| E.ulmoides(Eucommia ulmoides Oliver),a deciduous tree of Eucommia genus of Eucommiaceae.,is a dioecious and valuable gun-producing plant with important economic and medicinal value.The tea made from the male flower of E.ulmoides is a natural drink with abundant nutrition and natural active ingredients.Gutta-percha resistance to acid,alkali and seawater corrosion has good insulation,and it can produce high strength submarine cable and high strength adhesive.The seed coat of female E.ulmoides is rich in gutta-percha with high extraction rate and less impurities,and the seed bark of which gutta-percha is usually used to extract gutta-percha.Therefore,the cultivation of E.ulmoides had a certain gender preference according to production and application.However,E.ulmoides,the perennial woody deciduous trees,is usually not able to flower until 10 years after planting to identify their sex.Therefore,it is difficult to select male and female of E.ulmoides.Grafting is a kind of asexual reproduction technology,and gender selection by grafting has been introduced to production.The survival rate of grafting is affected by the degree of cell fusion between stock and scion.Relative research showed that β-1,4-glucanase can help the cell fusion between stock and scion of grafted plants.However,the extraction process of β-1,4-glucanase from plants is complicated and difficult with many impurities and high cost.While yeast engineering bacteria has some advantages such as easy operating and purification,low cost,high activity and mass production when expressing exogenous proteins.Pichia pastoris expression system is a commonly used as protein expression system,which has the advantage of performing post-translational modification for the expression of proteins and obtained via higher biological activity compared with prokaryotic expression system.In addition,it is relatively suitable for high volume protein production.Thus,the study aims to develop a yeast engineering bacterium to produce enzymes that can improve the survival rate of grafting and provide an auxiliary tool for grafting.To this end,this study intends to express E.ulmoides β-1,4-glucanase by pichia pastoris expression system and apply β-1,4-glucanase to E.ulmoides graft.The main results of this study are as follows:1.Cloning and bioinformatics analysis of E.ulmoides β-1,4 glucanase geneIn this study,the gene encoding β-1,4-glucanase in E.ulmoides was successfully cloned by RT-PCR.The total length of the gene encoding 638 amino acids was 1917 bp.The analysis showed that the predicted molecular weight of the protein encoded was 70.20 KDa,there was a transmembrane domain between 84-106 amino acids,and the functional domain was between 121-601 amino acids.There were seven glycosylation sites in total,but no signal peptide.The predicted results of secondary structure showed that the putative protein contained an α helix(31.94%),βrotation(1.88%)and random curl(52.82%).The predicted results of physicochemical properties of the protein showed that the theoretical isoelectric point of the protein was 5.63,the total negative charge residue base(Asp+Glu)was 71,and the total positive charge residue base(Arg+Lys)was 55.In terms of protein stability,the instability index was 41.74,indicating that the protein was unstable.The average hydrophilic index was-0.313,and it belongs to hydrophilic protein.2.Preparation of yeast engineering bacteriaBased on bioinformatics analysis to optimize E.ulmoides β-1,4-glucanase and create yeast engineering bacteria.The results showed that there was a transmembrane domain found outside the functional domain interval.In order to improve the expression success rate of yeast engineering bacteria,the target protein was expressed after removing the transmembrane domain.In order to have better expression effect in yeast engineering bacteria,the codon encoding E.ulmoides β-1,4-glucanase was optimized according to the codon preference of pichia pastoris.The optimized gene was constructed on the signal peptide vector p PIC9 K,and then transferred into the pichia pastoris strain GS115.In this study,a total of 10 positive clones were obtained,and they were used for fermentation and enzyme production.The protein expression of the ten positive strains were detected through Western Blot method.The results showed that E.ulmoides β-1,4-glucanase could be expressed under the conditions of temperature 28.5 ℃,rotation speed of 220 rpm and methanol addition of 1% in induction medium for 70 h.3.Expression and purification of E.ulmoides β-1,4-glucanase proteinThe molecular weight of the target protein was found to be between 130 and 250 KDa,while the predicted molecular weight was 60 KDa.The actual molecular weight of the target protein was higher than the predicted molecular weight,indicating that the expressed protein might exist in the form of a polymer.SDS-PAGE and Western Blot results showed that E.ulmoides β-1,4-glucanase protein was highly glycosylated protein.The results were consistent with the polymer and strong glycosylation of the enzyme under natural conditions.To further verify whether the expressed protein is the target protein,its protein spectrum analysis is performed.A total of 24 peptides were identified though mass spectrometry,and all analyses provided the protein sequence coverage of 67% in total.Combined with 6X His tag of the fused target protein as antigen,western blotting assay was performed to detect the expression level of the protein band via specific antibody,indicating that the expressed protein is the target protein.The fermentation conditions of β-1,4-glucanase secreted in pichia pastoris were optimized.The results showed that the induction of the fermentation could be terminated after 96 h to shorten the fermentation period.The optimal initial p H value was 6,and the optimum induction temperature was 28℃.The optimum methanol content is 1.5%,β-1,4-glucanase was induced by pichia pastoris and cultured for30 L under the optimum fermentation conditions.The crude enzyme liquid produced in fermentation was separated and purified via NI-NTA.SDS-PAGE results of purified protein showed that the protein was shown as a single band.Combined with6 X His tag of the fused target protein as antigen,western blot was performed to detect the expression level of the protein band by specific antibody,indicating that the purified protein is the target protein.The enzyme activity of the target protein was286.35 U/ml.The target protein purified from 30 L fermentation broth was lyophilized to obtain 29.46 g protein powder.4.Application of β-1,4-glucanase in grafting of E.ulmoidesGrafting is a form of asexual reproduction in plants.Studies have shown that β-1,4-glucanase contributes to the cell fusion of rootstock and scion cells in grafted plants,thus increasing the graft survival rate.In this study,β-1,4-glucanase produced by fermentation was applied to E.ulmoides graft,and commercial β –glucanase with mixβ-1,3-glucanase and β-1,4-glucanase was also applied to E.ulmoides graft for comparison.The results showed that compared with the control group,the experimental enzyme concentration could improve the survival rate of E.ulmoides grafting,and in the case of the highest germination rate,the concentration of β-1,4-glucanase was lower than that of β-glucanase. |
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