| Cyperus esculentus is a special economic oil crop.Its underground tuber tissue has a storage oil content of 25%-30% of dry weight,and is rich in high-quality oleic acid(60%-75%).As the only known oil-rich tuber plant today,C.esculentus is considered as an ideal model for studying lipid metabolism and its regulation mechanism in non-seed organs or vegetative organs.Application of molecular biology and genomics tools to further excavate key enzyme genes for controlling oil synthesis in tuber oil from C.esculentus,which can assemble lipid enrichment and metabolic pathways in plant vegetative organs,and increase the accumulation of oil and high-quality fatty acids in high biomass vegetative organs oil metabolism engineering provides better genetic resources.Triacylglycerols(TAG)are the major storage oil of plant fruits and seeds.Higher plant cells synthesize fatty acids de novo in the plastids and then transport them to the cytoplasm to synthesize TAGs on the endoplasmic reticulum.Diacylglycerol acyltransferases(DGAT)control the final acylation of TAG synthesis,which catalyzes acyl-Co A and diacylglycerol(DAG)to generate TAG.DGAT is considered to be a key rate-limiting enzyme for TAG biosynthesis.It has been found that DGAT enzyme proteins can be divided into at least three families,namely DGAT1,DGAT2 and DGAT3.During the tuber development of C.esculentus,how can each DGAT synergistically control oil synthesis and accumulation? Which DGAT has substrate specificity for oleic acid and promotes oleic acid enrichment? And can each DGAT function differently? These scientific questions remain to be answered.To this end,the genomics tool was used to identify the DGAT gene and isolate its c DNA clones.The spatiotemporal expression profile of the DGAT gene and the physicochemical properties of the encoded protein were analyzed.The yeast expression vector and plant expression vector of each DGAT gene were constructed separately.The biological function of these DGAT genes will be systematically demonstrated by Heterologous expression in TAG-deficient mutant H1246 yeast,phenotypic identification of transgenic yeast,and cultivation of homozygous transgenic tobacco strains and phenotypic analysis.The research is helpful to further analyze the molecular mechanism of oil biosynthesis in oils of tuber oil,and it can provide new insights into the regulation mechanism of fatty acid and oil biosynthesis in plant vegetative organs.The main findings are as follows:1.Dynamic analysis of tuber development and storage substance accumulation of C.esculentus showed that the tuber maturity of C.esculentus(Jinnong 1)planted in Taigu County area was about145 DAS(days after tuber sowing).Dynamic characteristics of oil accumulation are: low initial growth of oil content and slow growth in the early(50-90 DAS),rapid accumulation of oil in the middle period(90-120 DAS),high growth rate of oil content in the later period(120-145 DAS)slows down.The dry weight,soluble sugar and starch of C.esculentus tubers showed similar growth trends.However,the protein content increased slowly from the early to mid-stage of tuber development,and120 DAS began to decrease until it stabilized at maturity.2.By systematic analyzing the sequencing data of the transcriptome of the C.esculentus tuber in this laboratory,there were three C.esculentus DGATs with complete reading frames(ORF),namely one DGAT1(CeDGAT1)and two DGAT2(CeDGAT2-1 and CeDGAT2-2)genes.The length of CeDGAT1 is 1,509 bp,encoding 502 amino acids;CeDGAT2-1 and CeDGAT2-2 are 1,017 bp and954bp in length,encoding 338 and 317 amino acids,respectively.The CDD of NCBI was used to identify the conserved domains of CeDGATs.The results showed that CeDGAT1 and At DGAT1 belong to the MBOAT superfamily,and CeDGAT2 s and At DGAT2 belong to the LPAAT superfamily.Phylogenetic tree results show that CeDGAT1 protein is classified into the DGAT1 family and CeDGAT2 s is classified into the DGAT2 family.The three CeDGATs proteins do not seem to have high homology with Arabidopsis,and have homology with gramineous plants3.Detect the expression of CeDGATs in different tissues and developmental stages by qRT-PCR.The results showed that all three genes were expressed in roots,leaves and tubers,CeDGAT1 was highly expressed in roots,CeDGAT2-1 was highly expressed in leaves,and CeDGAT2-2 was highly expressed in tubers.At different developmental stages of tubers,all three genes showed a trend of first increase and then decrease,and the expression level was highest in the 90 DAS,but there were significant differences in their expression levels.The expression level of CeDGAT2-2 among the three CeDGATs genes was the highest in all periods,and the expression level at 90 DAS was about 3 times that of CeDGAT1 and about 11 times that of CeDGAT2-1.The coding sequences of CeDGAT1 and CeDGAT2-2 were successfully cloned from the tubers of C.esculentus by RT-PCR technology.4.Construct CeDGAT1,CeDGAT2-2 yeast expression vectors(pYES2-CeDGAT1 and pYES2-CeDGAT2-2)respectively,and heterologously express them in a yeast strain H1246 that cannot synthesize TAG,and obtain positive transgenic yeast strains.Through Nile red staining and thin-layer chromatography,it was found that CeDGAT1 and CeDGAT2-2 can restore the ability of H1246 yeast strain TAG biosynthesis,greatly increase the oil content of the yeast strain.The oil content of the transgenic yeast cells are 1.37 times and 1.48 times that of empty yeast cells,respectively.The transgenic yeasts accumulated more unsaturated fatty acids(palmoleic acid C16:1and oleic acid C18: 1).Especially for CeDGAT2-2 transgenic yeast,the substrate is preferably C18:1,whose content is as high as 49.98%,which is significantly higher than empty control,indicating that CeDGAT2-2 has higher substrate specificity for oleic acid.5.Construction of CeDGAT1,CeDGAT2-2 plant constitutive expression vectors(pBI121-CeDGAT1 and pBI121-CeDGAT2-2),respectively,and heterologous expression in common tobacco by Agrobacterium infusion method to obtain homozygous transgenic plants.Determination of oil,starch,protein and soluble sugar content in vegetative organs of transgenic tobacco lines.The results showed that the total oil content of CeDGAT1,CeDGAT2-2 overexpressed tobacco plants increased by 6.25 times and 7.15 times,respectively,compared with no load.The levels of unsaturated fatty acids(C18:1,C18:2)in transgenic CeDGAT1 and CeDGAT2-2 tobacco leaves increased,while the content of saturated fatty acids(C16:0 and C18:0)decreased accordingly;oleic acid(C18:1)was significantly It increased from 5.1% of no-load to 21.9% and 31.33%.The two DGAT enzymes may have similar substrate specificities,especially CeDGAT2-2 is more selective for 18: 1.The starch content of transgenic CeDGAT1 and CeDGAT2-2 tobacco leaves decreased significantly,the soluble sugar content increased,and the protein content was not significantly changed.In addition,the germination rate of tobacco seeds and chlorophyll content of the transgenic plants were not significantly changed,CeDGAT1 and CeDGAT2-2 heterologous overexpression did not adversely affect the growth and other agronomic traits of tobacco plants.In short,both CeDGAT from the tubers of Cyperus esculentus have diacyltransferase activity,which can significantly promote the biosynthesis of oil in vegetative organs and increase the content of oleic acid without significant negative effects.These two excellent genes can enrich oils content in high biomass nutrient organs through genetic engineering,and promote the sustainable green and efficient production of high-quality edible or industrial oils. |
