Multi-gene Vector Assembly And Co-expression Analysis Of FatB3,LPAAT And KASI From Coconut

Coconut(Cocos nucifera L.),an important oil-yielding tropical crop,contains 93%saturated fatty acid,60% medium fatty acid and 50% lauric acid in endosperm which has been becoming one of the best subjects to study medium fatty acid synthesis.Based on the previous results in our laboratory study,the research used three functional genes that paly one important role in medium-chain fatty acid synthesis of coconut endosperm: β-ketoacyl ACP synthetase I(KASI),lysophosphatidic acid acyltransferase(LPAAT)and fatty acid acyl carrier protein thioester(FatB3)for building one FatB3-LPAAT,one FatB3-KASI and one FatB3-LPAAT-KASI by Cre/LoxP multi-gene assembly and transformation vector system.Then three multi-gene systems were seed-specific expressed in wild Arabidopsis thaliana for further analyzing three genes co-expression effect in medium-chain fatty acid synthesis by detecting gene expression and comparing the content of fatty acids in transgenic seeds.Multi-gene co-expression vectors were constructed by using Cre/LoxP multi-gene recombination vector system.The expression modules of promoter/Gene/terminator were constructed for each gene.The seed-specific promoter Napin was selected for FatB3,At5400 and At16460 that expressed uniquely in embryo during seed development in Arabidopsis thaliana were selected for LPAAT and KASI.The NOS terminators of three genes were derived from common plant expression vector pCAMBIA1300 S.The FATB3 expression system and the LPAAT expression module were recombination to construct dual-gens coexpression system FatB3-LPAAT.Then the FatB3 expression system and the KASI expression system module were used to reassemble for the dual-genes co-expression system FatB3-KASI.Similarly,The FatB3-LPAAT expression system and the KAS expression module were used to produce the triple-genes co-expression system FatB3-LPAAT-KASI.Three different types transformation vectors were transformed into Arabidopsis thaliana by floral dip transformation method for functional verification.The T1 generation Arabidopsis thaliana leaf genomic DNA were extracted for PCR positive validation.The positive transgenic seeds total RNA was extracted for detecting each gene expression by the fluorescence quantification PCR.The lines with high expression were screened for the further fatty acid analysis.The fatty acid analysis of transgenic seeds was performed.Analysis of fatty acid in T3 homozygous transgenic seeds showed: 1.The C12:0(relative percent content)of transgenic FatB3-LPAAT,transgenic FatB3-KASI and transgenic FatB3-LPAAT-KASI increased by at least 395%,124% and 134% compared with wild type seeds;Similarly,the C14:0 of FatB3-LPAAT,FatB3-KASI and FatB3-LPAAT-KASI also increased by at least 383%,102% and106%,and it was no significant change in other types fatty acids.2.FatB3-LPAAT transgenic seeds increased 92% and 58% in C14:0 contrast to FatB3-KASI and FatB3-LPAAT-KASI transgenic plants seeds.Analogously,FatB3-LPAAT-KASI transgenic seeds increased 19%and 116% compare with FatB3-LPAAT and FatB3-LPAAT-KASI in C12:0.These results showed that the FatB3-LPAAT was expressed better than the other two systems in plants.3.The total fatty acid content of FatB3-LPAAT,FatB3-KASI and FatB3-LPAAT-KASI were increased by at least 47%,70% and 56% compared with wild seeds.The total fatty acid content of three types transgenic system was not significant difference.The result displayed that FatB3 and LPAAT played the main part in the fatty acid synthesis and accumulation while KASI did not play an important role in changing fatty acid composition or increasing total fatty acid content.In conclusion,the study built three types multi-gene systems FatB3-LPAAT,FatB3-KASI and FatB3-LPAAT-KASI by Cre/LoxP recombination system and transferred them into Arabidopsis thaliana for seed-specific co-expression.The preliminary analysis three genes combinations expression trend.This study can provide one possible solution for getting more medium-chain fatty acid in plants and afford some basis theoretical for the application of fatty acid metabolism engineering in further.