【作者】 陈圣林；

【导师】 张日清；

【作者基本信息】 中南林业科技大学 ， 森林培育， 2012， 博士

【摘要】 菲油果Feijoa sellowiana Berg系桃金娘科、菲油果属植物,是我国新近引进的世界新兴水果树种,兼具食用、观赏、绿化等用途,经济价值高。本学位论文开展了开花相关生物学特性研究,具有重要的科学意义和应用前景。论文首次对菲油果花器官发育进程进行了解剖观测、构建了菲油果花蕾cDNA文库和EST文库,克隆了菲油果FsLFY基因和MADS-box (FsPI、FsSEP1)基因,并进行了表达特征和生物信息学分析,获得了一系列开创性的研究结果,丰富了菲油果花发育的生物学理论,为进一步研究菲油果开花调控和开发利用菲油果提供了重要的科学依据。主要研究结果如下：1.菲油果花发育解剖学研究。菲油果的花芽分化可以划分为6个时期：即未分化期、前分化期、萼片形成期、花瓣形成期、雌雄蕊形成期和子房花药形成期。菲油果花药为4室：花药壁发育属基本型；腺质绒毡层；小孢子母细胞减数分裂中胞质分裂为同时型,四分体排列多为正四面体；开花前2-3天花粉成熟,成熟花粉为2-细胞型。大孢子母细胞减数分裂形成的4个大孢子呈直线排列,其中合点端的大孢子具功能,经过3次有丝分裂后,于开花当天形成成熟八核胚囊,胚囊发育属蓼型。在发育过程中,雌雄配子体与花形态特征之间有着相对稳定的关系。2.菲油果花器官cDNA文库构建。以6个不同发育阶段的混合菲油果花蕾为材料,提取总RNA,分离纯化mRNA,在SuperSciptTM Ⅱ RnaseH-Reverse Transcriptase等的作用下合成cDNA,与pBluescript Ⅱ SK(+)XR载体连接重组并转化感受态细胞DH10B,成功地构建了菲油果花器官发育时期的高质量cDNA文库。经检测,cDNA文库库容量2.02x106克隆子,重组率高达92.32%,插入片段长度在600bp-2000bp,平均长度超过800bp。该文库的构建为建立菲油果花器官EST文库、开展基因分离鉴定、制作菲油果基因芯片和基因表达检测等奠定了良好基础。3.菲油果花器官EST文库构建。对已构建的cDNA文库随机挑取3500个克隆进行5’端测序,获得了高代表性的表达序列标签EST文库。获得原始序列2856个,测序成功率81.6%,获得了2840条大于100bp的高质量EST序列,其中500bp-700bp的EST序列2520条,占88.73%。对2840条有效ESTs进行组装拼接,共获得1682个非重复序列,平均长度为698.76bp,其中重叠群为438个,单拷贝ESTs为1244个。从1682条非重复序列中预测得到1379个开放阅读框,其中1244条单拷贝ESTs中包含1120个开放阅读框,占总数的81.22%,438个重叠群有259个ORF,占18.78%。4.菲油果花器官EST生物信息学分析。1682条非重复序列在核苷酸数据库中进行同源性Blast,结果有637条能找到同源序列,占总数的49.53%,其中为推测功能基因的有367条；与非冗余核酸库中Blast结果有851条非重复序列找到同源序列,占总数的66.17%,其中612条序列为推测功能基因；与SWISSPROT蛋白库Blast结果有547条序列找到同源序列,占总数的42.53%。将非重复序列与COG库进行Blast分析,获到有注释的序列236条,按功能分为18类。其中,翻译、核糖体结构与生物发生,糖类转运与新陈代谢,转译后修饰与蛋白转换、脂类转运与新陈代谢等4类中注释的序列最多,数量分别为50、38、34、24,所占比例分别为21.19%、16.1%、14.41%、10.17%。5.菲油果FsLFY基因的克隆与生物信息学分析。根据已知菲油果LFY基因588bp EST序列信息,设计特异引物,分别通过5’RACE和3’RACE技术获得了FsLFY基因的5’和3’末端序列,通过拼接,获得FsLFY基因的全长cDNA克隆,再通过基因组PCR获得了FsLFY基因的基因组序列,将FsLFY基因cDNA和基因组序列提交至GenBank,接受号为JN562738。菲油果FsLFY基因cDNA全长1336bp,5’非转录区91bp和3’非转录区174bp,全部CDS1071bp,编码356个氨基酸。FsLFY基因含2个内含子,长度分别为63bp、109bp。FsLFY氨基酸序列含2个可变区和2个保守区。在DNA序列水平上,FsLFY基因与菊花ClLFY的相似性最低,为59.4%,与蓝桉EglLFY的相似性最高,为94.4%；在氨基酸水平上,FsLFY基因与兰花OiLFY基因的相似性最低,为60.3%,与蓝桉EglLFY的相似性最高,为95.2%。FsLFY蛋白预测分子量为40631.8Da,等电点为8.59,属于较不稳定蛋白质。FsLFY蛋白疏水指数从-3.933到1.456,整个蛋白质基本上是表现出亲水性。FsLFY蛋白不含信号肽,含31个α螺旋,30个β折叠链和34个卷曲。6.菲油果FsPI基因的cDNA克隆与生物信息学分析。根据已知菲油果PI基因626bp的EST序列信息,设计特异引物,分别通过5’RACE和3’RACE技术获得了FsLFY基因的5’和3’末端序列,通过拼接,获得FsPI基因的全长cDNA克隆,将其提交至GenBank,接受号为JN562739。菲油果FsPI的cDNA全长891bp,获得了部分5’非转录区6bp和完整的3’非转录区258bp,全部CDS为627bp,编码208个氨基酸。菲油果FsPI基因具有MADS-box B类PI基因的典型特征,含MADS结构域、K结构域以及PI结构域。在DNA序列水平上,菲油果FsPI与长春花CrGLO1的相似性最低,为66.7%,与白千层MqPI的相似性最高,为93.0%；在氨基酸水平上,与辣椒CaPI基因的相似性最低,为62.0%,与白千层MqPI基因相似性最高,为91.8%。菲油果FsPI蛋白预测分子量为24286.4Da,等电点为8.83,是一种弱碱性较不稳定蛋白。菲油果FsPI蛋白疏水指数位于-2.956到1.933之间,整个蛋白质基本上表现出亲水性。菲油果FsPI不含信号肽,属于非分泌性蛋白,其含25个α螺旋,21个β折叠链和20个卷曲。7.菲油果FsSEP1基因的cDNA克隆与生物信息学分析。根据已知菲油果SEP1基因338bp的EST序列信息,设计特异引物,分别通过5’RACE和3’RACE技术获得了FsSEP1基因的5’和3’末端序列,通过拼接,获得FsSEP1基因的全长cDNA克隆,将其提交至GenBank,接受号为JN562740。菲油果FsSEP1的cDNA全长1120bp,5’非转录区83bp和3’非转录区299bp,全部CDS为738bp,编码245个氨基酸。菲油果FsSEP1基因具有MADS-box E类SEP基因的典型特征,含MADS结构域、K结构域和SEP结构域。在DNA和氨基酸序列水平上,菲油果FsSEP1基因都是与拟南芥AthSEP1的相似性最低,分别为69.1%,66.7%,,与巨桉EgM3的相似性最高,为92.4%,为91.4%。菲油果FsSEP1蛋白预测分子量为28009.8Da,等电点为8.80,是一种弱碱性蛋白,属于较不稳定蛋白质。菲油果FsSEP1蛋白的疏水指数从-2.411到2.089,整个蛋白质基本上表现出亲水性。菲油果FsSEP1不含信号肽,属于非分泌性蛋白,其含23个α螺旋,25个β折叠链和26个卷曲。8.菲油果FsLFY基因的表达模式。通过RT-PCR扩增出菲油果钙调素基因Fscalm,以此为内参基因。半定量RT-PCR检测结果表明,菲油果FsLFY基因在生殖器官和营养器官中均有表达。总体上在整个花蕾发育期间的表达强烈,表达量要明显高于结果枝、营养枝、顶端嫩叶、嫩茎和腋芽等营养器官。在花蕾中,FsLFY基因在4月10日至4月22间的4个阶段表达量均非常强烈,表达量无明显差别,从26日开始,表达量逐渐呈下降趋势。FsLFY基因在结果枝、营养枝、顶端嫩叶、嫩茎和腋芽中表达量均较低,但相对而言,在结果枝和顶端嫩叶中的表达量稍高于营养枝、嫩茎和腋芽中。FsLFY基因的表达模式表明该基因无论在菲油果的生殖生长还是营养生长阶段均行使其功能,但与生殖生长间的关系更密切。更多还原

【Abstract】 Feijoa sellowiana Berg., a new kind of fruit tree species of Myrtaceae family, has edible and ornamental value. It has a relatively long juvenile phase, and the seedlings blossom and bear fruits after four to five years. Therefore, elucidation of flowering mechanism of F. sellowiana is of great significance to shortening and adjusting the juvenile phase, genetical modification, and new cultivars breeding. In this paper, systematic studies on the development of F. sellowiana floral organs and related expressed genes via comparative anatomy experiment, cDNA and EST libraries construction, cloning and expression pattern detection of floral organs-related genes, and bioinformatics analysis etc. The main results are as follows:1. Studies on comparative anatomy of F. sellowiana floral organs. The development of F. sellowiana’s flower bud differentiation was divided into six stages: predifferentiation, sepal formation, petal formation, stamen and pistil formation, ovary and anther formation and stamen and pistil maturation. F. sellowiana had four ventricles in each anther; the development of the anther wall was basic type; glandular tapetum; the meiosis in the microspore mother cells were belonged to a simultaneous type and the microspores were arranged in a tetrahedron shape in the tetrads; the pollen was matured in two or three days before blooming; The ripened pollen was of2-cell type. Megaspore mother cell meiosis to the formation of four spores were linear arrangement, which the chalazal megaspore had function. After three mitosis, the formation of mature eight-nucleate embryo sac was in the blossoming day. The embryo sac was a Polygonum type. Between male and female gametophyte and flower morphology had stably relationship in the developmental process.2. Construction of cDNA library of F. sellowiana floral organs. Total RNA was isolated from mixed floral buds of’Coolidge’in six different developmental stages. Then message RNA (mRNA) was purified from total RNA and was reverse transcribed to cDNA with SuperSciptTM II RnaseH-Reverse Transcriptase. The cDNA was ligated to pBluescript Ⅱ SK(+)XR vector and co-transformed into competent Escherichia coli cells DH10B. Eventually, a high-quality cDNA library of developing floral organs of F. sellowiana was successfully constructed. After the examination, the cDNA library aggregate the capacity of2.02×106clones, the recombination rate is92.32%, and the size of insertion segment range from600bp to2000bp. The cDNA library provieds a basis for EST library construction, isolation and identification of floral organs-related genes, gene miccroarry construction, and gene expression detection for F. sellowiana.3. Construction of EST library of F. sellowiana floral organs. Using the cDNA library of F. sellowiana floral organs as the material,3500positive clones was selected at random for DNA sequencing at the5’end, accordingly the EST library of F. sellowiana floral organs was constructed. After analysis and classification,2856original sequences were obtained with the sequencing seccess rate of81.6%.2840high-quality ESTs with the size of more than100bp were obtained, of which numbers of ESTs ranging from500bp to700bp were2520, accounting for88.73%. Assembling of the total of2840ESTs resulted in1682Unigenes that include438contigs and1244Singletons with the average size of698.76bp.1379ORFs (open reading frame) were predicted from1682Unigenes, of which1120ORFs were from1244Singletons accounting for81.22%, and259were from Contigs accounting for18.78%.4. Informatics analysis of EST sequences of F. sellowiana floral organs. Using EST sequences of1682Unigenes as queries for Blast analysis in Nucleotide Database,637could search out homologous sequences accounting for49.53%, of which367represented putative functional genes. Blast analysis in Non-redundant Database revealed that851Unigenes could search out homologous sequences accounting for66.17%, of which612represented putative functional genes. Blast analysis in SWISSPROT revealed that547Unigenes could search out homologous sequences accounting for42.53%. Blast analysis in COG database showed that236annotated sequences were obtained and were classified into18groups. Most of annotated sequences were found in four groups including Translation, ribosomal structure and biogenesis, Carbohydrate transport and metabolism, Posttranslational modification, protein turnover, and chaperones, and Lipid transport and metabolism. The numbers of the annotated sequences were50,38,34, and24, accounting for21.19%,16.1%,14.41%, and10.17%, respectively.5. Cloning and informatics analysis of FsLFY. Based on known588bp EST sequence for FsLFY gene, specific primers were designed for3’RACE (Rapid amplification of cDNA ends) and5’RACE PCRs. Accoringly, the full-length cDNA were obtained by assembling3’and5’teminal sequences. Genomic DNA sequences for FsLFY were also successfully amplified by genomic PCR and DNA sequencing. The FsLFY sequences were subbmited to GenBank and the Accession No. was JN562738. The full-length cDNA of FsLFY was1336bp with5’non-transcription region of91bp,3’non-transcription region of174bp and complete CDS of1071bp, encoding356amino acids. FsLFY gene contained two introns, the size of which was63bp and109bp, respectively. The amino acid sequence of FsLFY contained two viable regions and two conserved regions. At DNA sequence level, the FsLFY showed the lowest similarity with ClLFY (59.4%), the highest with EglLFY (94.4%). At amino acid sequence level, the FsLFY showed the lowest similarity with OiLFY (60.3%), the highest with EglLFY (95.2%). The predicted molecular weight and isoelectric points of FsLFY were40631.8Da and8.59, respectively, and belonged to unstable protein. Hydrophobicity index of the FsLFY varied from-3.933to1.456, exhibiting hydrophilicity in general. The FsLFY did not contain signal peptide. Secondary structure prediction showed that the FsLFY contained31Helixs,30Strands, and34Coils.6. Cloning and informatics analysis of FsPI. Based on known626bp EST sequence for FsPI gene, specific primers were designed for3’RACE and5’RACE PCRs. Accoringly, the full-length cDNA of FsPI were obtained by assembling3’and5’teminal sequences. The FsPI sequences were subbmited to GenBank and the Accession No. was JN562739. The full-length cDNA of FsPI was891bp with5’ non-transcription region of6bp,3’non-transcription region of258bp and complete CDS of627bp, encoding208amino acids. FsPI exhibited characteristic structual of B type, MADS-box, ie., contained MADS domain, K domain, and PI domain. At DNA sequence level, the FsPI showed the lowest similarity with CrGLO1(66.7%), the highest with MqPI (93.0%). At amino acid sequence level, the FsPI showed the lowest similarity with CaPI (62.0%), the highest with MqPI (91.8%). The predicted molecular weight and isoelectric points of FsPI were24286.4Da and8.83, respectively, and belonged to unstable protein. Hydrophobicity index of the FsPI varied from-2.956to1.933, exhibiting hydrophilicity in general. The FsPI did not contain signal peptide. Secondary structure prediction showed that the FsPI contained25Helixs,21Strands, and20Coils.7. Cloning and informatics analysis of FsSEP1. Based on known338bp EST sequence for FsSEP1gene, specific primers were designed for3’RACE and5’RACE PCRs. Accoringly, the full-length cDNA of FsSEP1were obtained by assembling3’ and5’teminal sequences. The FsSEP1sequences were subbmited to GenBank and the Accession No. was JN562740. The full-length cDNA of FsSEP1was1120bp with5’ non-transcription region of83bp,3’non-transcription region of299bp and complete CDS of738bp, encoding245amino acids. FsSEP1exhibited characteristic structual of E type, MADS-box, ie., contained MADS domain, K domain, and SEP domain. At DNA sequence level,the FsSEP1showed the lowest similarity with AthSEPl (69.1%), the highest with EgM3(92.4%). At amino acid sequence level, the FsSEP1also showed the lowest similarity with AthSEPl (66.7%), the highest with EgM3(91.4%). The predicted molecular weight and isoelectric points of FsSEP1were28009.8Da and8.80, respectively, and belonged to unstable protein. Hydrophobicity index of the FsSEP1varied from-2.411to2.089, exhibiting hydrophilicity in general. The FsSEP1did not contain signal peptide. Secondary structure prediction showed that the FsSEP1contained23Helixs,25Strands, and26Coils.8. Expression pattern analysis of FsLFY. Fscalm (F. sellowiana Calmodulin) gene was isolated by RT-PCR and used as a reference gene for expression analysis of FsLFY gene. Semiquantitative RT-PCR analysis revealed that FsLFY gene was expressed in both reproductive organs and vegetative organs. In general, the FsLFY gene was strongly expressed in reproductive organs with significantly higher expression level was than in vegetative organs including fruit-bearing shoots, vegetative shoots, stems, tender leaves, and axillary buds. A stable high expression level was detected from April10to April22, whereas the expression level began to decrease with floral buds approaching to maturity after April26. In addition, FsLFY mRNA accumulation was slightly higher in fruit-bearing shoots and tender leaves than in the other three organs. The expession pattern of FsLFY suggests that it should play a role in both vegetative and reproductive development in F. sellowiana, but appears to be more closely related to reproductive development.更多还原