Cloning And Functional Analysis Of DGAT1 Gene In Euphorbia Lathyris

Diacylglycerol acyltransferase(DGAT)belonging to the acyltransferase superfamily,is a key and rate-limiting enzyme in the final step of triacylglycerol(TAG)biosynthesis reaction in plants.At present,the gene of encoding DGAT1 has been cloned and performed functional from plants,such as Nicotiana tabacum,Brassica napus,Glycine max,and Ricinus communis.Euphoria lathyris L.is a new energy oil crop with promising prospects.It is an ideal raw material for biofuel development in China and its fatty acid composition is similar to that of an ideal diesel substitute.Related studies on the gene sequence and biological function of the Euphorbia lathyris DGAT1 have not been reported.In order to investigate its regulatory mechanism underlying seed oil synthesis in Euphorbia lathyris,we isolated and identified a DGAT1 gene(named El DGAT1)by RT-PCR based on transcriptome data,the sequence characteristics and expression characteristics of the gene were analyzed.The yeast and plant expression vectors of El DGAT1 gene were constructed,and the biological function of El DGAT1 gene was identified by heterologous expression,which was a comprehensive explanation of the molecular mechanism of the synthesis and accumulation of the oil,and the expression mechanism of its key genes provides basic data,and also provides a basis for the bioengineering transformation application of genetic engineering technology for the key genes that Euphorbia lathyris the high level synthesis of the oil.The main findings are as follows:1.Based on the transcriptome data,the El DGAT1 gene sequence was obtained,with a full length of2376 bp,a CDS length of 1530 bp,encoding a total of 509 amino acids;El DGAT1 protein isoelectric point of 8.89,an instability coefficient of 51.25,a fat solubility coefficient of 101.67,and a hydrophilicity coefficient 0.284,presumed to be a unstable hydrophobic membrane-bound protein;the protein is localized on the endoplasmic reticulum,does not have a signal peptide,contains 9 transmembrane helix regions,has a specific matching domain PLN02401,belongs to the MBOAT membrane protein family;The two-dimensional structure is mainly alpha helices and random coil and the three-dimensional structure is similar to the crystal structure of the template 6 bug.1 membrane protein,and has a diacylglycerolacyltransferase functional region,which is presumed to have an acyl transfer function;multiple sequence alignments are found: the El DGAT1 protein has an important functional site for the acyltransferase catalytic activity;the phylogenetic tree indicates that the Euphorbia lathyris has the closest genetic relationship with the same plant Triadica sebifera,the homology is as high as 86 %,and the homology with Hevea brasiliensis and Manihot esculenta is second.2.The results of semi-quantitative PCR and q PCR showed that the El DGAT1 gene was expressed in different organs of the Euphoria lathyris,and the expression level was the highest in the seed,and gradually increased;the expression in the roots and leaves was lower than other organs,and the expression of the root is the lowest,almost no expression;the expression in stems and flowers is slightly lower than the S1 period.The El DGAT1 gene of Euphoria lathyris was successfully cloned by using the seeds of 45 d after the flowering of the seeds as the test material.3.The successfully constructed yeast expression vector was heterologously expressed in Saccharomyces cerevisiae.The presence of oil bodies in the transgenic yeast stained with Nile Red fluorescence was observed by fluorescence microscopy.The TAG was present in the defective yeast H1246 of transfection p YES2.0-El DGAT1 by thin layer chromatography(TLC).The total fatty acid content in the transgenic yeast was determined,the results showed that the total fatty acid content in the yeast transformed with El DGAT1 was significantly increased.Analysis of fatty acid composition and content of transgenic yeast and control yeast by GC gas chromatography showed that the contents of Palmitoleic acid(C16:1)and stearic acid(18:0)were significantly reduced,while palmitic acid(16:0),oleic acid(18:1)content is significantly increased.Compared with the transgenic yeast H1246 mutant strain,the content of palmitic acid(16:0)in the fatty acid content of the transgenic yeast mutant strain was increased by about 1.3 times,and the content of oleic acid(18:1)was increased by about 10 %.4.The successfully constructed plant expression vector p CAMBIA1303-El DGAT1 was transfected into Nicotiana benthamiana leaves for transient expression,and the total fatty acid content and fatty acid composition in the tobacco leaves after transient expression were determined and analyzed.It was found that compared with the transgenic tobacco leaves,The total fatty acid content in tobacco leaves of transgenic El DGAT1 was significantly increased;GC gas chromatography analysis showed that there were six main fatty acid components in transgenic tobacco leaves.Compared with the control group,the content of oleic acid(18:1)in the fatty acid composition of transgenic tobacco leaves was significantly increasedby about 1.4 times;the content of the four fatty acid components of palmitic acid(16:0),stearic acid(18:0),linoleic acid(18:2)and linolenic acid(18:3)was lower than that of the control group,and the difference was significant;while the peanut acid(20:0)was in the experimental group and the control group no significant change occurred.The protein and starch content of transgenic tobacco leaves were decreased overall.