Identification And Analysis Of Scutellaria Baicalensis MicroRNA And Its Target Genes

MicroRNAs(miRNAs),with a length of 21-24nt,are a class of endogenous single-stranded non-coding RNAs and negative regulators of gene expression in eukaryotes.They mainly regulate gene expression at the transcriptional level by mediating mRNA methylation,cutting mRNA at the post-transcriptional level or affecting mRNA translation.Due to the rapid development of bioinformatics and high-throughput sequencing technology,many miRNAs in plants have been identified and analyzed functionally in recent years.Scutellaria baicalensis Georgi is a perennial herb of the labiformes family and the genus baicalensis,and its dried roots are one of the top ten traditional medicinal herbs in China.At present,its genome-wide sequence has been p ublished,but no research on miRNAs has been reported.Identifying its miRNA from the genomic level and studying the function of specific miRNA is of great significance to understanding the growth and development regulation mechanism of S.baicalensis and improving the content of specific secondary metabolites(such as flavonoids).In this study,we constructed small RNA libraries of different tissues such as root,stem,leaf and flower using the four-year S.baicalensis as the material,then known miRNAs and novel miRNAs were identified from the whole genome level of by small RNA(sRNA)sequencing and bioinformatics analysis.Target genes of miRNA were predicted and identified through the website of psRNAtarget and the degradation data.The expression patterns of differentially expressed miRNAs between tissues were analyzed by qRT-PCR.Candidate genes Sb-miR858,Sb-miR396,Sb-miRN3 and Sb-miRN14 were selected,and their overexpression vectors and STTM vectors were constructed.The specific research results are as follows:1.The sRNA sequencing libraries of root,stem,leaf and flower of S.baicalensis were constructed.Three replicates were set up for each tissue,and there were 12 libraries in total.After sequencing and analysis,removing the adapter,screening the low quality and too short length reads,we got clean reads.We regarded the sequence of 17-30nt as sRNA,and found that 24nt sRNA accounted for the highest proportion of the total reads through analysis of its characteristics.In the sRNA sequencing library,129 miRNAs were identified,including 99 known miRNAs from 27 miRNA families and 30 novel predicted miRNAs in S.baicalensis.The precursors of 129 miRNA sequences were analyzed,and the results showed that the precursors were mostly distributed in length of 55-230nt.Secondly,the length of all novel predicted miRNA mature sequences ranged from 21nt to 22nt,accounting for 43%and 57%,respectively,with the highest proportion of miRNA in length of 22nt.Finally,the length of the novel miRNA and the preference of the first base were analyzed,and the proportion starting with base U was the highest,reaching 77%.2.The website psRNAtarget was used to predict the target genes of 129 identified miRNAs and a total of 1472 targets were predicted,including 1275 known miRNA target genes and 197 novel miRNA target genes.We used qRT-PCR to verify the expression patterns of some known miRNA(Sb-miR159,Sb-miR390,Sb-miR403,Sb-miR858)and novel miRNA(Sb-miRN5,Sb-miRN9,Sb-miRN14,Sb-miRN17)in different tissues,and found that the results of qRT-PCR were consistent with the sequencing results.3.A library of S.baicalensis degradation group was constructed and high-throughput sequencing was conducted to obtain 27,733,898 clean tags.Tags of degradation fragments are more expressed in the positive chain of chromosomes.Degradation sequencing data were analyzed through Using CleaveLand software.503 miRNA target genes were abtained,62 of which scored 0 to 1 and were considered as reliable results.After the intersection of the 1472 target genes predicted by the psRNAtarget website and the 62 high-scoring target genes verified by degradation analysis,a total of 46 reliable results were obtained.Of these,41 target genes are from 13 families of known miRNAs,and 5 target genes are from 2 novel miRNAs(miRN8 and miRN10).Blast analysis of the 46 target genes revealed that most of them were transcription factors.For example,the target gene of miR858 corresponds to R2R3-MYB transcription factor,which may be related to the synthesis of flavonoids.4.Select the four candidate genes we are interested in:Sb-miR858,Sb-miR396,Sb-miRN3,and Sb-miRN14.Among them,miR858 may be related to the synthesis of flavonoids,and may induce the synthesis of flavonoids to protect plants in drought environment.MiR396 may regulate the growth and development of S.baicalensis,especially its roots.The expression levels of Sb-miRN3 and Sb-miRN14 were relatively high in roots.The precursor sequences of Sb-miR858,Sb-miR396,Sb-miRN3 and Sb-miRN14 were cloned into pJIM19 vector to obtain its overexpression vectors.Four candidate genes were constructed into pUC57 by STTM,and the interference vectors of four genes were successfully obtained,which laid a foundation for the functional research of the candidate genes.In summary,this study enriched the miRNAs data of S.baicalensis by sequencing small RNA and degradation group,analyzed the target genes of miRNA,and constructed the expression vectors of related genes,laying a foundation for the functional study of S.baicalensis miRNA.