| Ribes L.is a genus of the Grossulariaceae,which is also known as gooseberry.Deciduous,evergreen or semi-evergreen shrub dilute.There are more than 160 species of Ribes L.in the world.There are 59 species and 30 varieties of Chaenaceae in China,mainly distributed in the southwest and northwest to the northeast.The Ribes L.has high nutritional value,fruits,flowers,young leaves and buds are edible.In particular,the fruit is rich in unsaturated PUFAs,which can enhance the body's immunity and prevent disease such as hyperlipidemia and arteriosclerosis.The contents of PUFAs in Ribes L.'s fruits were variety differences.We analyzed the contents of PUFAs in eight varieties of Ribes L.in Northeast China.The PUFAs content of redcurrant and whitecurrant was higher than that in other species.The PUFAs content of blackcurrant is lower than that in other species.Redcurrant,whitecurrant and blackcurrant were used as the research material in this study.Cloning and expression of key genes in the biosynthesis of PUFAs,such as RnFAD3,RnFAD6,RnFAD8,RnNF-YB,RnABI3,and cloning and analyzed promoter activity of FAD3 promoter,were researched in this study.These researches were useful for understanding the molecular mechanism of the difference in the Ribes L.'s PUFAs synthesis at the molecular levels,which provides a theoretical basis for the production of PUFAs by genetic engineering.The results were as follows:1.The full-length cDNA sequences cloning of PUFAs-related genes in Ribes L.Three PUFAs pathway genes and two regulatory genes were cloned from the cDNA of Ribes L.by race technique.We achieved five genes which are RnFAD3 with the length of 1467bp?RnFAD6 with the length of 1599bp?RnFAD8?with the length of 1733bp?RnNF-YB with the length of 979bp?RnABI3 with the length of 2388bp.2.The phylogenetic analysis of PUFAs genes of the Ribes L.The study analyzed the genetic RnFAD3,RnFAD6,RnFAD8,RnNF-YB,RnABI3 genes under the help of various online softwares of biology,and then predicts primary and secondary structures of protein in six genes followed by systemic phylogenetic analysis.There are three evolutionary branches in the evolution of FAD3 gene.RnFAD3 and other dicotyledonous plants of Rosaceae's FAD3 gene,such as olives and cowpeas,belong to the same evolutionary branch.The origin of RnFAD6,RnFAD8 gene was close to that of three FAD6 genes and FAD8 genes of red currant.RnNF-YB gene is in the same evolutionary branch as the NF-YB gene of arabidopsis thaliana,cucumber,bean and microbroad bean.RnABI3 is closely related to grape's ABI3 gene and belongs to the same evolutionary branch.3.The expression patterns analysis of temporal and spacial of Ribes L.PUFAs genes by using Realtime PCRThe expression patterns analysis of temporal and spacial of RrFAD2,RnFAD3,RnFAD6,RnFAD8,RnNF-YB,RnABI3 by using Realtime PCR in different stages and different organizations:Six genes are expressed in the six organizations of three kinds Ribes L.,and there are differences in the organization.Only the expression of FAD3 gene in red currant was higher than that in white currant.The expression of FAD8 gene in black currant slowly increased with fruit development.The expression of FAD6 gene in red currant was proportional to the development of fruit.The expression levels of FAD3,FAD8 and ABI3 in white currant were inversely proportional to fruit development.4.Cloning and activity analysis the promoter sequence of the RrFAD3 geneUsing DNA of red currant as a template,primers were designed from the RrFAD3 gene sequence(cloned in the laboratory),and a 1753 bp RrFAD3 promoter sequence was obtained by chromosome walking.Online alignment and bioinformatics analysis showed that this sequence contained sequences such as the conserved CAAT box and TATA box,as well as many other functional elements.The FAD3 promoter recombinant vector was constructed and transformed into Arabidopsis thaliana seedlings.The results of the assay showed that the recombinant plasmid expression in the hypocotyl. |
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