Cloning And Function Analysis Of KASI、FatB、LPAAT From Coconut Endosperm

Coconut （Cocos nucifera L.）, an important oil-yielding tropical plantation crop, contains large amounts of medium chain fatty acids （MCFAs） in endosperm which has always been becoming the hotspot research in recent years. In this study, genes for KASI, FatB, LPAAT were cloned from coconut （Cocos nucifera L.） endosperm. Then, the bioinfomatic analyses of these genes were made. And we detected the expression of these genes during fruit development of coconut by Real-time fluorescent quantitative PCR. To confirm the functions of these genes in plant, the coding regions of these genes were cloned into the binary pCAMBIA1300S vector under the control of the seed-specific napin promoter, and were subsequently overexpressed in Nicotiana tobaccum following Agrobacterium tumefaciens-mediated transformation.The sequence analyses revealed that KASI （1869bp）, designated CocoKASI （GenBank Accession:JX275887）, contained an open reading frame （ORF） of1485bp, encoding a polypeptide of494amino acids （nucleotide positions134-1618）, which shared high sequence indentity of90%to KASI （XP₀02265207.1） from Vitis vinifera. Then, FatB （1938bp）, termed CocoFatBl （GenBank Accession:JX275886）, contained an ORF of1254bp, encoding a polypetited of417amino aicds （nucleotide positions356-1609）, which showed100%（JF338903） and95%（AAD42220.2） sequence similarity to CnFatBl and oil palm（Elaeis guineensis） acyl-ACP thioesterases, respectively. And LPAAT （1325bp）, termed CocoLPAAT, contained an ORF of927bp, encoding a polypetited of307amino acids （nucleotide positions259-1185）, which was the same with LPAAT （XP₀02522947.1） from coconut endosperm.Real-time fluorescent quantitative PCR analysis indicated that the CocoKASI transcript was most abundant in the endosperm during the fruit development while leves had～40%of this level. And the CocoFatB1transcript was most abundant in the endosperm from8-month-old coconuts while leaves and endosperm from15-month-old coconuts had～80%and～10%of this level. The CocoLPAAT transcript was most abundant in the leaves but both immature and mature endosperm during the fruit development displayed relatively low expression level.Analysis of the fatty acid composition of tobacco seeds showed that expression of transgenic CocoKAS1in tobacco seed increased levels of caproic acid （6:0）, caprylic acid （8:0） and capric acid （10:0） by at least100%,100%and150%compared with levels observed in untransformed tobacco plants, respectively. And the content of lauric acid （12:0）, myristic acid （14:0） and palmitic acid （16:0） displayed the increasing tendency.And the levels of myristic acid （14:0）, palmitic acid （16:0） and （stearic acid）18:0in CocoFatBl transgenic plants were increased by at least25.0%,34.3%and17.9%compared with levels observed in untransformed tobacco plants, respectively. These results indicated that CocoFatBl acts specifically on14:0-ACP,16:0-ACP and18:0-ACP and had an obvious impact on fatty acid composition in plants.Expression of transgenic CocoLPAAT in tobacco seed increased levels of caproic acid （6:0）, caprylic acid （8:0）, capric acid （10:0） and stearic acid （18:0） by at least66.7%、66.7%、100%and10.2%compared with levels observed in untransformed tobacco plants, respectively. And the content of palmitic acid （16:0） displayed the increasing tendency. In contrast, the levels of lauric acid （12:0） and myristic acid （14:0） in transgenic seed were reduced by different degrees. It was hypothesized that CocoLPAAT can’t work as described in coconut endosperm that catatlyzing12:0-CoA into sn-2of triacylglycerol in plants lacking lauric acid.In conclusion, we have cloned three genes involving the synthesis of medium chain fatty acids from coconut endosperm and verificated their functions in plants preliminarily. The study will be of value in efforts to engineer fatty acid metabolism in crop improvement programs.