The Chalcone Synthase Gene Cloning And Function Analysis In Dryopteris Erythrosora

Dryopteris erythrosora is a traditional medicinal fern，which is rich in bioactiveflavonoids, it have the effect of antiviral, antineoplastic, antisepsis andanti-inflammation. Now the study more concentrated in the determination of totalflavonoids and flavonoids types analysis of Diyopteris erythrosora, but the effectfactor of flavonoids content and formation is not have a systematic study. Undernatural conditions，seasonal changes have a significant impact on the flavonoidscontent, but research in this area yet reported. The fern flavonoids biosyntheticpathway has been in its infancy, now in ferns has been cloned chalcone synthase(CHS) gene, but its function is still not analyzed.Based on the above background, we obtained the following two aspects of theresults.1. The seasonal dynamics of the total flavonoids in various parts of D.erythrosora and their antioxidant activity were investigated. The total flavonoidscontent in vairous parts of D. erythrosora obviously showed a seasonal dynamicchange. The total flavonoids content in leaves was relative low with an averagecontent of about2.01%and a minimum content of about0.78%. However, the totalflavonoids content (from4.3%to12.5%) in stems was much higher than that in leaveswith an average content. In spring, the total lfavonoids content in stems was relativelylow, but increased rapidly from summer to winter. However, the total flavonoidscontent changes in leaves showed different model, the total flavonoids in leavesincreased in spring, then decreased rapidly after summer and keep low level. The totalflavonoids content in the stems showed a negative correlation with that in the leaves.The antioxidant activities of the extracts from leaves, rachis, roots and stems also altered in proportion to the change of collect time and total flavonoids content. Ingenera], the extracts from stems always showed highest antioxidant potentials and itwas suggested that the stems could be used as crude medicine.2. The chalcone synthase gene cloning and function analysis in Dryopteriserythrosora. The total RNA, DNA were extracted from sporophyte. Then the ORFsequence and genomic sequence of chalcone synthase were cloned by PCR.Bioinfonnatics analysis showed the ORF of DeCHS gene sequence was1212bp andencoding403amino acids, which contain an intron and obey gt/ag rule. BLASTshowed that the identity to other plants was very high, phylogcnctic analysis indicatedthat the DeCHS gene with Cyclosorus aewninatus’ Salvinia natans, Ceratopteristhalictroides and other ferns have close evolutionary relationship. Protein sequencealignment indicated the CHS active sit170e residues, Cys, Hi314s, A347sn and the83signature G3FGPG in DeCHS. Furthermore, the recombinant pET32-DeCHSplasmids was transformed into host E. coli BL21for expression. SDS-PAGE andWestern blotting analyse showed that the fusion protein was highly expressed in E.coli BL21with isopropyl-D-thiogalactoside (IPTG) induction, and the expressedDeCHS protein had a molecular weight of about63KD, a size matching that of thepredicted by bioinformatic analysis. In vitro enzyme activity test verified DeCHSprotein have activity, that could catalytic substrate generated the naringenin.In summary, flavonoids content in various parts of D. erytlu’osora obviouslyshowed a seasonal dynamic change. In spirng, leaves have high flavonoids contentand we can collect the leaves as crude mcdicine. Howcre, in other seasons flavonoidscontent in sterns was higher than that of other parts and can ucs it as crude medicine.Chalcone synthase is the first committed enzyme in the flavonoids biosyntheticpathway，functional analysis showed that DeCHS protein has biological activity. Thisreseach laid the foundation for study the flavonoids biosynthetic pathway? and it alsocan reveal the regulatory mechanism in ferns.