Cloning And Functional Evaluation Of Phenylalanine Ammonia-lyase Gene (PAL) And Capsaicin Synthase Gene (CCS) From Anoectochilus

Anoectochilus is a perennial plant of Orchidaceae.Among them,Anoectochilus roxburghii and Anoectochilus formasanus are used as medicinal plants because of their abundant content flavonoids and other pharmaceutical components.In recent years,the products of Anoectochilus on market are mostly cultivated artificially due to the wild resources have being gradually exhausted.However,the pharmaceutic value of cuyltivated plants are much less than the wild.It is important to improve cultivation conditions,promote the accumulation of pharmaceutical components and improve the quality of cultivated Anoectochilus products.Flavonoids are the main pharmaceutical components of Anoectochilus.They are synthesized through phenylalanine pathway.Phenylalanine ammonia-lyase(PAL)is the rate-limiting enzyme in this pathway.In other plants,the expression of the PAL gene was found to be induced by NaCl and other conditions.Capsaicin has a strong antioxidant effect and is a health component of Anoectochilus.Capsaicin synthase(CCS)is the rate-limiting enzyme in its synthesis pathway.Its expression can be induced by NaCl and light quality conditions.No genomic informatin is available for any psecies of genus Anoectochilus family Orchidaceae.In the present study,specific PCR primers were designed based on the full-length transcript data of A.roxburghii and A.formasanus,and used to amplify the sequences of the open reading frame(ORF)and the genomic DNA of the PAL and the CCS genes of these two species.After alignment analysis,subcellular localization,and transgenic verification,the relative expression levels of these two genes in response to different induction conditions were detected by real-time quantitative PCR(RT-qPCR).It was attempted to promote the synthesis and accumulation of pharmaceutical components and improve the medicinal value of cultivated Anoectochilus by induced expression of these two genes under artificial cultivation conditions.The main results are as follows:(1)Based on the assembled two full-length transcripts by using their transcriptome sequencing data,two pairs of PCR primers were designed and used to amplify the cDNA and DNA sequences of the PAL and CCS genes of A.roxburghii and A.formasanus for aligment analysis.The results showed that the PAL and the CCS genes were highly homologous between these two species.The full length was both 2733 bp.Their 2148 bp ORFs encoded 715 amino acids.Their putative proteins had the typical structure and catalytic activity sites of the PAL proteins.The ORF of the CCS gene of A.roxburghii was 1461 bp,encoding 486 amino acids,while that of A.formasanus was 1458 bp,encoding 485 amino acids.Homologous alignment,protein structure prediction,phylogenetic tree analysis and heterologous expression all comfirmed that the sequences we cloned were indeed the ORF and genomic seqeucnes of the PAL and the CCS genes of A.roxburghii and A.formasanus.The PAL proteins both of the two species were located in the nucleus.This result was verified by some recent reports,although it was different from the previous understanding of plant secondary metabolism.There are many kinds of flavonoids.Besides secondary metabolite of plants,they may be unknown new functions.(2)In response to 100 mmol/L NaCl induction,the expression of the PAL gene in the two Anoectochilus species was significantly up-regulated,reached 7.49 tiems of the control at 8 h in A.roxburghii and 81.53 times in A.formasanus.Combining with the content determination under the same conditions in our laboratory,it was indicated that of NaCl induction of appropriate amount during artificial cultivation could induce up-regulation expression of the PAL gene,promote flavonoids accumulation,and increased content of total flavonoids in these two species.In addition,the increased total flavonoids entered the flavonol and anthocyanin branches,which improved the medicinal value of the two species.(3)In response to 100 mmol/L NaCl induction,the expression of the CCS gene in A.roxburghii increased at the beginning(1 h),but began to decrease after 4 h.Its expression in A.formasanus was significantly upregulated with a large range.The response of the expression of the CCS gene to ultraviolet induction showed opposite between A.roxburghii(upregulated)and A.formasanus(downregulated).In response to red light induction,the expression of the CCS gene fluctuated in A.roxburghii and A.formasanus,varing at different time points.In conclusion,the PAL and the CCS genes play key roles in the synthesis of flavonoids and capsaicin in A.roxburghii and A.formasanus,respectively.In the artificial cultivation of Anoectochilus,NaCl supplement of appropriate amount could upregulated the expression of the PAL gene,promote the accumulation of flavonoids and increase the content of total flavonoids,especially the content of flavonols and anthocyanins with the best pharmacological and health effects.Therefore,in practical production and application,NaCl content could be increased appropriately in culture conditions to improve the medicinal value of Anoectochilus.However,the expression of the CCS genes in the two Anoectochilus species differed in response to NaCl,ultraviolet and red light.Further studies are needed.