Chalcone Isomerase(CHI) Gene Cloning And Expression Analysis Of Dryopteris Erythrosora

As a kind of secondary metabolites widely existing in the plants,flavonoid has large effects on establishing their own defensing system and participating the metabolism.It also has lots of functions,including anti-oxidation,anti-inflammation,inhibition of tumor cell activity,anti HIV activity.In addition,it is less toxic and has high medicinal value.The flavonoid metabolic pathway of seed plants is well understood,while the flavonoid metabolic pathway of the ferns,which possess higher flavonoid content in contrast to the seed plants,is unclear.Chalcone isomerase(CHI)is one of the rate-limiting enzyme in the flavonoids metabolic pathway.Its main function is transforming catalytic naringenin chalcone into naringenin.The naringenin is a precursor of the subsequent branch metabolic pathways.The CHI genes of seed plants belong to gene family of CHI.They can be divided into four types,i.e.Type ?,Type ?,Type ?,Type ?,which have different biological functions.However,it is uncertain about structures,types and funtions of the CHI genes among ferns.The investigations of CHI gene of ferns can elucidate the molecular mechanism of its metabolic pathways and have scientific significance on revealing the function and evolution of the fern CHI genes.Dryopteris erythrosora is chosen as experiment material in this study,in which the transcriptome sequencing,the PCR and high performance liquid chromatography(HPLC)techniques are used to clone and analyse the isomerase chalcone gene.Several related aspects have been analysised in a view of bioinformatics,including structure of the gene sequence,the physical and chemical properties,structure and function of the CHI protein.The experimental results of this study are mainly the following several aspects:1.The sequence of young leaf of Dryopteris erythrosora is determined by using Ilumina Hiseq 2000 platform and 8.5 G data volume has been got.Through sequence splicing and filtering we obtain a total of 143604 Unigene.According to the results of annotation,six CHI possibility genes are obtained.Three of the CHI genes were choosen as candidate genes according to the analysis.2.Three genes ORF sequences have been successfully cloned by the technology of transcriptome sequencing.The three genetic sequences were renamed as DeCHI1,DeCHI2 and DeCHI3.Bioinformatics analysis showed that the open reading frame(ORF)length of the gene sequence DeCHI1 was 651 bp and encoding 216 amino acids;DeCHI2 ORF span 822 bp,coded 273 amino acids;DeCHI3 ORF span 738 bp,encoding 245 amino acids.The cluster analysis indicated that DeCHI1 is parallel to Type ?,Type II,DeCHI2 belongs to Type IV type;DeCHI3 belongs to Type III type.3.The experiment successfully build three recombinant plasmids that pET32a-DeCHI1,pET32a-DeCHI2,pET32a-DeCHI3 of CHI,the recombinant plasmids were transed into BL21 cells,in order to build prokaryotic expression vector,and used IPTG induced fusion protein,protein size obtained with SDS-PAGE identification were 39.7 kDa,45.0 kDa and 45.0 kDa,which have the same size with the expected proteins.4.In this experiment,naringenin chalcone and isoliquiritigenin are choosed as substrates.The expressed proteins of three genes are used to catalyze the substrates.The high performance liquid chromatography(HPLC)is used to determine the product content.The results show that:DeCHI1,DeCHI2 and DeCHI3 protease can catalytze naringenin chalcone into naringenin,and also catalyze isoliquiritigenin into liquiritigenin,which is different to the result(1)that Type III CHI and Type IV CHI of seed plants have no catalytic activity;(2)DeCHI1 can not only catalyzec naringenin chalcone,but also catalyze isoliquiritigenin into liquiritigenin,which is different from the conclusion that only leguminous plant Type ? can catalyze isoliquiritigenin.Evolutionary tree clustering analysis showed DeCHI1 and seed plant Type ? and Type ? are clustered in the same branch.it is speculated that it has functions of Type ? and Type ?.The speculation of the result is consistent with the experimental results;(3)During the process of DeCHI1,DeCHI2 and DeCHI3 protease catalyzing naringenin chalcone into naringenin,intermediate product is formed,but the specific composition of the product needs further research.Above all,Dryopteris erythrosora have different types of CHI genes,but its function has not been differentiated clearly.The expressed protases of the genes can catalyze naringenin chalcone into naringenin and isoliquiritigenin into liquiritigenin,which reflects the relatively primitive features of fern flavonoids secondary metabolism.