【作者】 周宏；

【导师】 赵卫国；

【作者基本信息】 江苏科技大学 ， 特种经济作物饲养， 2017， 博士

【摘要】 自然条件下,植物在整个生命周期中经常受到多种胁迫。干旱是主要制约植物生长发育,作物产量减少的非生物胁迫之一(仅次于病虫害)。植物在分子、细胞和生理水平等多方面应答反应和防御系统来适应胁迫环境,许多基因都参与植物对非生物胁迫的应答。因此,非生物胁迫响应的相关基因研究具有重要理论和现实意义。转录因子参与生物和非生物胁迫的应答反应,在调节植物适应环境变化中起重要作用。转录因子通过调节下游基因的表达,实现对植物的形态建成、生长发育及抵抗生物和非生物胁迫的调节作用。转录因子除了可应答外界刺激和环境胁迫,还可控制目的基因的时空特异性表达,通过基因产物的作用对内、外界信号作出应答,引起植物的生理、生化发生变化,提高抗逆性。我国是蚕桑生产的发源地,桑树种质资源极其丰富。桑树不仅具有药用价值的经济作物,还形成了较强的适应性,抗逆境的特性,可用于受损生态环境的治理。多种逆境条件常常对桑树的生长产生重大的影响。桑树一些重要调控基因及其胁迫下分子机制的研究,对提高桑树产量、抗逆性及保存桑树种质资源有重要作用。目前,植物抗逆相关的转录因子研究主要集中在MYB、WRKY、b ZIP、AP2/ERF和NAC等几大类。但桑树中这些转录因子的研究还很贫乏。通过改变桑树基因抗逆性提高桑树的抗旱性,可以降低干旱自然条件对桑树生长和产量的不利影响,具有深远的意义。因此,本文重点对桑树抗旱相关的trihelix、b ZIP、MYB和ERF转录因子进行鉴定和分析,主要结果总结如下:1.桑树抗旱性相关的trihelix、b ZIP、MYB和ERF四个转录因子家族鉴定本文借鉴其他物种的主要抗旱转录因子基因的研究情况及桑树全基因组序列数据库,利用生物信息学方法对桑树抗旱性相关的trihelix、b ZIP、MYB、和ERF等四个转录因子家族进行鉴定和分析,并从基因组水平上对这四个基因家族的序列特征进行了系统分析与预测。研究结果如下:鉴定出桑树trihelix转录因子家族成员29个,序列聚类和功能结构域分析发现该家族均含有高度保守的、特征性的trihelix结构域;根据亲缘关系远近和结构域特点,将桑树trihelix转录因子家族分为5个亚家族。基于MEME程序分析,trihelix家族的保守基序与聚类分析结果具有较高一致性。鉴定出桑树b ZIP转录因子家族成员32个,序列聚类和功能结构域分析发现该家族均含有高度保守的、特征性的b ZIP结构域;根据亲缘关系远近和结构域特点,将桑树b ZIP转录因子家族划分为A、B、C、D、E、F、G、H、I和其他族等几个亚家族。基于MEME程序分析,b ZIP家族的保守基序与聚类分析结果具有较高一致性。鉴定出含2个及2个以上Myb_DNA-binding结构域的桑树MYB转录因子家族成员99个,其中含有2个Myb_DNA-binding的转录因子96个,含有3个Myb_DNA-binding结构域的转录因子3个。序列聚类和功能结构域分析发现该家族高度保守、特征性的DNA结合结构域,MYB结构域。序列特征motif1分析表明,含有2个motif1特征结构域的MYB家族成员24个,含有3个mofit1有3个,其余均含1个Motif1特征结构域。基于MEME程序分析,桑树MYB转录因子家族的保守基序与聚类分析结果具有较高的一致性。鉴定出桑树ERF转录因子家族成员85个,序列聚类和功能结构域分析发现该家族均含有高度保守的、特征性结构域;根据亲缘关系远近和结构域特点,并将桑树ERF转录因子家族划分为A、B、C、D、E、F、G、H、I、J、K、L等12个群及其他群,其中J群家族成员最多。基于MEME程序分析,桑树ERF其保守基序与聚类分析结果具有较高的一致性。4个转录因子聚类分析表明,桑树与拟南芥4个转录因子家族的分类相一致,但每个物种均含有不同亚家族的成员,表明该基因家族的分化早于物种的分化。以上研究结果初步明确了桑树trihelix、b ZIP、MYB、和ERF转录因子家族成员、结构和功能特点,为进一步揭示这四个转录因子家族的分子进化规律和生物学功能奠定了基础。2.桑树干旱胁迫转录组中转录因子鉴定采用桑树育71-1品种为材料,进行了干旱胁迫和正常生长条件下两个样本的转录组测序(RNA-seq)分析。通过分析,共鉴定到1051个差异表达基因。通过桑树trihelix、MYB、b ZIP和ERF转录因子家族基因的差异表达分析,发现10个差异表达基因,2个是下调差异基因,其余是上调差异基因。10个差异表达基因中,trihelix转录因子家族有1个下调差异基因;b ZIP转录因子家族有1个上调差异基因;MYB转录因子家族有3个是上调差异基因,1个是下调差异基因;ERF转录因子家族4个,均是上调差异基因。四个转录因子家族差异基因富集分析表明:桑树四个转录因子家族差异基因功能的执行主要集中生物过程分类下的各项。分子功能分类下的差异蛋白执行转录因子活性、序列特异性的DNA结合;核酸结合;DNA结合等功能。在细胞组分分类下的差异蛋白只执行核功能一项。3.桑树Mntrihelix基因的克隆和序列分析及表达模式成功克隆桑树Mntrihelix基因,其序列ORF长度为567bp,编码188个氨基酸残基。SMART分析其有一个SMART SANT结构域。SMARTblast进化分析表明,属于trihelix转录因子的GT1类亚家族;在非冗余的蛋白数据库中,Mntrihelix与川桑、可可树和木本棉亲缘关系较近,与川桑的关系最为密切;参考物种数据库中,其与拟南芥和大豆的亲缘关系较近。通过q RT-PCR分析Mntrihelix在干旱、高盐和低温的胁迫条件下的表达情况:基因表达量在胁迫条件下都有不同程度的上调。4.桑树Mnb ZIP基因的克隆和序列分析及表达模式.成功克隆桑树Mnb ZIP基因,其序列ORF长度为675bp,编码224个氨基酸的蛋白。SMARTblast进化分析表明,属于b ZIP转录因子的b ZIP_plant_GBF1家族;在非冗余的蛋白数据库中,Mnb ZIP与川桑、苹果和甜橙亲缘关系较近,与川桑的关系最为密切;参考物种数据库中,其与拟南芥和大豆的亲缘关系较近。通过q RT-PCR分析Mnb ZIP在干旱、高盐和低温的胁迫条件下的表达情况:基因表达量在胁迫条件下都有不同程度的上调。5.桑树Mn MYB基因的克隆和序列分析及表达模式.成功克隆了桑树Mn MYB基因,其序列ORF长度为822bp,编码293个氨基酸的蛋白。SMART分析发现含有两个典型的SMART SANT结构域。SMARTblast进化分析表明,属于MYB转录因子的myb_SHAQKYF类亚家族;在非冗余的蛋白数据库中,Mn MYB与川桑、金丝小枣和毛果杨亲缘关系较近,与川桑的关系最为密切;参考物种数据库,其与拟南芥和大豆亲缘关系较近。通过q RT-PCR分析Mn MYB在干旱、高盐和低温的胁迫条件下的表达情况:基因表达量在胁迫条件下都有不同程度的上调。针对桑MYB基因在抗旱中起到的重要作用,构建了桑转录因子基因的原核表达载体,在大肠杆菌中成功诱导表达,并获得了高纯度目的蛋白,为多克隆抗体的制备提供了优质蛋白来源,以完成后续的免疫组化、基因定位和蛋白互作研究。6.桑树Mn ERF基因的克隆和序列分析及表达模式.成功的克隆了桑树Mn ERF基因,其序列ORF长度为321bp,编码106个氨基酸的蛋白。SMART分析发现,含有一个SMARTAP2结构域。SMARTblast进化分析表明,属于ERF转录因子家族;在非冗余的蛋白数据库中,Mn ERF与川桑、构树亲缘关系较近;参考物种数据库中,其与拟南芥和大豆亲缘关系较近。q RT-PCR分析Mn ERF在干旱、高盐和低温胁迫条件下的表达情况:基因表达量在胁迫条件下都有不同程度的上调。更多还原

【Abstract】 Plants often encounter in a variety of adverse conditions between their growth and development in natural conditions.Drought is one of the major abiotic stresses that adversely affect plant growth and crop yield worldwide.Lots of genes are thought to be involved in responses to abiotic stress.Plants have various response and defense systems at the molecular,cellular,and physiological levels in order to survive.Therefore,the research on abiotic stress related genes has important theoretical and practical significance.The transcription factors are involved in responses against biotic and abiotic stress,and they play an essential role in regulation of plant adaptation to environmental changes.As upstream regulatory factors,transcription factors play an important role in the study of plant resistance,which can activate a variety of stress-related and functional gene expression.Transcription factors also regulate vast downstream genes and play an important role in regulation of development,metabolism and response to environment in plants.Our country is the main origin of mulberry and owns the most germplasm resources of mulberry in the world.Mulberry is an excellent ecological and economic tree.The growth and productivity of mulberry is adversely affected by abiotic and biotic stresses.Efforts to investigate the functional genes and their molecular adaptation mechanisms under stresses plays an important role in strengthening stress tolerance and improving production of mulberry,and are of fundamental importance to the preservation of these genetic resources.At present the main transcription factors,which are more researched in plant resistance,include MYB,WRKY,b ZIP,AP2/ERF and NAC,but the main transcription factors information on drought-resistant genes of mulberry is scare,which is of great significance to improve the drought resistance of mulberry trees by changing the gene resistance of mulberry trees and can reduce the adverse effects of mulberry trees on the yield and growth caused by drought and natural geographical conditions.Therefore,in this paper,we focused on the identification and analysis of trihelix,b ZIP,MYB,ERF transcription factors related to drought resistance in mulberry,the main results were summarized briefly as follows:1.Identification of drought-resistant related trihelix,b ZIP,MYB,ERF transcription factor family in mulberryThe bioinformatics methods were used to identify and analyze the drought resistance-related trihelix,b ZIP,MYB,and ERF families of four transcription factors in mulberry according toother species.The sequence characteristics of these four gene families were systematically analyzed and predicted from the genomic level.The results are as follows:In this study,twenty nine members of the trihelix family,which contain highly conserved and characteristic trihelix domain through sequence clustering and functional domains analysis,were identified in mulberry genome database using bioinformatics tools.These members could be classified into 5 subfamilies based on the evolutionary relationship and domain characteristics.Clustering analyses of the trihelix family in mulberry and Arabidopsis showed that each species contained different members of subfamily although the classification of the trihelix family were consistent in two species,which indicated that the differentiation of the trihelix gene family occur earlier than that of these species.The conserved motifs in the trihelix family of mulberry analyzed using the MEME program was highly consistent with the results of clustering analyses.Thirty two members of the BZIP family,which contain highly conserved and characteristic b ZIP domain through sequence clustering and functional domains analysis,were identified in mulberry genome database using bioinformatics tools.These members could be classified into A、B、C、D、E、F、G、H、I and other subfamilies based on the evolutionary relationship and domain characteristics.Clustering analyses of the b ZIP family in mulberry and Arabidopsis showed that each species contained different members of subfamily although the classification of the b ZIP family were consistent in two species,which indicated that the differentiation of the b ZIP gene family occur earlier than that of these species.The conserved motifs in the b ZIP family of mulberry analyzed using the MEME program was highly consistent with the results of clustering analyses.Ninety nine members of the MYB family,which contain more two highly conserved and characteristic Myb_DNA-binding domain through sequence clustering and functional domains analysis,were identified in mulberry genome database using bioinformatics tools.These members could be classified into A、B、C、D、E、F、G、H、I and other subfamilies based on the evolutionary relationship and domain characteristics.Clustering analyses of the b ZIP family in mulberry and Arabidopsis showed that each species contained different members of subfamily although the classification of the MYB family were consistent in two species,which indicated that the differentiation of the b ZIP gene family occur earlier than that of these species.The conserved motifs in the MYB family of mulberry analyzed using the MEME program was highly consistent with the results of clustering analyses.Eighty five members of the ERF family,which contain highly conserved and characteristic trihelix domain through sequence clustering and functional domains analysis,were identified in mulberry genome database using bioinformatics tools.These members could be classified into A、B、C、D、E、F、G、H、I、J、K、L and other subfamilies based on the evolutionary relationship and domain characteristics,and J subtribe gathered the most.Clustering analyses of the ERF family in mulberry and Arabidopsis showed that each species contained different members of subfamily although the classification of the ERF family were consistent in two species,which indicated that the differentiation of the ERF gene family occur earlier than that of these species.The conserved motifs in the ERF family of Mulberry analyzed using the MEME program was highly consistent with the results of clustering analyses.Our results preliminarily identified the evolution,functional characterization,which will provide a basis to further reveal the molecular evolution and biological function of the trihelix,b ZIP,MYB、and ERF transcription factors in mulberry.2.Identification of transcription factors in drought stress RNA-seq of mulberryRNA-seq was carried out for transcriptome analysis of mulberry 71-1 between two samples in regular and drought stress condition.A total of 10 differentially expressed genes were screened out based on the identification of differential expression genes of trihelix,MYB,b ZIP and ERF family in mulberry,of which 2 was down-regulated and 8 up-regulated.The b ZIP transcription factor family has 1 up-regulated differential gene.The trihelix transcription factor family has a down-regulated differential gene.There were 3 up-regulated genes and 1 down-regulated gene in the MYB transcription factor family.ERF transcription factor family 4,are up-regulated genes.GO enrichment of differentially expressed genes shows that The implementation of the function of differentially expressed genes of four TFs(Transcription factors)family mainly focuses on the classification of biological processes.The differentially expressed protein in molecular functional classification performs transcription factor activity,sequence-specific DNA binding,uucleic acid binding,DNA binding and so on.Differential expressed protein performs nuclear function in classification of cellular components.3.Cloning and sequence analysis of Mntrihelix from mulberry and its expression patternMntrihelixsequence was cloned with containing a 567 bp open reading frame(ORF),encoding 188 amino acid residues.SMART analysis showed Mntrihelix contained one typical SANT domains.Phylogenetic analysis showed that Mntrihelix belonged to the GT1 subfamily of the trihelix family and was most closely related to morusnotablis SMARTblast analysis indicated that Mntrihelix was close to trihelix of Arabidopsis thaliana and Glycine max from the reference species,and of Gossypium arboretum,Theobroma cacao and Morus notabilisfrom the non-redundant protein database.Further expression profile analysis using the quantitative real time-polymerase chain reaction technique(q RT-PCR)showed that most of the Mntrihelix gene from mulberry was induced by drough,cold and salt treatments.Expression levels have up-regulated in varying degrees under various stresses.4.Cloning and sequence analysis of Mnb ZIPfrom mulberry and its expression patternMnb ZIPsequence was cloned with containing a 675 bp open reading frame(ORF),encoding 224 amino acid residues.SMART analysis showed Mnb ZIP contained one typical SANT domains.Phylogenetic analysis showed that Mnb ZIP belonged to the b ZIP_plant_GBF1 subfamily of the b ZIP family and was most closely related to morusnotablis.SMARTblast analysis indicated that Mnb ZIP was close to Arabidopsis thaliana and Glycine max from the reference species,and Ziziphusjujuba,Malus domestica and Morusnotabilis from the non-redundant protein database.Further expression profile analysis using q RT-PCR showed that most of the Mnb ZIP gene from mulberry was induced by drought,cold and salt treatments.Expression levels have up-regulated in varying degrees under various stresses.5.Cloning and sequence analysis of Mn MYBfrom mulberry and its expression patternMn MYBsequence was cloned with containing an822 bp open reading frame(ORF),encoding 293 amino acid residues.SMART analysis showed Mn MYB contained two typical SMART SANT domains.Phylogenetic analysis showed that Mn MYB belonged myb_SHAQKYF subfamily of the MYB family and was most closely related to morusnotablis.SMARTblast analysis indicated that Mn MYB was close to Arabidopsis thaliana and Glycine max from the reference species,and Populustrichocarpa,Ziziphusjujuba and Morusnotabilis from the non-redundant protein database.Further expression profile analysis using q RT-PCR showed that most of the Mn MYB gene from mulberry was induced by drought,cold and salt treatments.Expression levels have up-regulated in varying degrees under various stresses.6.Cloning and sequence analysis of Mn ERFfrom mulberry and its expression patternMn ERFsequence was cloned with containing a 321 bp open reading frame(ORF),encoding 106 amino acid residues.SMART analysis showed Mn ERF contained one conservative AP2 domains.Phylogenetic analysis showed that Mn ERF belonged ERF subfamily of the AP2/ERF superfamily.Phylogenetic analysis indicated that Mn ERF was close to morusnotablis and Broussonetiapapyrifera from the reference species and of Gossypium arboretum,Theobroma cacao and Morusnotabilis from the non-redundant protein database by SMARTblast.Further expression profile analysis using q RT-PCR showed that most of the Mn ERF gene from mulberry was induced by drought,cold and salt treatments.Expression levels have up-regulated in varying degrees under various stresses.更多还原