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Chalcone synthase( CHS) and chalcone isomerase( CHI) are key enzymes in the biosynthesis pathway of flavonoids. In this study,unigenes for CHS and CHI were screened from the transcriptome database of Arisaema heterophyllum. The open reading frame( ORFs) of chalcone synthase( Ah CHS) and chalcone isomerase( Ah CHI) were cloned from the plant by RT-PCR. The physicochemical properties,expression and structure characteristics of the encoded proteins Ah CHS and Ah CHI were analyzed. The ORFs of Ah CHS and Ah CHI were 1 176,630 bp in length and encoded 392,209 amino acids,respectively. Ah CHS functioned as a symmetric homodimer. The N-terminal helix of one monomer entwined with the corresponding helix of another monomer. Each CHS monomer consisted of two structural domains. In particular,four conserved residues define the active site. The tertiary structure of Ah CHI revealed a novel open-faced β-sandwich fold. A large β-sheet( β4-β11) and a layer of α-helices( α1-α7) comprised the core structure. The residues spanning β4,β5,α4,and α6 in the three-dimensional structure were conserved among CHIs from different species. Notably,these structural elements formed the active site on the protein surface,and the topology of the active-site cleft defined the stereochemistry of the cyclization reaction. The homology comparison showed that Ah CHS had the highest similarity to the CHS of Anthurium andraeanum,while Ah CHI had the highest similarity to the CHI of Paeonia delavayi. This study provided the basis for the functional study of Ah CHS and Ah CHI and the further study on plant flavonoid biosynthesis pathway.

Plant type Ⅲ polyketide synthases (PKSs), the pivotal enzymes in the biosynthesis of polyketides, produce backbones of many structurally diverse and functionally different polyketides. So far, a variety of functionally diverse plant type Ⅲ PKSs have been cloned and identified from plant origin. Site-directed mutagenesis is a useful technique to study the complex relationship between protein structure and function. This review summarized advances in the structure-function relation of plant type Ⅲ polyketide synthases by site-directed mutagenesis in recent years, including the modification of the amino acid residues influencing enzyme architectures (such as controlling the specificity of starter substrates, the number of condensation reactions, and the cyclization reactions of the intermediate product). This review provides information to study the structure-function relation of plant type Ⅲ polyketide synthases.

Based on the transcriptome data, we cloned the open reading frame of IiHCT gene from Isatis indigotica, and then performed bioinformatic analysis of the sequence. Further, we detected expression pattern in specific organs and hairy roots treated methyl jasmonate( MeJA) by RT-PCR. The IiHCT gene contains a 1 290 bp open reading frame( ORF) encoding a polypeptide of 430 amino acids. The predicted isoelectric point( pI) was 5.7, a calculated molecular weight was about 47.68 kDa. IiHCT was mainly expressed in stem and undetectable in young root, leaf and flower bud. After the treatment of MeJA, the relative expression level of IiHCT increased rapidly. The expression level of IiHCT was the highest at 4 h and maintained two fold to control during 24 h. In this study, cloning of IiHCT laid the foundation for illustrating the biosynthesis mechanism of phenylpropanoids in I. indigotica.

Plant type III polyketide synthase (PKS) generates backbones of a variety of plant secondary metabolites with diverse functions, and has long been models to elucidate the relationship between the three-dimensional structure and function. More than 80 type IIII PKS crystal structures with different functions have been reported in Protein Data Bank, including the crystal structures of the well-studied Chalcone Synthase of plant type III PKS, as well as the 6 other kinds of PKSs in the family, which are critical for understanding the structural basis for diverse starter molecule selectivity, polyketide chain length and the cyclization reaction. Structure-based analysis and site-directed mutagenesis are foundation for the investigation of enzyme engineering, genetic and metabolic engineering. This review summarized 7 plant-specific type III PKS in the aspects of their crystal structures and functions.

Objective: To get Scutellaria viscidula CHS gene sense and antisense plant expression vectors,and explore the mechanism of CHS's effect on the flavonoid biosynthetic pathway through the transformation of Scutellaria viscidula., Methods: CHS gene fragment was amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR) from total RNA of Scutellaria viscidula and cloned into PMD18-T vector, sequenced and bioinformaticly analyzed., Results: The DNA sequence was correct and we simultaneously guessed the amino acid sequence and the three-dimensional structure of CHS. Cloned fragments and pCAMBIA1304+ vector were digested using Blg II and BstE II at the same time, then they were connected with CHS gene fragment and pCAMBIAI304+ big skeleton fragment to get CHS sense and antisense plant expression vectors., Conclusion: The successful construction of Scutellaria viscidula CHS gene sense and antisense plant expression vectors, and the transformation into C58C1 and LBA4404 through the freeze-thaw method, lay the foundation for Agrobacterium-mediated transformation of Scutellaria viscidula CHS gene in plant heredity.

To clarify the important functional residues in the active site of N-myristoyltransferase (NMT), a novel antifungal drug target, and to guide the design of specific inhibitors, multiple sequence alignments were performed on the NMT family and thus evolutionary trace was constructed. The important functional residues in myristoyl CoA binding site, catalytic center and inhibitor binding site of NMT family were identified by ET analysis. The trace residues were mapped onto the active site of CaNMT. Trpl26, Asn175 and Thr211 are highly conserved trace residues and do not interact with current NMT inhibitors, which are potential novel drug binding sites for the novel inhibitor design. Pro338, Leu350, Ile352 and Ala353 are class-specific trace residues, which are important for the optimization of current NMT inhibitors. The trace residues identified by ET analysis are of great importance to study the structure-function relationship and also to guide the design of specific inhibitors.

042BM noeA was obtained by PCR. It is identical to that of S. meliloti 1021 at 99% level, and similarity of their NoeA is 97%. In addition, it was found that this protein shares significant homology with the SAM-dependent methyltransferase of Mesorhizobium sp. BNC1 (32% similarity), and the similarity of its 303-362 region to the 160- 220 region of Ll11 methyltransferases of E. coli (PrmA) is 41%. Compared to 042BM, the noeA deletion mutant 042BMA-Km showed different degrees of increase in number of nodule, fresh weight of nodule and plant top dry weight on alfalfa cultivars of Putong Zihua, Baoding, Ningxia, Baifa and Aohan, but decrease on Milu. However, this mutant has no significant change in ability to nodulate cultivars of Huanghou and Zahua. Hence, noeA is involved in alfalfa cultivar-specific nodulation.

Our research showed that propionyl acylase gene is on 4.16kb insert DNA fragment originated from S. mycarofaciens in recombinant plasmid pIJM9. For localization of this gene on 4.16kb insert DNA fragment, sub-cloning has been carried out with religation of BamHI digested plasmid pIJM9. Among the transformants, molecular weight of recombinant plasmid pIJM95 harbouring in No. 5 transformant is 5.0kb. Molecular weight of insert DNA fragment is 0.53kb in this plasmid. In bioconversion experiment for spiramycin of No. 5 transformant, the bioconversion product was analysed with TLC, bioautography, HPLC and mass spectrum (FAB). Results showed that the bioconvert product is propionylspiramycin. No. 5 transformant is able to transform spiramycin into propionylspiramycin. Propionyl acylase gene was locolized on 0.53kb insert DNA fragment of recombinant plasmid pIJM95. Analysis result of DNA sequence showed that content of G + C is 68.2% for 0.53kb insert DNA fragment. More than 70 kinds of restriction endonuclease have cut sit on this fragment. From No.54-No.393 nucleotide, there is an open reading frame, which codes a polypeptide consisted of 122 amino acids. Start codon is ATG, stop codon is TGA.