Cloning And Analysis Of Chalcone Synthase Superfamily Genes In Dryopteris Erythrosora

As a kind of secondary metabolites widely existing in the plants,flavonoids have large effects on establishing their own defensing system and participating the metabolism.They also have lots of functions,including anti-oxidation,antiinflammation,tumor cell inhibition activity,anti HIV activity.In addition,they are less toxic and have high medicinal value.The flavonoid metabolic pathway of seed plants has been well understood,while the flavonoid metabolic pathway of the ferns,which possess higher flavonoid content in contrast to the seed plants,is still unclear.CHS is the first critical enzyme in flavonoid metabolic pathway and plays an important role in the metabolism of plant flavonoid.With the process of evolution,CHS genes have been inordinately duplicated and differentiated in different plant lineages,resulting in CHSlike genes formed in most plant genomes.The CHS and CHSlike genes makes up the chalcone synthase gene superfamily.So far,we have separated many kinds of chalcone synthase superfamily genes in spermatophyte,fern and bryophyte,but the function of fern chalcone synthetase superfamily genes is still unclear.It has important significance for elucidating the mechanism of flavonoid metabolism,and revealling the function and evolution of chalcone synthetase superfamily genes.Dryopteris erythrosora is chosen as experiment material in this study.Techniques of the transcriptome sequencing,the PCR and high performance liquid chromatography(HPLC)are used to clone and analyse the chalcone synthetase superfamily genes.Structure of the gene sequence has been analysised using bioinformatics.The experimental results of this study are mainly the following several aspects:1.The sequence of young leaf of Dryopteris erythrosora is determined using Ilumina Hiseq 2000 platform and 8.5 G data volume is got.Through sequence splicing and filtering,143604 Unigene are obtained.According to the results of annotation,twenty eight CHS possibility genes are obtained.Four of the CHS genes were choosen as candidate genes according to the analysis.2.Four genes ORF sequences have been successfully cloned by technique of transcriptome sequencing.The four genetic sequences were renamed as DeCHS,DeCHSlike1,DeCHSlike2 and DeCHSlike3.Bioinformatics analysis showed that theopen reading frame(ORF)length of the gene sequence DeCHS was 1212 bp and encoding 403 amino acids;DeCHSlike1 ORF span 1371 bp,coded 456 amino acids;DeCHSlike2 ORF span 1506 bp,encoding 501 amino acids.DeCHSlike3 ORF span1332 bp,coded 443 amino acids.3.Using in-fusion connection technology,the experiment successfully build four recombinant plasmids that pET28a-DeCHS,pET28a-DeCHSlike1,pET32aDeCHSlike2,pET32a-DeCHSlike3 of CHS,the recombinant plasmids were transfered into BL21 cells to build prokaryotic expression vector.IPTG is used to induce fusion protein.The protein sizes obtained with SDS-PAGE identification were 44.2 kDa,48.3 kDa68.5 kDa and 64.6 kDa,which are equivalent to the expected proteins.4.In this experiment,Malonyl CoA,Coumaroyl CoA,Phenylacetyl CoA,Acetyl CoA,Isovaleryl CoA are choosed as substrates.The expressed proteins of four genes are used to catalyze the substrates.The high performance liquid chromatography(HPLC)is used to determine the product content.The results show that: DeCHS protease can catalytze Coumaroyl CoA and Malonyl CoA into naringenin,and also catalyze Phenylacetyl CoA and Malonyl CoA into naringenin,which is functionally the same to the CHSs of other plants reported.DeCHSlike1 protease can catalytze Coumaroyl CoA and Malonyl CoA into naringenin,but can not catalyze the reaction of Phenylacetyl CoA and Malonyl CoA.However,products can be detected when Acetyl CoA and Malonyl CoA,Isovaleryl CoA and Malonyl CoA are used as substrate,but the products are unknown and need further investigation.DeCHSlike2 and DeCHSlike3 protease could catalytze Coumaroyl CoA and Malonyl CoA,Phenylacetyl CoA and Malonyl CoA into naringenin;catalytze Isovaleryl CoA and Malonyl CoA into 6-isobutyl-4-hydroxy-2-pyrone.Meawhile,they could catalytze Acetyl CoA and Malonyl CoA into an unknown products.However,there are some function differences between them and need further researches.This study finds that Dryopteris erythrosora have different types of CHS genes,but their functions are not differentiated clearly.According to the present experiment,DeCHS has the same function as the CHS gens in other reported plant;DeCHSlike1has both CHS gene and CHSlike gene function;DeCHSlike2 and DeCHSlike3 can catalytze Isovaleryl CoA and Malonyl CoA into 6-isobutyl-4-hydroxy-2-pyrone,which has the function of VPS,indicating that both DeCHSlike2 and DeCHSlike3 are CHSlike types.All results demonstrated that the chalone synthetase superfamily genes of Dryopteris erythrosora had multiple functions.