Cloning,expression And Function Of Endogenous Glycoside Hydrolase 9 Family Genes In Procambarus Clarkii

Procambarus clarkii is environmentally adaptable and omnivorous,making full use of the abundant natural plant and animal bait in the paddy environment.Cellulases are a class of glycoside hydrolases（GH）capable of hydrolyzing cellulose and include a variety of enzymes that work in concert to effectively degrade polysaccharides such as plant cell walls.Cellulases are essential for the digestive metabolism of omnivorous and herbivorous animals.Glycoside hydrolases family 9（GH9）genes encode cellulases that are widely distributed in crustaceans,but studies are still limited to cloning,sequence analysis and preliminary enzymatic functional exploration.Previous studies in our laboratory have shown that a large number of GH9 genes exist in the genome of P.clarkii and gene family duplication events occur,but the function of this family of genes in the digestive process of P.clarkii and the effect of related gene duplication on crayfish are still unclear.In this study,we investigated the molecular characteristics,phylogeny and enzymatic properties of the GH9 family（PcGH9）of P.clarkii,analyzed the gene evolution and functional characteristics of the GH9 family.The main results of this study are as follows:1.Cloning and bioinformatics analysis of PcGH9sBased on the results of the genome sequence analysis of P.clarkii in our laboratory,nine PcGH9 genes were cloned,with open reading frames ranging from 913 to 1734bp in length and 304 to 578 amino acids in number.PcGH9s have a typical GH9 catalytic structural domain,in addition that the N-terminus of PcGH9＿1 also contains a carbohydrate-binding module family 2（CBM＿2）,the N-terminus of PcGH9＿3contains a serine protease gastrulation defective N-terminus（GD＿N）.However,the GH9 structural domain of PcGH9＿4 and PcGH9＿10 was incomplete,missing key catalytic sites and conserved motifs.These differences in structural domains between different genes may have arisen from genetic recombination and duplication of PcGH9s during evolution.The occurrence of these events increased the structural diversity of PcGH9 and may have led to the neofunctionalization of the PcGH9s protein.The results of phylogenetic tree construction showed that the crustacean GH9 genes formed a separate branch,forming a sister group with insects,and the PcGH9s of P.clarkii were within the crustacean branch.2.Analysis of the expression of PcGH9s in plant digestionReal-time quantitative PCR results showed that the expression of PcGH9s was significantly higher in P.clarkii continuously fed plant-based baits than continuously feding animal-based baits（P<0.05）,indicating that PcGH9s is involved in and plays an important role in the digestion and utilization of plant-based baits of P.clarkii.3.Prokaryotic expression and activity verification of PcGH9＿1,PcGH9＿4,PcGH9＿12 and PcGH9＿13In this study,the recombinant expression plasmids of PcGH9＿1,PcGH9＿4,PcGH9＿12 and PcGH9＿13 were successfully constructed and introduced into BL21（DE3）Escherichia coli hosts,and the recombinant proteins were successfully induced to be expressed.p ET-30a-PcGH9＿1,p GEX-4t-1-PcGH9＿4 and p ET-32a-PcGH9＿13 recombinant proteins could be expressed in the supernatant in a soluble form,and p ET-28a-PcGH9＿12 recombinant protein could only be expressed in the precipitate as inclusion bodies.The soluble protein of p ET-30a-PcGH9＿1 was detected by DNS method with endoglucanase activity of 0.194 U/m L using sodium carboxymethylcellulose as substrate,while the soluble proteins of p GEX-4t-1-PcGH9＿4 and p ET-32a-PcGH9＿13 and the inclusion body complex protein of p ET-28a-PcGH9＿12 were inactive.4.Protein purification and enzymatic properties of PcGH9＿1In this study,the induction conditions of the active recombinant protein p ET-30a-PcGH9＿1 was optimized,and the results showed that the p ET-30a-PcGH9＿1recombinant protein had an initial induction OD of 1.1 at 600 nm,an induction temperature of 16°C,and the highest soluble protein expression after 16 h of induction.The p ET-30a-PcGH9＿1 soluble protein was purified by nickel column affinity chromatography and anion exchange chromatography,respectively,to obtain pure enzyme with high purity.The enzymatic properties of PcGH9＿1 showed that with carboxymethyl cellulose as the substrate,the optimum p H for PcGH9＿1 to participate in the enzymatic reaction was 5.5 and the optimum temperature was 55℃,and it retained 62.92%after 3 h incubation at 55℃.The relative enzyme activity of Mn²⁺and Fe²⁺significantly increased 181.75%and 159.45%of the endoglucanase activity,respectively（P<0.001）.Fe³⁺and Cu²⁺inhibited the endoglucanase activity,with Cu²⁺significantly inhibiting the activity（P<0.001）and retaining only 32.70%of the activity.Besides,PcGH9＿1 was able to hydrolyze five substrates,carboxymethyl cellulose,microcrystalline cellulose,birch wood xylan,locust bean gum and apple pectin,with specific activities of 1.32±0.03,1.32±0.04,1.26±0.02,0.71±0.09 and 0.33±0.05U/mg,respectively,but p-nitrophenyl-α-D-glucopyranoside was inactive.This result suggests that PcGH9＿1 is a specific multifunctional enzyme with endoglucanase,exoglucanase,endo-xylanase,mannanase and pectinase.In summary,this study revealed that the GH9 gene family of P.clarkii may be newly functionalized and involved in the digestion and utilization of plant bait by C.cristata through gene rearrangement and duplication;PcGH9＿1 has multiple functions of endoglucanase,exoglucanase,endo-xylanase,mannosidase and pectinase activities.The results elucidated the structure and function of the GH9 family of P.clarkii,enriched the crustacean GH9 gene species,and revealed the mechanism of digestion and utilization of natural baits by P.clarkii in the paddy environment.