Genome-wide Analysis Of Methyl Jasmonate-regulated Isoform Expression In Medicinal Plant Andrographis Paniculata

The alternative splicing of the precursor mRNA(pre-mRNA)produces multiple transcripts,thereby increasing the diversity of the transcriptome and proteome.Depending on the location of the splice sites,the types of alternative splicing are currently divided into four broad categories: intron retention(IR),alternative 3'acceptor sites(AA),alternative 5' donor sites(AD),and exon skipping(ES).In addition to these four splicing types,there are some splicing types that occur less frequently,such as alternative polyadenylation.The alternative splicing of genes involves many biological processes such as plant growth and development,especially when plants are subjected to abiotic stresses,and alternative splicing can help plants cope with changes in various environmental factors.IR event is the most variable splicing event in plant genes,and the expression levels of IR isoforms are regulated by a variety of environmental factors.The generation of secondary metabolites is one of the forms of coping when medicinal plants are under pressure from the environment.When the medicinal plant exogenous methyl jasmonate(MeJA)is administered,the amounts of secondary metabolites in the plant increases.Currently,studies have also reported that alternative splicing involves the biosynthesis of important secondary metabolites in plants.The aerial part of Andrographis paniculata(Burm.f.)Nees is used as a traditional Chinese medicine for the treatment of various body diseases.The main active metabolic component is andrographolide,which is increased after MeJA treatment.Accurate identification of the A.paniculata gene isoforms requires transcriptome sequencing.Second-generation sequencing(SGS)technology is limited by short read lengths.Three-generation sequencing(TGS)technique such as single-molecule real-time(SMRT)sequencing technology can achieve construst full length transcript,but the error rate of base is higher and throughput is lower.Therefore,a combination of SGS technology and SMRT technology can be used to obtain a full-length transcriptome.In this study,a complete full-length transcriptome of medicinal plant A.paniculata was obtained by using both Illumina-and SMRT-based RNA-seq.All IR isoforms in the genome level of the A.paniculata were then identified usingbioinformatics analysis software.The A.paniculata seedlings were then treated with MeJA and transcriptome sequencing was performed on samples at 0h,24 h and 48 h.The IR isoform was then subjected to differential expression analysis and GO enrichment analysis at 0h and 24 h,0h and 48 h.Finally,the expression pattern of IR isoform in the andrographolide biosynthesis pathway was analyzed,and some IR isoforms were verified by RT-PCR and qRT-PCR.A total of 4,846 IR isoforms were identified in this study.At 24 h,a total of 310 IR isoform expression levels were up-regulated and 629 IR isoform expression levels were down-regulated.At 48 h,a total of 296 IR isoform expression levels were up-regulated and 659 IR isoform expression levels were down-regulated.Overall,the expression levels of more IR isoforms were down-regulated after MeJA processing.The GO enrichment analysis results show that the differentially expressed IR isoforms corresponding genes has enrichment on GO terms.In the andrographolide biosynthesis pathway,8 genes from the geranylgeranyl diphosphate(GGPP)synthetic pathway were alternatively spliced,resulting in a total of 25 isoforms,12 of which belonged to IR isoform.After MeJA treatment,there are 4 IR isoforms with significant differential expression that may be involved in the regulation of andrographolide biosynthesis.RT-PCR and qRT-PCR experiments confirmed the existence of 5 IR isoforms.This study can deepen the understanding of the transcriptome of A.paniculata and can help the future study of andrographolide biosynthesis.