Construction And Characterization Of BIBAC Library Of Jatropha Curcas L. And Identification Of Microsomal ω-3Fatty Acid Desaturase

Jatropha curcas L., a member of the Euphorbiaceae, is bio fuel crop and widely distributed in tropical and subtropical areas all over the world. The Jatropha kernel contains high amounts of lipids which with21%saturated fatty acid and over70%unsaturated fatty acid and its seed oil with low viscosity, that it has been considered as one of the substitutes for fossil fuels. In order to investigate Jatropha curcus genome, gene cloning and characterization, physical mapping and large-scale genome sequencing of Jatropha L., Large-insert DNA libraries are fundamental and useful tools and platforms. Genetic engineering technology is one of the effective ways to alter seed oil composition and breed new varieties. Microsomal ω-3fatty acid desaturase (FAD3) of plant is a member of fatty acid desaturase family and catalyze the reaction of linoleic acid to linolenic acid, studies shown that FAD3protein is the major ω-3desaturase in non-photosynthetic tissues and it accounts for nearly80%of C18:3syntheses. Therefore, the study of Jatropha microsomal ω-3fatty acid desaturase will not only lead to a better understanding of seed oil syntheses but also provide the thesis for altering the Jatropha seed oil composition through genetic engineering.The BIB AC library of Jatropha L. which constructed by BamH I with the vector pCLD04541, consisting of30,720clones.214clones in the library have been random sampled and analyzed. Results showed that the library has an average insert size of131.9kbp and more than80%of the clones had inserts larger than100kb, which ranged from141to160kbp. As7.9%of its clones are estimated to none inserts, the BIBAC library had approximately28,293clones containing Jatropha L. genomic DNA inserts. Since the haploid Jatropha L. genome is around416Mbp, the library has a genome coverage equivalent to approximately8.9x haploid genomes and provides more than99%probability of identifying any single-copy gene. Five BIBAC clones were randomly picked and analyzed their stability. Results revealed that the insert DNA was stable in the vector for long terms of cultivation. A subset (8.0x) of the Jatropha L. BIBAC library double-spotted onto three high-density nylon filters, containing27,648clones in total with9,216clones on each filter, representing90%of the whole library. Then the filters were screened by overgos designed nine fatty acid metabolism related genes in Jatropha (FAD3, FAD6, FAD7, ACCase, KASâ… , KASâ…¡, KASâ…¢, acyl-ACP thioesterase and chloroplast acyl-ACP thioesterase). The filters Hybridization results showed that from one to fourteen positive clones were identified for each gene, except acetyl-CoA carboxylase. A total of forty-two positive clones were identified, with an average of5.3positive clones per gene. This result indicates that the Jatropha BIBAC library is suitable for gene cloning and a powerful tool for physical map construction and genome sequencing.The Southern result of nine fatty acid metabolism related genes in Jatropha shows that all of the nine genes are single copy in Jatropha genome.Positive clones of clone22G19which contains FAD3gene and clone64D4which contains FAD7gene in Southern hybridization had been sequenced and data spliced, and NCBI blast shows that FAD7is2655bp, containing8exons and7introns and encoding525amino acids. The transcriptional start site is base G locating at-385bp and promoter area is-1730--385bp. Analysis of cis-acting element of FAD7promoter shows that FAD7has many light responsiveness elements, others were heat stress and low-temperature stress resposiveness, drought-inducibility and endosperm expression cis-acting elements. FAD3is1797bp, containing8exons and7introns and encoding377amino acids. The transcriptional start area is-93～-23and the start site is base A locating at-54bp. Analysis of cis-acting element of FAD3promoter shows that cis-acting elements in FAD3are mainly involved in light responsiveness and, defense and stress responsiveness, anaerobic induction, zein metabolism regulation and heat stress responsiveness.Jatropha FAD3gene was cloned from Jatropha leaves, it was named JcFAD3with an open reading fame of1134bp which encoding377amino acid residues with the predicted molecular mass of41.1kD. The deduced amino acid sequence showed high identities with FAD3from Vernicia fordii, Populus tomentosa, Ricinus communis, Glycine max and Linum usitatissimum by homologous sequences blast. The phylogenetic analysis of various FAD3indicated that JcFAD3was close to FAD3from Vernicia fordii. The structural analysis of FAD3protein shows that FAD3has Deltall2-like domain, Membrane-FADS domain and three conserved His motifs which are located at94(HDCGH),130(HRTHH) and297(HVIHH), respectively.The expression patterns of JcFAD3in different tissue of Jatropha were investigated by real-time PCR which showed that JcFAD3was expressed in all the organs of Jatropha and the expression level was the highest in the root and the lowest in the leaves. During the development of Jatropha seed, JcFAD3in the seed was expressed decreasingly in the early and middle stage and increasingly in the late stage. The amount of trienoic fatty acids in the cell are the direct result response to temperature in plant. After exposing Jatropha at4℃and40℃, respectively, it was found that JcFAD3expressed at different temperature and its expression was increased at low temperature while decreased at high temperature.INVScl-pYes2.0yeast transformation system was used to study JcFAD3gene expression in yeast cell and then the fatty acid contents in transformed yeast cell with empty vector and JcFAD3were also investigated by GC analysis, the result showed that a novel fatty acid methyl ester peak corresponding to linolenic acid methyl ester standard was detected in the yeast cell transformed with JcFAD3, which was absent in the yeast cell containing empty vector. This indicated that JcFAD3gene was expressed correctly and JcFAD3protein catalyzed linoleic acid to linolenic acid in yeast cell.The expression vector pVKH-JcFAD3was constructed based on the character of pVî™'H-35S-GUS and JcFAD3gene and then transformed into wild-type Arabidopsis by Agrobacterium-mediated method.5transgenic plants with JcFAD3were obtained by PCR analysis. The fatty acid was isolated from wild Arabidopsis leaves and transgenic Arabidopsis showed there proportions were21%and37.5%in total fatty acids respectively which detected by GC analysis.