Effect Of Induction Conditions To Accumulation Of Flavonoids And Expression Of Chalconesynthasegene In Anoectochilus

Anoectochilus formasanusand and Anoectochilus roxburghiiare are the two major medicinal species of genus Anoectochilus.In traditional Chinese medicine,they are used to treat tumors,fatty liver,hepatitisand other diseases because of their abundant pharmaceutical constituentsof flavonoids,and to use as health food because of aboundant content of polysaccharides.In recent years,wild Anoectochilusrecources have been gradually exhausted duo to excessive collection and ecological deterioration.Artificial cultivation and tissue culture techniques have been applied to increase production to meet market demand.However,the content of the pharmaceutical constituentsmay be different between the two Anoectochilusspeciese.The accumulation of the pharmaceutical constituent under artificial cultivation ortissue culture conditions is much different from that of wild plants.Therefore,in-depth study on the differences of the pharmaceutical constituents between the two species,as well as the effectsof artificial cultivation ortissue culture conditions to the accumulation of the pharmaceutical constituents and to the expression of key genes for synthesis,can provide a reference for the improvement of the pharmaceutical constituents of the plants of artificial cultivation or tissue culture.In plants,flavonoids are biosynthesized through the phenylpropanoid pathway.Chalcone synthase(CHS)are the key speed limiting enzyme in this pathway.It is beneficial to the improvement of the accumulation of the pharmaceutical constituents to study the expression difference of its coding gene between the two species and under different cultivation or culture conditions,and its effect on the accumulation of flavonoids.However,all species of Anoectochilusgenus have not been studied for their genomics information,even theirchromosomal ploidy is not unkown.Homologous cloning of this gene is very difficult,and there are few reports on gene expression.In the present study,the effect of different induction conditions on the accumulation of major flavonoids in two medicinal Anoectochilusspecies was analyzed by high performance liquid chromatography.After chromosome karyotype analysis,the coding sequence of the CHS gene was cloned according to the assembled transcript from transcriptome analysis.After function evaluation by heterologous expression,its expression difference under different induction conditions was detected by real-time quantitative PCR.It was attempted to explore the probability of promoting the expression of the synthesis key gene and increasing the accumulation of the flavonoids in tissue cultured or artificial cultivated Anoectochilus.The main results are as follows:(1)The content of total flavonoids and the rate of free radical scavenging in A.roxburghii were 1.73 and 1.52 times that in A.formasanus.The contents of rutin,quercetin and kaempferide in A.roxburghii were 25.76,5.48 and 6.02 times that in A.formasanus.Under non-induction conditions,A.roxburghii accumulated more total flavonoids,had stronger antioxidant activity and medicinal value.(2)Under the induction of ultraviolet light and phenylalanine,the content of total flavonoids and the rate of free radical scavenging increased,the content of the three flavonols decreased,and the content of anthocyanin increased in the two Anoectochilus species.These results indicated that the increased flavonoids mainly entered the anthocyanidin branchof flavonoids biosyntesis,did not increase the medicinal value of Anoectochilus,but might have inhibitory effect on plant growth because of the increased malondialdehyde content.Under NaCl induction,the content of total flavonoids and the rate of free radical scavenging increased in the two Anoectochilus species,and the increase in A.roxburghii was greater than that in A.formasanus.The contents of the three flavonols and anthocyanins increased in varying degrees except that the content of rutin remained unchanged in A.formasanus.Even more,the content of rutin in A.roxburghiiincreased by21.28 times,while the content of malondialdehyde only increased slightly.These results suggested that the induction of NaCl was more effective than that of ultraviolet light and phenylalanine in increasing the content of flavonoids in cultured or tissue-cultured Anoectochilus plants,and the increased content mainly entered the flavonol branch,which could effectively improve the medicinal value of Anoectochilus.As long as the induced concentration is appropriate,it would not cause obvious damage to the plants.(3)The CHS gene sequences of A.formasanus and A.roxburghii were amplified by the specific primers designed according to chromosome ploidy identification and transcriptome sequencing analysis.Their nucleic acid sequences and deduced protein sequences were highly similar to those of their related species of Orchidaceaefamily.Theirputtive proteins contained all the conserved structural domains and functional sites of the CHS proteins.Their heterologous expression in Escherichia coli conferred activity of CHS enzyme.Their heterologous expression in rice increased the tcontent of total flavonoids.All these evidences confirmed that these two cloned sequences were indeed the CHS genes of A.formasanus and A.roxburghii.They were submitted to GenBank for registration.(4)The CHS proteins of the two Anoectochilus species were found to contain a nuclear localization signal(R⁶⁶-K⁶⁷-R⁶⁸-H⁶⁹)at their near N-terminal of the primary structure,and actually localized in the nucleus.This result was contrary to most of previous reports,but similar to the subcellular localization of the CHS protein in Arabidopsis.It was speculated that the Anoectochilus CHS proteins might catalyze flavonoids synthesis of the phenylpropane metabolism pathway in the nucleus,and their products might be involved not only in the basal metabolism,stress response,reproductive development and many other growth and development processes,but also in the transcriptional regulation of related genes.(5)The expression of the CHS gene had significant organic specificity in A.formasanus and A.roxburghii.Under the induction of ultraviolet light,phenylalanine and NaCl,the expression of the CHS genes of the two species were up-regulated in varying degrees,although their expression patterns might be different.This result could explain the increase of total flavonoids content in the two Anoectochilus species under these three induction conditions,and also showed the key rate-limiting role of the CHS enzyme in the synthesis metabolism of flavonoids in Anoectochilus.The cis-acting elements such as promoter enhancers,photoresponsive elements and anaerobic responsive elements were found in the promoter sequences of the two Anoectochilus CHS genes,explaining the mechanism of the expression response of these CHS genes to ultraviolet light and NaCl induction.To sum up,the accumulation of flavonoids in A.roxburghii is greater than that in A.formasanus.Ultraviolet light and phenylalanine induction could up-regulate the expression of the CHS gene and increase the content of total flavonoids,but the increased flavonoids mainly entered the anthocyanin branch,did not improve the medicinal value,and inhibited plant growth because of the increase of malondialdehyde content.However,NaCl induction could not only increase the content of total flavonoids through its up-regulation to expression of the CHS gene expression,but also induce the increased flavonoids mainly into the flavonol branch,and is beneficial to improve the medicinal value.As long as an appropriate induction concentration,NaCl will not cause obvious damage to the plants,and is easy for application to production.