【作者】 郭晋隆；

【导师】 许莉萍；

【作者基本信息】 福建农林大学 ， 作物栽培学与耕作学， 2013， 博士

【摘要】 甘蔗(Saccharum officinarum L.)是重要的糖料作物和能源作物,蔗糖分别占世界食糖和我国食糖总产量的70%和90%以上,甘蔗燃料乙醇产量约占世界生物质燃料乙醇总产量的40%。干旱是造成甘蔗等作物减产的重要因素,我国甘蔗85%种植在盐碱地或旱坡地,甘蔗水分需求与供给不平衡的矛盾尤为突出。挖掘甘蔗抗旱基因是甘蔗抗逆性育种的重要基础工作。转录水平调节是作物从分子水平上适应干旱胁迫的一个重要机制,利用干旱胁迫应答基因在转录水平的表达差异来筛选并鉴定抗旱相关基因,是挖掘作物抗旱基因的有效手段。斑茅(Erianthus arundinaceus)是甘蔗近缘属植物,具有优异的抗逆基因资源,已成为甘蔗抗逆育种的重要基因来源。有性杂交虽然也是一种有效的途径,而且,经过了十几年研究,目前也已培育出甘蔗与斑茅杂交的BC3和BC4后代。但是,在斑茅优异的抗逆基因资源的杂交利用与“高贵化”回交过程中,还面临较多的困难,如杂交后代花粉不育、计划杂交的组合花期不遇和优良目标性状的多基因聚合问题,同时,在杂交后代广泛变异群体中,具有优良目标性状聚合基因型单株的鉴定与选择的准确性和效率依然不高。因而,即便对于甘蔗品种间的杂交,一般认为在10万个杂交后代变异群体中,经过10年的鉴定与选择,有希望培育出1个优良栽培品种。基于上述考虑,利用综合性状好且具有斑茅血缘的甘蔗BC2材料,进行模拟干旱逆境胁迫,以期在解析甘蔗应答干旱逆境胁迫分子机理的同时,挖掘重要的抗逆基因和转录调控元件,为后续抗逆育种奠定物质基础、理论依据与技术储备。本研究以甘蔗与斑茅杂交选育的BC2无性系(S. complex)崖城05-179(YCE05-179)为实验材料,采用Illumina GA/HiSeq第二代测序平台进行RNA-Seq高通量测序,通过收集、比对与分析在模拟干旱胁迫条件下(25%PEG800,24h)的甘蔗根中的转录本数据,高通量的分离和筛选差异表达基因,并对部分PEG胁迫应答基因进行了功能验证。主要结果与结论如下：1.以抗旱斑茅蔗无性系YCE05-179为植物材料,基于Illumina/Solexa二代测序平台的RNA-Seq技术,成功获得YCE05-179的根中响应PEG胁迫的转录本序列信息,处理和对照2个样本的测序总量分别达到514,274,992个和556,342,815个总碱基对(Total base pairs);其中,胁迫处理样本得到10,495,408个clean reads,对照样本得到11,353,935个clean reads。测序质量的多重评估分析结果表明,本研究中RNA-Seq测序质量良好,测序深度充分,测序结果已经基本覆盖了2组样本细胞中全部表达的基因。2.以NCBI的高粱基因组数据库以及高粱基因数据库数据为参考,对上述两组RNA-Seq数据进行比对和分析,共检测到23,550个基因。从两组样品中,共筛选到12,035个差异表达基因,其中上调表达的基因有6,480个,下调表达的基因有5,555个。表达显著差异(差异表达基因为FDR≤0.001且倍数差异不低于2倍)的基因共有597个(其中上调表达329个,下调表达268个)。本研究从上述差异表达基因中挑选出部分基因做进一步的功能验证,包括参与细胞次生代谢途径中木质素合成的dirigent蛋白基因、参与重金属绑定和细胞活性氧清除的金属硫蛋白基因,以及参与转录调控的R2R3-MYB转录因子家族基因。3. Dirigent和dirigent-like家族蛋白与植物细胞的木质素合成相关,参与了植物对病原体和非生物胁迫的应答。本研究在对RNA-Seq和甘蔗茎cDNA全长文库数据分析的基础上,分离克隆了1个dirigent-like基因,命名为ScDir (GenBank Accession number:JQ622282)。 ScDir的cDNA全长819bp,包含1个564bp的开放读码框,编码187个氨基酸。ScDir蛋白的氨基酸序列N端的第1到25个氨基酸残基和第7到26个氨基酸残基的位置分别包含一个信号肽和跨膜结构区。用His标签标记ScDir蛋白,并成功在大肠杆菌(Escherichia coli)中表达了该重组蛋白(理论分子量大小为27.4kDa).ScDir基因在大肠杆菌中的表达提高了宿主菌对PEG和NaCl的耐受能力。以GAPDH基因为内参基因的实时荧光定量PCR研究表明,ScDir基因在甘蔗的茎中高度特异表达,其在茎中的相对表达量分别是在根、叶和芽中的8.64±0.48倍,25,635.16±2,966.03倍和721.50±8.17倍。实时荧光定量PCR研究结果表明,H2O2、PEG和NaCl胁迫提高了ScDir基因在甘蔗组培苗中的表达水平,其中ScDir基因的表达受PEG胁迫的诱导而显著上调,在处理12h时间点其表达量最高,为对照的35倍以上。上述实验结果说明,ScDir基因是一种茎高度特异表达基因,该基因在大肠杆菌中的作用及其在甘蔗中的表达模式,揭示了该基因在甘蔗应答干旱、高盐和氧化等非生物胁迫方面起到积极的作用。4.金属硫蛋白基因是一类富含半胱氨酸的小分子量金属结合蛋白。前人的研究显示,植物金属硫蛋白具有可逆结合有毒离子和基本金属离子的能力,在解毒、维持金属离子代谢平衡和调节金属离子转运等方面起重要作用。本研究在对RNA-Seq和甘蔗茎cDNA全长文库数据分析的基础上,分离克隆了一个金属硫蛋白基因,命名为ScMT2-1-3(GenBank Accession number:JQ627644)。ScMT2-1-3基因全长700bp,包含一个240bp的开放读码框,编码79个氨基酸。在大肠杆菌中成功表达了带His标签的ScMT2-1-3重组蛋白(分子量大小约为19.01kDa),并通过MALDI-TOF-TOF MS验证了该重组蛋白。ScMT2-1-3基因在大肠杆菌中的表达提高了宿主菌对Cd2+、Cu2+、PEG和NaCl的耐受能力。实时荧光定量PCR研究表明,CuCl2、H2O2、PEG和NaCl胁迫均提高了ScMT2-1-3基因在甘蔗组培苗中的表达水平,而CdCl2胁迫抑制了ScMT2-1-3基因的表达水平；ScMT2-1-3基因在甘蔗芽和根中的相对表达量是在茎和叶中的14倍以上。ScMT2-1-3基因在大肠杆菌中的作用及其在甘蔗中的表达模式,提示了该基因在甘蔗应答干旱、高盐和氧化等非生物胁迫时,扮演着抗氧化的角色,并起积极的作用。此外,ScMT2-1-3基因还参与了铜离子在细胞中的解毒和富集过程,但其对甘蔗细胞中镉的解毒和富集过程的作用机理还需进一步研究。5.R2R3-MYB基因是MYB转录因子基因家族的主要成员,已被证明在次生代谢和非生物逆境胁迫应答中起重要作用。本研究在RNA-Seq高通量测序的基础上,综合应用电子克隆、RT-PCR和RACE技术,分离克隆了3个甘蔗R2R3-Myb基因/亚家族Sc2RMyb1、 Sc2RMyb2和Sc2RMyb3s。这些基因的表达特性或功能验证实验结果如下：(1)Sc2RMyb1(GenBank Accession number:JQ823165)的基因组DNA全长1,807bp,由3个外显子和2个内含子构成,编码区长度为1,284bp,编码427个氨基酸。构建含有Sc2RMyb1基因的原核表达载体并转入大肠杆菌,经IPTG诱导产生的重组蛋白相对分子量约为52kDa。在NaCl胁迫的LB培养基上,重组菌生长明显优于对照菌。实时荧光定量PCR分析表明,甘蔗中Sc2RMyb1基因的表达受H202和NaCl抑制而明显下调；Sc2RMyb1基因对PEG的应答除了在处理12h时略有上调以外,在其余的6个理处时间点均明显下调。推测该基因作为负调节因子参与了NaCl等胁迫应答相关基因的调控过程。(2)Sc2RMyb2基因通过不同剪接方式,产生至少两种转录本,即Sc2RMyb1和Sc2RMyb2。其中Sc2RMyb2S1的cDNA全长为1,066bp,开放阅读框长度为687bp,共编码228个氨基酸；Sc2RMyb2S2无明显开放读码框,不编码功能蛋白。序列分析表明,串联内含子的结构特征是Sc2RMyb2剪接调控的结构基础。Sc2RMyb2转录基因子基因通过剪接调控,调节细胞中Sc2RMyb2S1和Sc2RMyb2S2两种转录本的相对表达量,从而对PEG胁迫引起的生理生化变化进行应答。实时荧光定量PCR研究表明,Sc2RMyb2S1基因的表达受PEG胁迫的抑制而显著下调,该基因在烟草叶片中的瞬时表达导致叶片变黄,提示了该基因参与了PEG胁迫引起的细胞衰老的调控过程。(3)本研究还分离克隆了Sc2RMyb3s亚家族的7个成员基因(Sc2RMyb3-1～Sc2RMyb3-7),并对Sc2RMyb3-1、Sc2RMyb3-2和Sc2RMyb3-3等3个基因进行了初步的功能验证。在烟草叶片中的瞬时表达实验表明,Sc2RMyb3-1和Sc2RMyb3-2基因没有引起烟草叶色等表型的明显变化,而Sc2RMyb3-3基因在烟草叶片中的瞬间表达导致了叶色明显变黄,这提示了Sc2RMyb3-3基因在细胞衰老等相关生理生化过程中发挥重要的调控作用；Sc2RMyb3s亚家族各成员基因在甘蔗响应环境因子的过程中执行不同的功能,体现了R2R3-Myb转录因子调控机制的复杂性。序列分析表明,氨基酸序列中的C端序列结构在决定甘蔗Sc2RMyb3s转录因子的功能上起到重要的作用。更多还原

【Abstract】 Sugarcane(Saccharum officinarum L.) is one of the more important sugar and energy crops of the world. Cane sugar accounts for more than70%of the sugar production in the world and90%of that in China. Moreover, sugarcane fuel ethanol accounted for about40%of total biomass fuel ethanol production in the world. Drought is one of the most important yield-limiting factors of sugarcane plants. In China, due to85%of the sugarcane planting in saline or sloping dryland, the problem of insufficient water supply became more prominent.Mining for drought tolerance gene sources is an important work for sugarcane resistance breeding. Identification of the genes responsible for drought tolerance by monitoring the changes in gene expression at the transcriptional level is an effective method to target drought-tolerance genes. Erianthus arundinaceus is a closely related genus of S. officinarum L. With its outstanding stress-tolerance and high rooting ability, E. arundinaceus has proved to be one of the every important genetic resources for improvement of stress-tolerance in sugarcane. Although conventional hybridization is an effective way, and the BC3or BC4fertile hybrids from intergeneric crosses between S. officinarum L. and E. arundinaceus have been generated over the past ten years of work. Still, there are several difficulties in the process of making use of the outstanding stress-tolerance gene resources of E. arundinaceus and "noblelization" backcross, such as pollen sterility, flowering asynchronism, and target trait polymerisation problems, etc. Thus, it usually takes more than ten years for a cycle of sugarcane breeding to develop a good variety. In this study, an intergeneric BC2hybrid (S. complex) with good comprehensive characters named YCE05-179from S. officinarum X E. arundinaceus, was taken as the experimental material in imitation of drought stress. Analysis of molecular mechanism of sugarcane in response to drought stress and mining important functional protein genes and transcriptional regulatory elements are our main purpose. A high-throughput technology of RNA-Seq based on Illumina/Solexa platforms (Illumina GA/HiSeq) was used to produce a substantial expressed sequence tags dataset from the roots of YCE05-179under the treatment of PEG (25%PEG8000,24h). The main results and conclusions are as follows:1. RNA-seq based on the Illumina GA/HiSeq, was used to generate an extensive map of YCE05-179(S. complex) roots transcriptome under the treatment of PEG. In total,514,274,992basepairs raw sequence data corresponding10,495,408clean reads and556,342,815basepairs raw sequence data corresponding11,353,935clean reads were generated from the treatment and the control samples, respectively. The quality of these sequence reads was assessed by multiple analyses, and the information indicated that the high-quality RNA sequencing data in this study could fully cover the expressed genes in cells from each sample.2. Clean reads were aligned to sorghum genome and unigenes. In total,2.18million reads were aligned to the genome and unigenes, and23,550genes were identified. A total of12,035genes,6,480up-regulated genes and5,555down-regulated genes, were found to be differentially expressed in roots. Among them,597genes (329up-regulated genes,268down-regulated genes) were defined as highly significant differential expression filtered by conditions on FDR≤0.001and|log2Ratio|≥1. In this study, three types of gene including dirigent-like protein gene, metallothionein gene and R2R3-Myb transcription factor gene, which were clustered based on gene ontology (GO) terms, possess functional signatures identify as secondary metabolism (lignin synthesis), heavy metal binding (oxygen and reactive oxygen species metabolic process) and regulation of transcription, respectively, and all these genes were picked out for further functional verification.3. Dirigent and dirigent-like family proteins contain a number of proteins involved in lignification or in the response to pathogen infection and abiotic stress in plants. In this study, a full-length cDNA sequence of a dirigent-like gene designated ScDir (GenBank Accession Number JQ622282) was obtained from sugarcane based on RNA-Seq and bioinformatics analysis using the data of sugarcane stem full-length cDNA library. The ScDir gene was819bp long, including a564bp ORF encoding187amino acid residues. The protein N-terminus contained signal peptides at amino acid residues of1to25and trans-membrane regions at7to26aa. A His-tagged ScDir protein with an estimated molecular mass of27.4kDa was expressed in Escherichia coli system. The expressed ScDir protein had increased the host cell’s tolerance to PEG and NaCl. When an endogenous GAPDH gene was used as the internal control, results from Real-time qPCR demonstrate that the ScDir mRNA amount in sugarcane stalks was significantly higher than that in the roots, leaves and buds, as18.64±0.48-,25,635.16±2,966.03-,721.50±8.17-fold, respectively. The ScDir transcript levels in sugarcane seedling increased under H2O2, PEG or NaCl stress. The expression level of ScDir was significantly upregulated under PEG stress, and the highest level (>35-fold) was observed at12h after stress. Thus, both the ScDir hosted cell performance and the enhanced expressions in sugarcane imply that the ScDir gene is involved in the response to abiotic stresses of drought, salts, and oxidation. The transcription of the ScDir gene is highly stalk-specific, as revealed by Real-time qPCR.4. Metallothioneins (MTs) are cysteine-rich, low-molecular-weight, metal-binding proteins. Due to their ability to reversibly bind both toxic and essential metal ions, previous studies indicated that plant MTs play important roles in detoxification, metal ion homeostasis and metal transport adjustment. In this study, a novel metallothionein gene, designated as ScMT2-1-3(GenBank Ace:JQ627644), was obtained from sugarcane based on RNA-Seq and bioinformatics analysis using the data of sugarcane stem full-length cDNA library. ScMT2-1-3gene was700bp long, including a240bp ORF encoding79amino acid residues. A His-tagged ScMT2-1-3protein with an estimated molecular mass of19.01kDa was successfully expressed in Escherichia coli system and was identified at the same time by MALDI-TOF-TOF MS. The expressed ScMT2-1-3protein had increased the host cell’s tolerance to Cd2+, Cu2+, PEG and NaCl. The expression of ScMT2-1-3was up-regulated under Cu2+, H2O2, PEG and NaCl stress, but down-regulated under Cd2+stress. Real-time qPCR demonstrated that the expression levels of ScMT2-1-3gene in bud and root were over14times higher than those in the stem and leaf. Thus, both the E. coli assay and sugarcane plantlets assay suggested that ScMT2-1-3gene act as antioxidants when it functions in sugarcane response to drought, high salinity and oxidative stress. Moreover, ScMT2-1-3gene is significantly involved in the copper detoxification and storage in the cell, but its functional mechanism in cadmium detoxification and storage in sugarcane cells need more testification though its expressed protein could abviously increase the host E. coli cell’s tolerance to Cd2+.5. R2R3-MYB gene is a principal member of MYB transcription factor superfamily, which has been showed to play an important role in secondary metabolism and abiotic stress responses. Based on high-throughput RNA sequencing, three sugarcane R2R3-Myb genes/sub-family genes were isolated in the present study. (1) Genomic DNA sequence of Sc2RMybl gene (GenBank Acc:JQ823165) was1,807bp in length, including3exons and2introns, and the complete coding sequence was1,248bp encoding427amino acids. The recombinant protein with an estimated molecular mass of52kDa was produced in positive prokaryotic strain induced by IPTG The recombinant E. coli cells exhibited better growth than the negative strain in liquid LB medium with addition of NaCl. When25srRNA gene was used as the internal control, the expression profiles of Sc2RMybl gene responsive to different stresses were detected by Real-time qPCR in sugarcane. The transcript levels of Sc2RMybl gene in sugarcane seedling decreased under both H2O2and NaCl stresses. Moreover, when treated with PEG, Sc2RMybl gene was only slightly up-regulated at the treatment time point of12h and down-regulated visibly in the other six treatment time points. It suggested that the Sc2RMyb1gene, as a negative adjustment factor, may involve in the response to salt stress, etc.(2) Sc2RMyb2gene was able to generate at least two distinct transcripts, Sc2RMyb2S1and Sc2RMyb2S2, by alternative splicing. Sc2RMyb2S1gene was1,066bp long, including a687bp ORF encoding288amino acid residues. Sc2RMyb2S2had no significant open reading frame and did not encode a functional protein. Sequence analysis showed that series introns structure in the gDNA sequences of Sc2RMyb2gene was the structure foundation for alternative splicing. The alternative splicing of Sc2RMyb2gene is responsive to PEG stress, by means of regulating the relative transcript level of Sc2RMyb2S1and Sc2RMyb2S2in cells. The transcript level of Sc2RMyb2S1gene was inhibited by PEG with obvious down-regulation during the whole period of treatment. The tobacco leaves turned yellow when the Sc2RMyb2S1gene expressed transiently in it, which suggested that the Sc2RMyb2Sl gene was involved in cellular senescence induced by PEG(3) In the present study,7genes termed as Sc2RMyb3-1to Sc2RMyb3-7were cloned and classified into the same sub-family genes:Sc2RMyb3s. Three of them, Sc2RMyb3-1, Sc2RMyb3-2and Sc2RMyb3-3, were chosen for a transient expression assay in the tobacco leaf. The transient expression of Sc2RMyb3-1or Sc2RMyb3-2gene in the tobacco leaves, did not cause any significant phenotypic changes, such as colors. On the contrary, the tobacco leaves turned yellow notably when the transient expression assay of Sc2RMyb3-3gene was performed under the same conditions. It was suggested that the Sc2RMyb3-3gene was involved in cellular senescence induced by PEG, and the complex role played by Sc2RMyb3s sub-family members in regulating multiple aspects of different stresse responses. Sequence analysis showed that the C-terminal amino acid sequence may play an important role in determining the function of Sc2RMyb3s transcription factors in sugarcane.更多还原