| Camelina sativa L.Crantz is a characteristic oil crop.The oil content of seeds is as high as 40% to 50%.It’s fatty acid composition mainly polyunsaturated linoleic acid(18:2)and linolenic acid(18:3),widely used in edible oil,animal feed,industrial raw materials and biofuels.C.sativa has short growth period,barren resistance and high resistance to various adversity.It has strong cold tolerance especially in the seedling and vegetative growth period.It is also an ‘environmentally friendly’ crop.Analyzing the regulation mechanism of lipid synthesis and the molecular basis of stress resistance has always been the main field of C.sativa seed genetic improvement,new variety cultivation and high-value oil products.Stearoyl-acyl carrier protein △~9 desaturase(SAD)can catalyze the dehydrogenation reaction of palmitoyl-ACP(C16:0-ACP)and stearoyl-ACP(C18:0)to produce palmitoyl-ACP(C16:1-ACP)and oleoyl-ACP(C18:1-ACP).It is a key enzyme that determines the content of plant saturated fatty acids and unsaturated fatty acids.To date,there is no research on the identification,functional analysis and participation in stress response of C.sativa SAD family and its members.Therefore,we conducted the genome-wide identification of Cs SAD genes family and analyzed the expression patterns of Cs SAD3,Cs SAD4 and Cs SAD5 genes in various tissues and different development stages of seeds of C.sativa.The Cs SAD3,Cs SAD4 and Cs SAD5 genes were obtained from the seeds(20 days after flowering)by molecular cloning technology,and we constructed recombinant yeast expression vector and recombinant plant expression vector respectively.The three genes were heterologously expressed in wild yeast INVSc1,unsaturated fatty acid-deficient yeast BY4389,and transiently expressed in tobacco leaves.In order to identify the SAD encoded protein enzyme activity,substrate specificity and physiological function,the total fatty acid content and fatty acid composition of transgenic yeast and tobacco were detected.In addition,to exploring whether Cs SAD3,Cs SAD4 and Cs SAD5 are involved in plant stress response.We detected the expression profile of target genes under cold stress conditions and the physiological phenotype changes of the C.sativa seedlings.Finally we cloned the promoter sequence of the Cs SAD3,Cs SAD4,Cs SAD5 genes and analyzed its activity.The main research results are as follows:1.Using the Arabidopsis At SADs protein sequence as the search sequence,BLAST C.sativa genome,16 Cs SAD genes were identified.Using bioinformatics tools to analyze the structure of genes and the cis-acting elements in the promoter region,the physical and chemical properties of encoded proteins,high-level structure,multiple sequence alignment and phylogenetic tree construction.The Conserved Domain Database(CDD)revealed that the SAD-encoded proteins all have Acyl-ACP-d ESAT domains and belong to the Ferritin-like superfamily.The Cs SAD genes were distributed on different chromosomes of C.sativa,and the structure of introns and exons of genes also differs according to the length of the gene sequences.Multiple sequence alignment and phylogenetic tree analysis showed that the members are relatively conservative.The Cs SAD6,Cs SAD8,Cs SAD16 proteins were highly related to the 16:0-ACP specific Arabidopsis At SAD3 protein,while the Cs SAD3,Cs SAD4,Cs SAD5 proteins were closely related to the 18:0-ACP substrate specific Arabidopsis At SSI2 protein.According to the results of conservative structure and phylogenetic tree,the expression characteristics analysis and functional analysis of Cs SAD3,Cs SAD4,Cs SAD5 genes were selected to inverse the mechanisms of participating in linen oleic acid synthesis.According to the results of the conservative structure and phylogenetic tree analysis,the Cs SAD3,Cs SAD4,Cs SAD5 genes were selected,and the expression profile and function analysis were further used to explore the mechanisms involved in the synthesis of camelina oleic acid.2.The expression profiles of Cs SAD3,Cs SAD4,Cs SAD5 in different tissues and developing seeds were detected by RT-PCR and q RT-PCR.The results showed that Cs SAD3,Cs SAD4,Cs SAD5 were expressed in all tissues,and the expression levels were higher in seeds(20 d and 30 d after flowering).speculating that Cs SAD3,Cs SAD4,Cs SAD5 participate in the regulation of seed oil synthesis during seed development.3.Cs SAD3,Cs SAD4,Cs SAD5 were cloned from the developing seeds of camelina.In this study,yeast recombinant expression vectors p YES2.0-Cs SAD3,p YES2.0-Cs SAD4,p YES2.0-Cs SAD5 and recombinant plant expression vectors p CAMBIA1303-Cs SAD3,p CAMBIA1303-Cs SAD4,p CAMBIA1303-Cs SAD5 were constructed for subsequent yeast transformation and tobacco transient expression.Finally,we analyzed the enzyme activity,substrate specificity,physiological function of encoded proteins.4.The Cs SAD recombinant yeast expression vector was heterologously expressed in wild yeast INVSc1 and unsaturated fatty acid-deficient mutant BY4389 cells in order to detected the total fatty acid content and fatty acid composition of the transgenic yeast.The results showed that Cs SAD3,Cs SAD4,Cs SAD5 all had dehydrogenase catalytic activity.The total oil and unsaturated fatty acids content in the transgenic BY4389 yeast of Cs SAD3,Cs SAD4,Cs SAD5 were significantly increased.All the three SAD enzyme genes preferred stearyl-ACP(C18:0-ACP)as substrate to catalyze its dehydrogenation to form oleoyl-ACP(C18:1-ACP).The Cs SAD recombinant plant expression vector was transiently expressed in Nicotiana benthamiana leaves by agrobacterium infection,PCR detection of target genes are effectively expressed.Cs SAD transient expression tobacco leaves oil content and fatty acid composition analysis showed that Cs SAD3,Cs SAD4,Cs SAD5 all have SAD function and higher selectivity to C18:0 substrate,which significantly promotes monounsaturated fatty acids(oleic acid)and total oil accumulation in tobacco leaves.5.C.sativa seedlings were treated with 4℃ cold stress,and samples were taken at 0,3,6,12,24,48,72 h,then analyzed the expression profiles of Cs SAD3,Cs SAD4,Cs SAD5 under cold stress and the unsaturated fatty acid content of the seedlings.The results suggested that the expression levels of Cs SAD3,Cs SAD4,Cs SAD5 were increased in C.sativa seedlings under cold stress conditions,and were positively correlated with the increase of unsaturated fatty acid content in seedlings under cold stress.It proved that Cs SAD3,Cs SAD4,Cs SAD5 play an important role in the expression of cold resistance of C.sativa seedlings.The gene promoter sequences of Cs SAD3,Cs SAD4,Cs SAD5 were successfully cloned,further study on their promoter functions will provide new evidence for comprehensively analyzing the mechanism of Cs SAD3,Cs SAD4,Cs SAD5 participating in plant stress response.In summary,all the results revealed the new function of Cs SAD in response to plant stress,which provided new knowledge for further analysis of oil synthesis and regulation mechanism of C.sativa seeds,and provided a new molecular target for genetic improvement of yield and quality of C.sativa oil. |
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