Establishment Of Asexual Propagation System And Cloning Of ChST2A Gene In Cardamine Hupingshanensis

Selenium,the essential trace element of human body,ⁱs the same groups the same group element of sulfur and has similar chemical properties with sulfur element,they share most of the absorption and metabolic pathways in plants.The use of3'-phosphorylated adenosine 5'-phosphoryl-selenate as a donor to transfer selenate to hydroxyl-containing biomolecules is said to be selenization,the chemistry and process being similar to sulfonation.The sulfotransferase?ST?that catalyzes the sulfonation process in plants can sulfonate hydroxyl-containing biomolecules with adenosine3?-phosphate sulfate as the sulfonyl donor.The transcriptome sequencing and transcriptional differential analysis showed that the ChST2A gene was sensitive to high concentration of selenium,which combined with the function of ST family gene and the results of preliminary basic research.It is inferred that ChST2A may be involved in selenization of Cardamine hupingshanensis.In order to study the function of Ch ST2A in selenization of Cardamine hupingshanensis,the asexual reproduction system of Cardamine hupingshanensis was established,then the ChST2A sequence was cloned.After preliminary bioinformatics analysis,the expression product of ChST2A was purified after prokaryotic expression.The purification conditions were also studied.The main findings of the study are as follows:1.Establishment of asexual propagation system of C.hupingshanensis.1)Callus induction of C.hupingshanensis.The callus was induced by using roots,stems and leaves of sterile seedlings as explants,MS medium as the basic medium,plant hormones such as TDZ,6-BA,NAA and 2,4-D as inducements.The results show that:_?1?The induction efficiency of four different media combinations was as follows:TDZ+NAA>6-BA+NAA>TDZ+2,4-D>6-BA+2,4-D;_?2?The induction rate of MS+TDZ?0.8?g/mL?+NAA?0.4?g/mL?was the highest 89.37%;_?3?The seedling age of C.hupingshanensis had a significant effect on callus induction.With the increase of seedling age,the highest rates of callus mortality and browning were46.75%and 64.94%,respectively;_?4?The callus induction rates of different explants were different.The induction rates of leaves and stems to roots were significantly increased to 75.25%and 52.45%,respectively.2)Induction of adventitious buds.The sterile seeds and callus were cultured in MS medium,the former was supplemented with TDZ and NAA,the latter was supplemented with TDZ,NAA and 6-BA to induce adventitious buds.The results showed that the MS+TDZ?0.4?g/mL?+NAA?0.3?g/mL?concentration can achieve the best differentiation rate of 79.54%and the callus was more favorable for adventitious bud differentiation in MS+TDZ?0.4?g/m L?+NAA?0.4?g/mL?+6-BA?1?g/m L?.3)Adventive buds take root.The MS medium was used as the base medium,with different concentrations of NAA,sucrose,to explore the rooting of adventitious buds of C.hupingshanensis.The results showed that at 1/2MS+IBA?1.5?g/mL?,the average number of rooting shoots was 10.5 slip/bottle,the average root length was12.42 mm/root and the rooting index was 4.63 with 1/2MS+IBA?1.0?g/m L?,which was the best medium.2.Cloning of ChST2A Gene from C.hupingshanensis.The full-length cDNA of ChST2A was cloned using rapid amplification of cDNA ends?RACE?,and the fragments were spliced using Contig Express software.The full-length Ch ST2A was amplified by RT-PCR and sequence analysis was performed using MEGA 7.0 software maximum likelihood method,and the open reading frame was analyzed by GENSCAN.The physical and chemical properties,hydrophilicity,subcellular localization and transmembrane structure,signal peptide analysis,and spatial structure and activity centers were predicted by ProtParam,PSORT II Prediction,ProtScale,NetPhos 2.0 Server,BioEdi,SignalP 4.1 Server,TMHMM Server v.2.0 software,SWISS-MODEL and Maestro The results show that:1)Molecular phylogenetic analysis showed that ChST2A was closely related to AtST2A,so the sulfotransferase was renamed ChST2A.2)The total length of the gene is 1797 bases,and the open reading frame encodes 365 amino acid,and the 3'and 5'untranslated regions with 377 bases and 322 bases,respectively.3)The molecular size of ChST2A protein is 42.08 kDa,the theoretical isoelectric point is8.09,there are 40 possible phosphorylation sites,without transmembrane domain and signal peptide,this protein is a hydrophilic protein located in the cytoplasm.There are four characteristic structural regions with highly conserved sulfotransferase family.4)Because the structure of AtSOT18 gene in Arabidopsis is the most similar,the three level structure is modeled on the basis of the 5MEK.1 structure.The catalytic site of enzyme activity center analysis is Lys¹⁰⁵?Thr¹⁰⁸?Thr¹⁰⁹?His¹⁷²,and the binding site with small molecule PAP is Lys¹⁰⁵?Gly¹⁰⁷?Thr¹⁰⁸?Thr¹⁰⁹?Trp¹¹⁰?Arg¹⁹⁴?Ser²⁰²?Tyr²⁶⁰?Arg³²⁶?Lys³²⁷and Gly³²⁸.3 Prokaryotic expression and product purification of ChST2AThe cloned ChST2A was codon-modified according to the preference of Escherichia coli,and the overexpression vector was constructed with pET-30a vector and transformed into E.coli BL21?DE3?.The recombinant protein molecular weight was analyzed by SDS-PAGE electrophoresis of the supernatant and precipitate of the supernatant collected from the supersonic broken cells induced by IPTG at 16_?for16 hours.The results showed that ChST2A could be successfully expressed by the engineered strain,the molecular weight of the protein was about 42k by SDS-PAGE with inclusion body expression,and the optimum concentration of imidazole elution by Ni²⁺gel affinity chromatography was 60 mm,and the protein content could reach50 mg/L.