Identification And Expression Vector Construction Of Chalcone Synthase (CHS) Gene Family In Batrachium Bungei From Qinghai-tibet Plateau

In order to adapt to the extreme environment on the Qinghai-Tibet Plateau,plants have evolved complex adaptation mechanisms.Batrachium bungei a representative species of Ranunculaceae,is a perennial aquatic plant,widely distributed in the Qinghai-Tibet Plateau,and an important component of the aquatic vegetation group on the Plateau.It is mainly used to prevent water pollution and purify water quality,and is an indicator plant for wetland water quality monitoring.It is an ideal material for exploring the adaptation of aquatic plants to extreme plateau environment.Chalcone synthase(CHS)gene family also known as plant type III polykeone synthase gene family.It is the first rate-limiting enzyme in the synthesis of flavonoids and plays an important role in plant growth and development and adaptation to the environment.At present,most studies on CHS gene have been focused on terrestrial plants but the characteristics,functions and evolution of CHS gene in aquatic plants are still unclear and need further investigation.Previous transcriptome studies showed that the flavonoid metabolism pathway was significantly enriched in Batrachium bungei under the plateau environment,and some CHS genes were highly expressed with the change of altitude gradient,which verified that CHS gene was involved in the regulation of Batrachium bungei adaptation to various extreme environments on the Plateau from the molecular level.In this study,Batrachium bungei from Qinghai-Tibet Plateau was selected as the research object.Based on the early transcriptome data of Batrachium bungei.The chalcone synthase gene family of Batrachium bungei was identified and analyzed.Bb CHS4 gene was cloned and its sequence characteristics,protein structure and function were analyzed.Plant overexpression vector and prokaryotic expression vector were constructed.Transgenic Arabidopsis thaliana Bb CHS4 gene was obtained.The main results are as follows:1.Identification and analysis of Bb CHS gene familyBased on the transcriptomic data of Batrachium bungei,Bb CHS1?Bb CHS6 were identified by bioinformatics methods.The length of CDS is about 1200 bp,the number of amino acids is 380?396 aa,the molecular weight of protein is 40?43 KD,the isoelectric point is 5.50?6.04,and the consistency of amino acid sequence among members is 38%?85%.Comparison of Bb CHS1?Bb CHS6 protein tertiary structure model showed that Bb CHS1-5 had higher structural similarity,and Bb CHS6 obviously lacked part of helix structure.Motif10 was lost in Bb CHS1,and motif1,7,and 10 of Bb CHS6 were all lost.Bb CHS2,Bb CHS3,Bb CHS4,and Bb CHS5 were clustered into one branch,and Bb CHS1 and Bb CHS6 were separate branches,which were consistent with the results of structural analysis,Batrachium bungei were highly conserved structurally and had low variability.Phylogenetic tree was constructed for nine species with 121 CHS sequences.The CHS were distributed in four subgroups,and Bb CHS3 and Bb CHS4 were clustered into one branch,indicating that the CHS were conserved and homologous in the evolution process.2.Cloning and homology analysis of Bb CHS4 geneWe designed specific primers to isolate and clone Bb CHS4 gene from c DNA of Batrachium bungei.Which has Chal?sti?synt?N and Chal?sti?synt?C family characteristic domains.NCBI online blast function was used to search and analyze the homology of nucleotide and amino acid sequences of Bb CHS4.The results showed that Bb CHS4 gene had the highest homology with CHS gene in both nucleotide and amino acid levels,up to more than 90%.3.Structural characteristics and functional correlation of Bb CHS4 proteinThe CDS sequence of Bb CHS4 gene was 1182 bp,encoding 393 amino acids.The protein weight was 42.87 Ku,the theoretical p I was 5.92,and the instability index was 39.29.It was a stable protein,and the hydrophilic and hydrophobic analysis results were biased to hydrophilic protein.The results of secondary structure showed that the?-helix ratio of Bb CHS4 protein was 43.51%.It is speculated that Bb CHS4protein has no signal peptide and belongs to non-secretory protein.The results of tertiary structure prediction showed that Bb CHS4 protein could form homologous dimer,and the catalytic cavity was located in the outer part.The monomer model was compared with Chain A of At CHS-A in Arabidopsis thaliana.The spatial structure of Bb CHS4 polypeptide chain was highly overlapping,indicating that Bb CHS4 and At CHS protein were highly similar in structure and functionally conserved.By comparing the amino acid sequence of Bb CHS4 with homologous species,it was found that Bb CHS4 retained the active site of Cys-His-Asn catalytic triad in CHS protein catalytic center,and the active functional site of CHS amino acid was"RLMMYQQGCFAGGTVLR".Characteristic sequence"GVLFGFGPGL"and phenylpropyl amino acid residues(Phe215 and Phe265)substrate specific binding site.4.Genetic transformation of Bb CHS4 gene in Arabidopsis thaliana and prokaryotic expressionBy seamless cloning,Bb CHS4 gene was inserted into plant overexpression vector PBI121-EGFP and prokaryotic expression vector PET-32a.The plant overexpressed recombinant plasmid was transferred into wild type Arabidopsis thaliana by agrobacterium-mediated inflorescence immersion method,and 20 T₁p BI121-EGFP-Bb CHS4 transgenic lines and 5 T₃ homozygous overexpressed lines were obtained by resistance screening,fluorescence labeling,g DNA level identification and RNA level identification.The results of subcellular localization showed that Bb CHS4 gene was located in cytoplasm of tobacco.The flavonoid content of Arabidopsis thaliana overexpressing Bb CHS4 gene in T₃ generation was compared with that of wild-type Arabidopsis thaliana.The content of flavonoid in Arabidopsis thaliana overexpressing Bb CHS4 gene was lower than that of wild-type Arabidopsis thaliana.The analysis may be related to the mutual inhibition of homologous genes,which needs further analysis and verification.When the recombinant plasmid PET-32a-Bb CHS4 was transferred into E.coli BL21 for induction,Bb CHS4 could normally encode about 42 KD protein expression,but existed in the form of inclusion body.The induction conditions need to be further optimized to lay a foundation for further exploring the functional mechanism of Bb CHS4 gene in Batrachium bungei.