Construction Of MiRNAs Expression Profiles Of Alfalfa And Maize Straw And Expression Verification And Screening Of Known Cross-border MiRNAs

The expression of miRNAs related to nitrogen conversion efficiency in mammary gland of dairy cows was affected by feeding high quality alfalfa compared with low quality corn straw.And it is known that the exogenous plant miRNAs in food can cross-border control the target gene of mammals to affect their physiological functions.So the difference of miRNAs in alfalfa and straw may be one of the main reasons for the difference.Therefore,in this present study,the miRNAs of alfalfa and corn straw were studied,and the miRNAs gene library of alfalfa and corn straw was constructed by using high-throughput omics technology,and the target genes and signal pathways of miRNAs were predicted by bioinformatics method.Differential miRNAs with known transboundary regulatory functions were selected for quantitative analysis and verification of their expression patterns by qRT-PCR.Finally,differential miRNAs with high expression were screened out,which laid a foundation for further revealing the mechanism of alfalfa miRNAs regulating lactation in dairy cows.The experiments and results wer as follows:1.High throughput Omics sequencing analysis of maize straw miRNAsThe miRNAs expression spectrum of maize straw was constructed by the Illumina sequencing platform for high-throughput sequencing.409 miRNAs were obtained,of which 199 miRNAs were known and 210 were newly predicted.393 of 409 miRNAs predicted target genes,with a target number of 14700.There were 2 miRNAs with significant differences,one was up regulated and another was down regulated.Finally,according to the sequencing results of maize straw and the reported cross-border miRNAs,six miRNAs,miR 156a,miR164a,miR166a,miR167a,miR168a and miR319a-3p,were found to be expressed in maize straw,and their expression levels ranged from 607.20 to 8101.28,among which miR 167a was the highest and miR 164a the lowest.2.High throughput sequencing analysis of miRNAs of MsZm1The miRNAs expression spectrum of MsZm1 was constructed by using the Illumina sequencing platform.759 miRNAs were obtained,519 of them were known,and 240 were newly predicted.707 miRNAs of 759 were predicted to be target genes,and the number of target genes was 11002.Among the 86 miRNAs with significant difference were found in fresh and dry samples of MsZml,46 were up regulated and 40 were down regulated.Finally,according to the sequencing results of alfal,combined with the reported miRNAs with cross-border function,it was found that miR156a,miR164a,miR166a,miR167a,miR168a and miR319a-3p were expressed in alfal,and the expression level ranged from 276.38 to 63178.43,among which mir166a was the highest and miR164a was the lowest.3.Quantitative analysis of miRNAs difference between maize straw and alfalfaSix miRNAs were selected from maize straw and alfalfa,and they were known to have cross-border function.The expression patterns were verified and analyzed by qRT-PCR.The results showed that the expressions of miR164a and miR1 67a in maize straw were higher,which were consistent with the sequencing results.The expressions of miR166a and miR168a in MsZmlF alfalfa were higher,which were consistent with the sequencing results.The expressions of miR167a,miR168a and miR319a-3p in the MsZm1G alfalfa were higher,which were consistent with the sequencing results.In the comparison of MsZm1F and MsZm1G alfalfa samples,the expressions of miR168a-in the dry samples were higher than that of the fresh one.In the comparison between MsZml alfalfa and maize straw,miR166a was screened out,which were highly expressed as the miRNAs.Combined with the results of high-throughput sequencing and qRT-PCR quantitative analysis of maize stalk and MsZm1 alfalfa,miR166a was selected.If miR166a enters into dairy cows,it may regulate the immune performance of dairy cows.