| 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. |
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