Cloning And Functional Analysis Of FATB Gene Of Litsea Pungens

Medium-chain fatty acids MCFAs,including octanoic acid?8:0?,decanoic acid?10:0?,lauric acid?12:0?,and myristic acid?14:0?,is an important material for industrial production of detergents,soaps,cosmetics,surfactants and lubricants.At present,the production of MCFAs can not meet our growing demand.The key enzymes from different plants especially in Cuphea which regulating MCFAs have been transformed into crops through genetic engineering in foreign countries.However,these studies are still in the initial stage in China,where many seeds contain MCFAs in Lauraceae.To make good use of these resourses,Litsea pungens,which rich in about 50%lauric acid and 20%decanoic acid in kernel,was a good material for discovering the key enzymes account for improving MCFAs in plant.During de novo fatty acid synthesis,FATB acyl-acp thioesterase FATB is the key enzyme to control the carbon chain length of fatty acid.One FATB was cloned from kernel of Litsea pungens by homologous cloning.Then,the LpFATB gene overexpression vector was constructed and transform into Arabidopsis thaliana wild type.Functional analysis of cloned gene was carried out in transformed lines.In order to further explore the mechanism of MCFAs accumulation,we analyzed the transcriptome of the kernel in the middle and late stage of development in Litsea pungens.The main results of this study are as follows:1)A FATB gene was cloned from Litsea pungens with a total length of 1257bp.Through comparison with the fatty acid-acyl ACP thioesterase of known species,it was found that the cloned genes were clustered into FATB group instead of FATA,and the amino acid 166Q,196W,198V,225G,226K,227N,228G in our FATB,together constitute the active site of thioesterase,called 4HBT.2)The LpFATB overexpression vector,which constructed by enzyme digestion connection technology was transformed into wild-type arabidopsis Col-0,and the fatty acid content of T₃ generation positive transformed seeds were analysed by gas chromatography,the content of palmitic acid?16:0?was obviously improved,oleic acid and eicosenoic acid were reduced in a certain degree.Compared with untransformed arabidopsis wild-type Col-0,the ratio of palmitic acid was increased by 2-3 times and the highest was 25.3%.This result indicates that the LpFATB gene is more favorable to the substrates of palmitate-ACP than that of laurate-ACP in the de novo fatty acid synthesis pathway.3)In order to explore whether the FATB obtained is the only one in the kernel,Illumina sequencing was used to sequence the transcriptome of the seed embryo tissue in the middle and later stages of the development of Litsea pungens,and 38897 high-quality Unigene sequences were finally obtained.By comparing and analyzing the gene sequences of Unigenes and the known model plant arabidopsis thaliana,we found that 247 Unigenes were involved in the lipid metabolism pathways?including fatty acid synthesis,triglyceride assembly,and enzymes and transcription factors related to lipid metabolism?.4)The protein sequences were analyzed between LpFATB and California bay UcFATB,we found that the N-terminal sequence of the protein between two sequences is bigger,which maybe the cause of functional differences.So we use structure swap methods to replace N-terminal sequence of Litsea pungens into California bay N-terminal sequence,but the result is similar with LpFATB.The results showed that the difference of N-terminal sequence between Litsea pungens and California bay may not be determining factor of substrate difference.Moreover,different N-terminal sequences may affect the activity of FATB.