The Study On Cloning And Function Of Seven Genes Involved In The Fatty Acid Metabolism Of Camellia Oleifera Seeds

As a principal species of woody oil plants in China, Camellia oleifera is one of the four most famous edible oil woody plants including Elaeis guineensis, Olea europaea and Cocos nucifera, which are planted in acidic red soil areas in the South Downs.Camellia oil is mostly composed of unsaturated fatty acids （UFA）, such as oleic acid and linoleic acid, and the average content of UFA is above90%, So Camellia oil is a high quality edible oil. Druing the fatty acid metabolism process of Camellia oleifera seed, there are many enzymes and genes to regulate the process ofphysiological and biochemical metabolic, such as the lipids biosynthesis’s enzymes including malonyl-CoA:ACP transacylase and3-hydroxyacyl-CoA dehydratase, the fatty acid β-oxidation’s enzymes including acyl-CoA dehydrogenase/multifunctional protein and acyl-CoA thioesterase, the stress resistance’s enzymes including aldehyde dehydrogenase and fatty acid hydroperoxide lyase.In this paper, state trial oil-tea variety ’Huashuo’as material, on the basis of the digitized transcriptome and expression profiling database, the full-length cDNA and bioinformatic analysis of seven genes of Camellia oleifera were carried out. Expression vectors and RNA interference vectors were constructed,then were transformed into host cells to express. The correlation between the three genes（acyl-CoA dehydrogenase, aldehyde dehydrogenase and fatty acid hydroperoxide lyase） relative expression abundance and different developmental period of Camellia oleifera seeds was analyzed. The main results are as follow:1. Seven genes cloning and bioinformatics analysis of Camellia oleifera.According to the analysis of transcriptome, designed degenerate or specificprimers respectively,combined the technology of RT-PCR and RACE, seven genes’full-length cDNA were cloned:（1） The full-length cDNA of CoMCAT gene was found to be1867bp,GenBank accession number forKJ910337. This sequence had an open reading frame of1131bp, encoded376amino acid residues. The molecular mass of the CoMCAT protein was39.9797kDa,with theoretical pI of6.84and targeting sequence of58amino acid residues,and it contained two highly conserved motif, which were’GQGXQ’and’GXSXG’,locating in malonyl-CoA binding site and ACP binding site respectively;（2） The full-length cDNA of CoHCD gene was found to be1145bp and its open reading frame was666bp, encoded221amino acid residues,GenBank accession number forKJ910336. The molecular mass of the CoHCD protein was25.2045kDa,with theoretical pI of9.40and hydrophobic residues’rate of48.9%,showing a hydrophilic protein.CoHCD proteinhas four obvious transmembrane domains and contained a protein tyrosine phosphatase motif HGXXGXXRS’;（3） The full-length cDNA of CoACAD gene was found to be2702bp and its open reading frame was2487bp, encoded828amino acid residues,GenBank accession number forKJ910338. The molecular mass of the CoACAD protein was94.4113kDa with theoretical pI of8.47. CoACAD proteinhas two obvious transmembrane domains and contained a typical tyrosine protein kinase （PTK） active site’LVHGDFRIDNLVF’,whose structure domain were protein kinase domain,aminoglycoside phosphotransferase（APH） domain,ACAD_N domain, ACAD_C domain and ACAD Center domain;（4）The full-length cDNA of CoMFP gene was found to be2541bp and its open reading frame was2178bp, encoded725amino acid residues,GenBank accession number forKJ910342. The molecular mass of the CoMFP protein was79.0333kDa with theoretical pI of9.11. CoMFP proteinhas six obvious transmembrane domains.CoMFP protein not only contained highly conserved sequences,such as a ECH signal sequence’VAAIDGLALGGGLEVAMAC-HA’,aHCDH signal sequence’NCTGFAVNRMFFPYTQAAILLVERGV, a ECH motif-N25-P26-P27’,a phosphoric acid binding motif319G-X-G321-X-X-G324’,but possessed highly conserved salt bridge "’R500Nε and E638Oε’and’R500Nη1/η2and E645Oε1/ε2’" and active site’S428、H449、E461、N499、D523’, whose structure domain were ECH domain,3HCDH_N domain,NAD （P） binding Rossmann-fold domain,3HCDH_C domain and phosphogluconic acid dehydrogenase binding domain;（5） The full-length cDNA of CoACOT gene was found to be1588bp and its open reading frame was1164bp, encoded387amino acid-residues,GenBank accession number forKJ910339. The molecular mass of the CoACOT protein was43.5478kDa with theoretical pI of7.14. CoACOT protein not only contained a conserved sequences of cNMP₁binding domain’VVREGEAGDGVYFIWDG’,but possessed highly conserved triple peptides of peroxidase signal sequence’SKL’in the C-terminal, whose structure domain were cyclic nucleotide binding domain and Similar RmlC-jelly roll fold binding domain;（6） The full-length cDNA of CoALDH gene was found to be1809bp and its open reading frame was1506bp, encoded501amino acid residues,GenBank accession number forKJ910340. The molecular mass of the CoALDH protein was54.5137kDa with theoretical pI of5.50. CoALDH proteinhas three obvious transmembrane domains and contained a cytoplasmic transit peptides of36amino acid residues. CoALDH protein not only contained three highly conserved structural domain of the aldehyde dehydrogenase gene families,including a glutamic acid activation site’LELGGKSP’, a cysteine activation site’LYNKGEICVAGS’and a coenzyme binding site’GFGPTAG’,but possessed a highly conserved sequences sequences including a decapeptide’VSLELGGKSP’,’FTGSTE’,’GPWPR’and’IIPM-NFP’andso on, whose structure domain was aldehyde dehydrogenase domain, belonging to the aldehyde dehydrogenase family depended on the NAD;（7） The full-length cDNA of CoHPL gene was found to be1648bp and its open reading frame was1476bp, encoded491amino acid residues,GenBank accession number forKJ910341. The molecular mass of the CoHPL protein was54.8581kDa with theoretical pI of8.04. CoHPL proteinhas five obvious transmembrane domains and contained a chloroplast transit peptides of38amino acid residues and rich in proline. CoHPL protein contained four highly conserved structural domain of the Cytochrome P450protein families,including a A domain（I coiled coil region）’LFMLGFNAYGGY-SIF’, B domain（K coiled coil region）’LSFDSVKEMELVKSFVYETLRLNPP’, C domain’RDSKVFDDPEKFIFDRFTKEK’and D domain（Hemecombination zone）’P-SESNKQCAAKDYVTLACL’.2. Prokaryotic expression of Seven genes from Camellia oleifera.The recom-binant expression vector,including pET30a-MCAT, pET30a-HCD, pET30a-ACAD, pET30a-MFP,pET30a-ACOT,pET30a-ALDHand pET30a-HPL, were conducted successfully by the pET30a and BL21（DE3） prokaryotic expressionsystem,and were overexpressed the target product,whose apparent molecular weight were approxim-ately40kD、25kD、93kD,79kD、44kD、55kD and55kD respectively.3. The relative expression abundance of Co ACAD, CoALDH and CoHPL genes of different developmental period from Camellia oleifera. The relationship between the relative expression abundance of CoAC AD, CoALDH and CoHPL and thirteen different developmental stages of Camellia oleifera seed had been analyzed by the real-time quantity PCR technology.（1）The expression of CoACAD were three peaks, which were the highest in the early of August,the second at the end of September and next in the middle of October.（2）The expression peaks of CoALDH were the highest in the middle of October; next in the early and middle of August and at the end of September,they are almost the same.（3）The efficient expression of CoHPL were mainly in August, and the highest was in the early of August.4. Constructing the eukaryotic expression vector and selecting the vector of CoALDH and CoHPL genes to transform. The eukaryotic expression vector （overexpression vectors and RNA interference Gateway vector） of CoMCAT, CoHCD, CoACAD, CoMFP, CoACOT, CoALDH and CoHPL were constructed.The CoALDH and CoHPL vectors had been transformed to wild Arabidopsis thaliana and the TO and T1transgenic Arabidopsis thaliana had been obtained by the filter of antibiotic and the test of PCR technology.