Identification Of MiRNA Involved In Erucic Acid Metabolism In Brassica Napus L. And AmiRNA Regulate Research

Brassica napus L.is an important source of edible oil,and it’s also crucial chemical materials,which way of rapeseed was used decided by the content of erucic acid in the rapeseed oil.Becacuse the edible oil need the erucic acid in rapeseed oil as low as possible,on the other hand,which used for chemical material need the erucic acid content as high as possible.So it is very importan to understand the way of erucic acid metabolic process and regulate the erucic acid content for various application Brassica napus L..Erucic acid metabolic process and regulate it were all focused on antisense inhibiton,RNA interference（RNAi）or gene overexpression in the previously study,but about the miRNA involving in erucic acid metabolic and its regulate erucic acid content were rarely be studied.So we predicted the miRNA involving in Erucic Acid metabolic with bioinformatics.On the other hand,identification of miRNA involving in Erucic Acid metabolic with sRNA and degradome high-through sequencing.At the same time,we design special amiRNA target the genes BnFAE1 and BnFAD2,then construct seed special vector to identify its function and role.We got the following results:1.Our study used the gene participate in the way of erucic acid matabolic process as the target to find conserved miRNAs,which target transcripts are these genes.And then blast the miRNA sequences with Brassica napus genome to select potential pre-miRNA.At last,we get the novel conserved miRNA sequences and using q-PCR to expect it.We found that miR156、miR161、miR840、miR2923、mi R5380、miR1044、miR7504、miR5079、miR6116、miR5143、miR9563 could participate in the process of erucic acid metabolic.The selected miRNA target Brassica napus 3-ketoacyl-CoA synthase（KCS）、Brassica napus 3-ketoacyl-CoA thiolase（KAT）、Brassica napus acyl-[acyl-carrier-protein]desaturase、Brassica napus acyl-coenzyme A oxidase 4、Brassica napus omega-6 fatty acid desaturase（FAD2）families one or more genes.These target gene participate in fatty acids chain extente,fatty acids desaturate and fatty acidsβoxidase,etc.All of the above illustrate that miRNA play important role in the way of erucic metabolic.ThemiRNAs could take part in these ways trough regulate it’s target gene expression and play a crucial role.2 We empoyed the miRNA and degradome high-through sequencing to find the miRNAs and its target transcripts in different development rapeseeds.As a result,we identified 936miRNA sequences,including 151 conserved miRNAs,246 novel conserved mi RNAs and 539newly miRNAs.And using Targetfinder web software to predict the target gene,20354 mRNAs were selected,then show the gene funtional annotation for all predict mRNA with KOG,GO and KEGG database。Extract the RNA of samples separately,and mixed them equally for digradome sequencing.Through analyse we got 589 mRNA could be targeted by 236 miRNAs.Analyse these mRNAs and their target transcript,we found that miR838、miR156e、miR159c、bna-5p-163957₁8、bna-5p-163957₁8、bna-5p-396192₇、miR156a、mi R172b、miR395b-p3、mi R9563a-p3and miR9563b-p5 couled target long-chain acyl-CoA synthetase,enoyl-CoA hydratase（3-hydroxyacyl-CoA dehydrogenase）、stearoyl-CoA desaturase（delta-9 desaturase）acyl-CoA oxidase、pyruvate dehydrogenase phosphatase、dolichyldiphosphatase和choline-phosphate cytidyl transferase etc..These target mRNAs participate in the extention of carbon chain,regulation of very long chain fatty acids,desaturation of fatty acids and assemble of lipids,which are all important process in erucic acids metabolic.As a result,no matter the predicted novel conserved mi RNAs or newly miRNAs from high through sequecing,they are all could involve in the erucic acid metabolic through their target gene.On the other hand,all target gene play important role in the process of erucic acid metabolic,so miRNAs could regulate erucic acid metabolic and play important role.3.We desigh special amiRNA aim to the key gene Bn FAE1 and BnFAD2 in the process of erucic synthesis and clone it into the pre-mi RNA through overlapping PCR.Using the seed special promoter Napin and Nos terminator to structure expression vector and separate transgenic it into high and low erucic acid Brassica napus by Agrobacterium impregnation method,at last we got the trasgenic plant.We got the T₀ seeds from two raprseed varieties which transform pCENND2 expression vector,test the Fad2 gene expression and the content of oleic acid,linoleic acid and erucic acid,analyse the silencing effect to Fad2 and the change of erucic acid in trangenic rapeseed.As a result,we found that amiRNA have well effect to silencing Fad2 gene and lead to change the content of erucic aced.At the same,the ami RNA also have well effect to the improve the oleic acid content and decrease the linoleic acid content,which illustrate that amiRNA is a good way to regulate the composition of rapeseed fatty acid and improve the quality of rapeseed oil.