Study On The Regulation Mechanism Of Bulbil Formation Of Pinellia Ternata Based On Transcriptome Sequencing

Pinellia ternata(Thunb)Breit.is a perennial herb in the genus Araceae,the underground tuber can be used as a traditional medicine in China after concoction,with the effects of drying dampness,resolving phlegm,lowering rebellion and stopping vomiting.In recent years,the wild resources of P.ternata have been depleted due to excessive collection.The production of P.ternata was severely restricted due to the low reproduction efficiency and continuous cropping obstacles in the artificial planting process.Therefore,improving the propagation efficiency of P.ternata is an important way to increase the production of P.ternata and meet the market demand.The propagation of pearl buds of P.ternata has significant advantages over seed and tuber propagation in terms of the growth cycle,survival rate and quantity.However,the mechanism of the occurrence and development of bulbils of P.ternata has yet to be fully explored and elucidated.In this study,we explored the mechanism of bulbil formation and development in P.ternata from several aspects,including morphological observation,endogenous auxin(IAA)content and enzyme activity determination,transcriptome sequencing and key gene expression profile analysis in the early period of bulbil formation and induction.The main results are as follows:1.According to the morphological characteristics of bulbil formation and development,the early bulbil development of P.ternata can be divided into four periods(P1-P4).The content of endogenous auxin(IAA)during the bulbil development of P.ternata was determined by HPLC with the petiole of the same period as a control.The results showed that the changes of auxin content had apparent tissue and period specificity.The auxin content was relatively stable in P1 and P2 but decreased significantly after a slight increase in P3.The auxin content in petiole increased in P1-P3 but decreased significantly in P4(P<0.05).Using the petiole of the same period as the control,the enzyme activities of the antioxidant enzyme system during the development of the bulbil were measured by enzyme label.The results showed that the enzyme activities of SOD,POD,CAT,PAL,PPO and APX showed significant differences between periods and tissues.The variation of MDA content in peroxidation products was relatively stable during the development of bulbil.2.qRT-PCR technique and ge Norm,Norm Finder,Best Keeper and ref Finder programs were used to evaluate the expression stability of 8 candidate reference genes(CRGs)in leaves,petioles and roots during the four periods of P.ternata bulbil development(P1-P4),and to find the best reference genes.The results showed that GAPDH ranked the highest in the stability of gene expression in different tissues,different periods and all samples.Subsequently,qRT-PCR was used to verify the best reference gene GAPDH by using six genes related to the development of bulbils.GAPDH showed accurate normalization of the expression data of the six genes,indicating the reliability of GAPDH as an internal reference gene.3.m RNA and miRNA sequencing were performed on the bulbil and petiole of P.ternata in the early period of bulbil development(P1).The results showed that 17381 differentially expressed genes were significantly enriched in eukaryotic pathways such as ribosome biosynthesis,spliceosome,and fatty acid extension,phenylpropyl biosynthesis,flavonoid biosynthesis and photosynthesis.miRNA sequencing showed 20 miRNAs with significant differential expression(P<0.01),and a total of 257 target genes were predicted.Analysis of target gene GO enrichment showed that the number of transcriptional regulation-related GO term enriched target genes was high.The enrichment analysis of target genes KEGG pathway showed that the enrichment degree and enrichment significance of plant hormone signaling transduction,starch and sucrose metabolism and MAPK signaling pathway were relatively high.Among them,the expression profiles of miR393,miR160 and the corresponding target genes TIR1 and ARF17 involved in growth hormone signaling were identified by qRT-PCR,which were consistent with the spatiotemporal variation of auxin(IAA)content.LAX5 in the phytohormone signaling pathway showed the same trend out of IAA content variation,and ARF5,IAA25 and IAA31 also showed inter-period and inter-tissue expression differences The expression pattern of miR171 b and its target gene SCL6 involved in gibberellin signal transduction was like that of auxin.The results suggest that the starch and sucrose metabolism as well as the regulation of plant hormone signal transduction pathway were crucial to the occurrence and development of bulbil.miRNA is involved in P.ternata bulbil occurrence and development by regulating the expression of genes related to these two pathways.4.The expression profiles of miR171,miR393,miR160 and the corresponding target genes SCL6,TIR1 and ARF17 were analyzed in different tissues during four periods of bulbil development under the induction of exogenous auxin(NAA)using qRT-PCR.The results showed that miR393 and miR160,which are involved in the auxin signaling transduction pathway,were induced to be expressed during the development of bulbils,and the expression levels of the target genes TIR1 and ARF17 were also up-regulated at different levels during bulbils development,especially at P1 period.5.Plant hormone(NAA+6-BA)was used to induce the formation of bulbil structure in isolated petioles of P.ternata.Subsequently,m RNA and miRNA sequencing were performed on bulbils and petioles at the early bulbil induction period(P2)and combined with qRT-PCR to verify the expression profiles of key genes during bulbil induction to explore the mechanism of bulbil induction in P.ternata.m RNA sequencing resulted in8602 genes with significantly different expression levels(P<0.05).Starch and sucrose metabolism and phenylpropanoid biosynthesis pathways were significantly enriched in the KEGG pathway enrichment analysis of differentially expressed genes.miRNA sequencing showed 15 miRNAs with significant differences(P<0.01),and a total of 624 target genes were predicted.In the KEGG pathway enrichment analysis of target genes,plant hormone signaling transduction and starch and sucrose metabolism pathways was enriched.qRT-PCR expression identification of miRNAs involved in plant hormone signaling transduction pathways and their target genes showed that the expression levels of miR167 and its target gene ARF6 involved in the auxin signaling transduction pathway were significantly upregulated,and the expression levels of miR171 a,and its target gene SCL6 involved in the gibberellin signaling pathway were significantly upregulated.miR171 a and its target gene SCL6 also showed period and tissue expression differences.miR156 was significantly up-regulated during bulbil induction and its target gene SPL6 expression level was significantly down-regulated.Among the starch and sucrose metabolic pathway genes,the expression level of sucrose synthase SUS2 was consistently increased with the induction of bulbil development.The results further suggested that miRNAs were involved in the regulation of P.ternata bulbil formation and development by targeting the expression of genes related to plant hormone signal transduction pathway and starch and sucrose metabolism pathway.