摘要
油桐是我国特有的木本油料树种,所产桐油经济价值特高,广泛应用于高级油漆、涂料、油墨等制造业,又可供制生物柴油、树脂、人造橡胶、皮革、塑料、颜料及医药等。油桐种质资源是我国特有的基因资源,在基因表达水平开展对油桐品质及产量形成相关基因的研究,对油桐分子育种具有重要的理论意义和实践价值。本文以油桐对年桐品种为试材,构建种子cDNA文库、EST文库,并对EST序列进行全面分析,对重要基因全长cDNA克隆等方面进行了研究,主要结果有：
(1)油桐近成熟种子cDNA文库的构建。采用Unizol方法提取总RNA,经Oligotex mRNA kit分离并纯化得到mRNA,在SuperSciptTM ⅡRnaseH-Reverse Transcriptase等的作用下合成cDNA,与pBluescriptⅡSK(+)XR载体连接重组并转化感受态细胞DH10B,成功地构建了油桐对年桐近成熟种子的cDNA文库。文库总容量为8.53×106,重组率为88.95%,插入片段长度500-1500bp。构建的文库质量高,为进一步建立油桐EST文库、开展基因分离鉴定、制作油桐基因芯片和基因表达检测等奠定了良好基础。
(2)油桐近成熟种子EST文库的构建。以构建的油桐对年桐近成熟种子cDNA文库为材料,随机挑选3203个阳性克隆进行5’端测序,获得了具代表性的表达序列标签(EST)。经分析、整理,获得3202有效EST序列,平均长度554bp,500-700bp的ESTs占73.6%；在线CAP3拼接,共获得876个非冗余基因序列,其中重叠群202,单一EST序列674。重叠群的EST拷贝数从2个到106个不等,拷贝数为2的最多,达81个；拷贝数为3的有29个；拷贝数7个以上的出现频率多为1或2。油桐对年桐品种的EST文库系国内首次构建,为油桐功能基因组及遗传改良等的进一步研究提供了资源基础。
(3)油桐EST序列功能注释。3202个有效EST序列中,1954个为功能已知基因序列,423个为功能未明确的基因序列,760个为相似性较低的基因序列,44个为没有重要相似性的基因序列,21个非植物类基因序列。1954个功能已知的基因序列中,非冗余基因序列467个,包括重叠群序列176个,单一序列291个,涉及379种功能基因；其中与油桐本种同源的序列133个,与同属千年桐同源的序列349个；与大戟科变叶木等11种植物同源的序列1347个。与其他科属植物同源的序列125个。
(4)油桐种子EST文库已知功能基因表达分析。油桐近成熟种子表达的379个已知功能基因归为物质代谢、能量代谢、细胞生长发育、转录、蛋白质合成、蛋白质定位与贮藏、载体、细胞结构、信号转导、抗病与防卫、次生代谢及未分类基因等12大类,蛋白质合成相关基因的表达最丰富,占49.6%,蛋白质定位与贮藏蛋白相关基因占21.9%,抗病和防卫相关基因占8.1%；其他各类的相关基因表达量均不足5%。说明材料采集时油桐种子正处蛋白质合成与贮藏高峰期。其中与脂肪酸代谢相关的功能基因有Δ12油酸脱饱酶(FAD2)、B-酮脂酰ACP合成酶(KASI)、烯脂酰基酰基载体蛋白还原酶(EAR)、参与柠檬酸穿梭的苹果酸脱氢酶、2,4-二烯脂酰CoA还原酶、3-酮脂酰辅酶A硫解酶、脂肪酸β一氧化多功能蛋白等,其中Δ12油酸脱饱酶(FAD2)基因属高丰度表达。与油脂贮藏相关的油体蛋白Oleosin基因高丰度表达。与贮藏蛋白相关的基因有蛋白A3前体、豆球蛋白B前体、11S球蛋白β亚基前体、豆球蛋白类蛋白、种子贮藏蛋白等,多为高丰度表达。与抗性相关的基因35种,主要有金属硫蛋白、热激蛋白、亲环素、脱水素、翻译控制肿瘤蛋白、富含脯氨酸蛋白、谷胱甘肽过氧化物酶等高丰度表达的基因。与能量代谢相关的基因主要有果糖二磷酸醛缩酶、磷酸甘油酸激酶、磷酸葡萄糖变位酶、二氢硫辛酰胺脱氢酶、丙酮酸脱氢酶、琥珀酸脱氢酶等低丰度表达的基因。与蛋白质合成相关基因64种,主要有18S核糖体RNA、26S核糖体RNA、小亚基核糖体RNA、30S核糖体蛋白、40S核糖体蛋白、50S核糖体蛋白、60S核糖体蛋白、翻译起始因子等；与氨基酸代谢相关的序列4条。与蛋白质结构相关基因17条,主要有前折叠素相关蛋白、蛋白质二硫键异构酶、泛素等。与信号转导相关的基因序列32条,涉及23种功能基因。与基因转录相关的基因序列36条,涉及24种功能基因。与次生代谢相关基因23种44条序列。与物质转运相关基因序列23条。
(5)油桐FAD2基因的全长cDNA克隆。所克隆的FAD2基因序列长1537bp,含长度为1146bp的完整编码序列,编码383个氨基酸。其氨基酸序列有3个多变区,N端的起始段6个氨基酸为信号肽序列,3个组氨酸簇高度保守。酶蛋白分子量44144.4Da,等电点p18.57,有5个跨膜结构域,说明FAD2为膜结合酶,在N端、C端及中间各有一强亲水区,为膜外结构区；中间若干区段为疏水区,为膜内结构区,组氨酸簇位膜表面,二级结构中α螺旋主要位于肽链C端,β折叠链较短,卷曲主要位于肽链N端。
(6)油桐核糖体蛋白及rRNA基因的全长cDNA克隆。60S核糖体蛋白L8的基因序列长1086bp,含有长783bp的完整编码序列,编码261个氨基酸。氨基酸序列近N端和近C端高度保守,C端尾部变化较大。酶蛋白分子量28175.3Da,pI11.44,为碱性蛋白。N端、C端各具强亲水性,中间则亲水区与疏水区相间。二级结构上,卷曲主要位于肽链的中部,β折叠链较短,α螺旋较少,主要位于N端和C端。18SrRNA基因序列全长2523bp,26SrRNA基因序列长2933bp。不同产地和不同生存环境的物种,其rRNA基因有不同的碱基组成,亲缘关系越近的物种间碱基相似性越大,构建rRNA基因进化树是检测物种基因同源性和鉴别物种有力的辅助工具。26SrRNA和18SrRNA分别参与到核糖体大小亚基的自组装形成中,rRNA分子内有大量的反向互补序列,形成大量局部双链区并自卷曲和折叠而形成稳定的“茎环状”结构。rRNA分子结构有大量链内双链区,双链间有较小的内凸环,侧凸环成链的拐点,较大环则成为双链定向的环岛,双链末端为发夹结构,呈长臂状的“吸臂”,这有利于rRNA分子与核糖体蛋白结合形成结构稳定的核糖体亚基。
Vernicia fordii is peculiar arboreal oil tree in China, its product the tung oil has high economic value, widely applies in high-quality paint, coating, printing, ink and otherwise in manufacturing industries, and supplys to produce biology diesel oil, resin, synthetic rubber, leather, plastic, pigment and medicine etc. The germplasm resource of V. fordii is a unique genetic resource in China, carrying out the relate genes reseach about the quality and yield formation in gene expression level, it has important theoretical significance and practical value on molecular breeding of V. fordii. Taking the seeds of'Duinian tung' oil tree as the experiments material in this thesis, had constructed cDNA library and EST library of seeds, and carried on the overall analysis about the EST sequences, and reseached the full-length cDNA clone of the important gene. The main result includes:
(1) Construction of the cDNA library with near-mature seeds of V. fordii. Using the Unizol method to extract total RNA, obtaining the mRNA after separation and purification by Oligotex mRNA kit, synthesize cDNA under the SuperSciptTM Ⅱ RnaseH-Reverse Transcriptase, then recombination with the vector pBluescript Ⅱ SK (+)XR and transformation competent cell DH10B, have successfully constructed the cDNA library of near-mature seeds of'Duinian tung' oil tree. The capacity of cDNA library has8.53×10clones, the recombination rate is88.95%, the size of insertion segment between500-1500bp. Constructing the high quality library has laid a good foundation to further construct the EST library, separate and identify the gene, make the gene chip and detect the gene expression of V. fordii.
(2) Construction the EST library with near-mature seeds of V. fordii. Taking the cDNA library of near-mature seeds of V. fordii as material, choosing3203positive clones randomly to sequence at5'end, have obtained the representative expression sequence tags(EST). After analysis and classification, obtaining3202effective EST sequences, the average length is554bp, the ratio of ESTs between500and700bp was73.6%; Carrying on CAP3online splicing, obtaining876non-redundant gene sequences altogether, which consist of202contigs and674singletons. In202contigs, the copies of EST from2to106, the contigs which contain2copies are most, reaches81; and contain3copies have29; and contain more than7copies appear1or2. The EST library of'Duinian tung'oil tree is constructed interiorly for the first time, which provide the resource to further research the function genome and heredity improvement for V. fordii.
(3) Annotation the function of the EST sequence of V. fordii. In3202effective EST sequence, there are1954sequences which function has known,423sequences which function is ambiguous,760sequences which function is low similarity,44sequences which function is no significant similarity,21sequences are non-plant gene. In1954gene sequences which function is known, have467non-redundant gene sequences, consisting of176contigs and291singletons, which relate to379kinds function genes. There are133homologous sequences with V. fordii;349homologous sequences with V. montana;1347homologous sequences with11species of Euphorbiaceae as Codiaeum variegatum.125homologous sequences with other family plants.
(4) Analysis the gene expression of known function in EST library of V. fordii. The379known function genes of near-mature seeds of V. fordii are classified in12classes, include material metabolism, energy metabolism, cell growth/division, Transcription, protein synthesis, protein destination and storage protein, transporters, cell structure, signal transduction, disease/defence, secondary metabolism, and unclassified gene. The quantity of gene expression related to protein synthesis is richest to49.6%, the gene related to protein destination and storage protein is21.9%, disease/defence gene is8.1%. The quantity of gene expression is insufficient5%in other classes. It was the peak time of protein synthesis and protein storage at the seeds of V. fordii was gathered as experiment material. The function genes in EST library relate to fatty acid metabolism have△12oleic acid desaturase (FAD2), beta-ketoacyl-ACP synthase I (KASI), enoyl-acyl-carrier-protein reductase (EAR), malate dehydrogenase which take part in citriate shuttle,2,4-dienoyl-CoA reductase,3-ketoacyl-CoA thiolase B, glyoxysomal fatty acid beta-oxidation multifunctional protein and so on. The FAD2gene belongs to high abundance expression. The Oleosin genes relate to the oil storage are high abundance expression. The genes relate to storage protein includes glutelin type-A3precursor, legumin B precursor,11S globulin subunit beta precursor, legumin-like protein, seed storage protein and so on, are high abundance expression. The genes relate to resistance of disease/defence have35kinds, the high abundance expression genes include metallothionein, heat-shock protein, cyclophilin, dehydrin, translationally controlled tumor protein, proline-rich protein, glutathione peroxidase and so on. The genes relate to energy metabolism are low abundance expression, mainly include fructose-bisphosphate aldolase, phosphoglycerate kinase, phosphoglucomutase, dihydrolipoamide dehydrogenase, pyruvate dehydrogenase, succinate dehydrogenase and so forth. The genes relate to protein synthesis have64kinds, mainly include18S rRNA,26S rRNA, small subunit rRNA,30S ribosome protein,40S ribosome protein,50S ribosome protein,60S ribosome protein and eukaryotic translation initiation factor.4sequences relate to amino-acid metabolism. The gene relate to protein structure have17kinds. Mainly include prefoldin-related KE2-like protein, protein disulfide isomerase, ubiquitin and so on. The gene relate to signal transduction have23kinds (32sequences), and transcription24kinds(36sequences), secondary metabolism23kinds (44sequences), material transportation23sequences.
(5) Cloning of the full-length cDNA of FAD2of V. fordii. The sequence length of the FAD2gene clone is1537bp, contains the complete CDS its length1146bp, that code383amino acids. There are3changeable sectors at amino acid sequence of FAD2. The initial sector which contain6amino acids at N end is the signal peptide sequence of transmembrane, the3histidine clusters is highly conservative. The molecular weight of zymoprotein is44144.4Da, isoelectric point is8.57. There are5transmembrane structure domain, that explaine FAD2is the membrane-conjugating enzyme, There are strongly hydrophilic section at N end, C end and center-section respectively, which is structure outside the membrane; The middle sectors are some hydrophobic section, which is the section inside the membrane. The histidine clusters is located at membrane surface. In secondary structure of the protein, the helices is mainly located at the peptide chain C end, the strands is short, the coils is mainly located at the N end of peptide chain.
(6) Cloning of the full-length cDNA of ribosome protein and rRNA of V. fordii. The sequence length of60S ribosome protein L8gene is1086bp, which includes the complete CDS its length783bp, codes261amino acids. It is highly conservative near N end and near C end, but change in rear part of peptide chain at C end. The protein MW28175.3Da, pI11.44, which is an alkalinity protein. The N end and C end of protein is strongly hydrophilic respectively, hydrophilic section and hydrophobic section is alternate at middle. In secondary structure of protein, the coils is mainly located at middle of peptide chain, the strands is short, the helices are few, mainly located at N end and C end. The full-length sequence of18SrRNA gene is2523bp, the26SrRNA gene sequence length is2933bp. The species which come from different habitat and different living environment have different base composition in rRNA gene. The nearer the relationship of the species, the smaller the change at base, the bigger the similarity of rRNA. Constructing the evolution tree of rRNA gene is one of powerful auxiliary means to detect the homology of species gene and differentiate the species.26SrRNA and18SrRNA participate into the self-assembly process of the ribosome large and small subunit respectively. The abundance reverse supplementary sequences in rRNA bring on formation of massive double strand, which curl and fold to form the stable secondary structure as'stem ring' shape. There is lots intrachain double strands in rRNA molecule, the small ring in jog is between the double strands, the ring side jog is the flex point of the strands, the big ring is the rotary island that direction double strands, the terminal of double strands show hairpin structure as long brachial'drag arm'in periphery, that is advantage to form the stable structure of ribosome subunit with rRNA molecule and ribosome protein.
引文
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