Screening And Preliminary Identification Of MicroRNAs Related To Piperine Biosynthesis In Pepper (Piper Nigrum L.)

Black pepper(Piper nigrum L.)is a perennial evergreen liana,belonging to Piperaceae,known as the"King of Spices",and has important economic and medicinal values.MicroRNA(miRNA)is a class of classical endogenous small noncoding RNAs that are known as the regulator of gene expression at the posttranscriptional level and widely functioned in regulating plant growth,biotic and abiotic stress processes via its targets.Recently studies have confirmed that miRNAs are widely involved in the regulation of biological processes such as the growth and development of grain,oil,and cotton crops,as well as biotic and abiotic stresses;but few reports on miRNA regulating the piperine biosynthesis in black pepper were documented.In this research,a small RNA sequencing strategy was performed in the root,stem,leaf,flower and pepper fruits with growth months of 2,4,6 and 8(after pollination)to identify the known and novel miRNAs differentially expressed in tissues in black pepper.The main results are showing as follows:1.In this experiment,three biological replicates were set up for each sample,and Small RNA library construction and sequencing were performed.After the raw reads obtained were screened according to the conditions,about 84.8%of clean reads were obtained,and then the clean reads were compared to the latest pepper Genomics and identification of known and newly discovered miRNAs.Finally,a total of 109 known miRNAs and 153 newly discovered miRNAs were obtained,for a total of 262 miRNAs.We screened low expressed miRNAs under the condition of TPM<5,and finally obtained 68 known miRNAs and 149newly discovered miRNAs,totaling 217 miRNAs.2.Randomly select 15 miRNAs(10 known miRNAs,5 newly discovered miRNAs)from highly expressed miRNAs for q RT-PCR detection and analysis to verify the accuracy of sequencing.After performing linear regression analysis on the expression data obtained by q RT-PCR and the sequencing data,it was found that the accuracy of sequencing data is high(R~2=0.86).3.The 262 pepper miRNAs obtained by sequencing were used for genome-wide target prediction through ps Robot and Target Finder software,and 1,757 target genes were obtained.Subsequently,GO annotation and KEGG analysis of the target revealed that the target genes were mainly enriched in aromatic compound synthesis,phenylpropane metabolism,and cyclic compound biosynthesis.In addition,according to the annotations and clustering results of KEGG,it was found that 43 miRNAs were involved in the synthesis and regulation of secondary metabolites of pepper,mainly related to:phenylpropanoid biosynthesis;terpenoids,piperidine and pyridine alkaloids biosynthesis;phenylalanine Acid,tyrosine and tryptophan biosynthesis;lignin biosynthesis and anthocyanin biosynthesis,etc.,for example,mi R396 targets and regulates 4 homologous PER genes(Pn24.105,Pn7.365,Pn16.989 and Pn4.2519)involved in catalyzing the synthesis of lignin monomers.mi R169 is involved in the biosynthesis of anthocyanins by regulating UGT79B1.4.In order to further screen the miRNAs and target genes involved in piperine biosynthesis,using the two direct precursor sources of piperine biosynthesis as clues,combined with RNA-seq data,a total of 7 involved in the metabolism of phenylpropanine and lysine were screened.5’RLM-RACE and q RT-PCR analysis verified that four miRNAs:mi R166,mi R397,mi R396 and novel＿mi R43 are involved in the biosynthesis of piperine by regulating the target genes 4CL,PER,CCR and AOC2/3.