, the main function is energy metabolism and translation; sugarcane ScUBc E2 gene 997 bp, ORF 447 bp, coding amino acids 148, coding a non-signaling peptide, located in the cytoplasm of the instability SceIF5A gene of sugarcane is 1174 BP long, ORF is 483 BP long, and encodes 160 amino acids. The encoded protein is a non-signaling peptide, located in the cytoplasm of stable, hydrophilic, acidic, non-secreting proteins. The secondary structure of protein is mainly irregular curl, and the main function is as follows: Translations and energy metabolism; sugarcane ScSAM gene is 1788 BP long, ORF 1196 BP long, encoding 396 amino acids, is a signal-free peptide, located in the cytoplasm of stable, hydrophilic, acidic, non-secretory protein, protein secondary structure is mainly irregular curl, the main function of energy metabolism and translation.
Finally, the expression of these six genes in leaf, lateral bud, sugarcane pulp and root tissues and their expression characteristics under SA, MeJA and ABA stress were analyzed by real-time fluorescence quantitative PCR. The results showed that the transcripts of these six genes, except for Sc14-3-3 gene and ScADH gene, were detected in lateral buds of sugarcane. In YC05-179, the expression of Scb5R gene in sugarcane showed a trend of "rising-inhibiting-rising" under the stress of Smut Fungus and SA, MeJA and ABA, the expression of Sc14-3-3 gene in sugarcane was strongly induced by Smut Fungus, SA, MeJA and ABA, and the expression of ScADH gene in sugarcane was induced by ABA and inhibited by Smut Fungus at the same time. Sugarcane ScUBc E2 gene was induced by SA and MeJA, sugarcane SceIF5A gene was induced by Smut Fungus, SA, MeJA and ABA, and sugarcane ScSAM gene was expressed in a trend of "rising-inhibiting-raising" after inoculation with Smut fungus.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：S435.661;Q943.2
【参考文献】
相关期刊论文 前10条
1 袁文龙;康占海;陶晡;赵春辉;霍建飞;耿硕;;基因芯片技术及其在植物中的应用[J];安徽农业科学;2010年26期
2 姜述君;马建;范文艳;戴凌燕;张国庆;于涵;刘朝;;纹枯病菌诱导水稻表达的WRKY基因克隆及分析[J];安徽农业科学;2011年17期
3 赵晋锋;余爱丽;朱晶莹;王高鸿;赵根友;;玉米S-腺苷甲硫氨酸合成酶基因(SAMS)逆境胁迫下的表达分析[J];河北农业大学学报;2010年05期
4 王新超;杨亚军;陈亮;马春雷;姚明哲;;茶树休眠芽与萌动芽抑制消减杂交文库的构建与初步分析[J];茶叶科学;2010年02期
5 陈旭;齐凤坤;康立功;李景富;;实时荧光定量PCR技术研究进展及其应用[J];东北农业大学学报;2010年08期
6 双宝;韩英鹏;李明;李文滨;;真核细胞翻译起始因子5A(eIF5A)研究进展[J];东北农业大学学报;2010年08期
7 徐晨曦;姜静;刘甜甜;王玉成;刘桂丰;杨传平;;柽柳泛素结合酶基因(E2s)的序列分析及功能验证[J];东北林业大学学报;2007年11期
8 李娟娟;陈福龙;李鑫;王蕾;贾俊忠;陈远良;陈芳;高剑峰;;黄瓜芽黄突变体抑制消减杂交文库的构建及初步分析[J];西北植物学报;2010年05期
9 谢伟伟;王凭青;杨青川;刘博;李志中;;植物差异表达基因克隆技术及研究进展[J];重庆大学学报(自然科学版);2005年12期
10 许莉萍,陈如凯;甘蔗黑穗病及其抗病育种的现状与展望[J];福建农业学报;2000年02期
相关博士学位论文 前1条
1 黄秀丽;玉米弯孢菌叶斑病抗性相关蛋白质鉴定及功能研究[D];上海交通大学;2009年
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