Expression Of The Thermophilic Endoglucanase Gene (E1) In Plants

Through modern bioengineering technology,the use of large-scale production of cellulolytic enzymes by transgenic plants has provided feasibility for the efficient conversion and resource utilization of lignocellulose.Expressing cellulase in plants can promote cell wall depolymerization,effectively reducing the addition of commercial enzymes and the cost of lignocellulose degradation.In the face of technical challenges of cellulase activity at room temperature interfering with the growth and development of host plants,genetic transformation using thermophilic cellulase genes is an important alternative strategy.In order to promote the high expression of cellulase in plants,five plants were constructed using the thermophilic endoglucanase gene(E1;EC 3.2.1.4)derived from Acidothermus cellulolytics.Five plant expression vectors can promote the expression of cellulase in different subcellular organelles including cytoplasm,extracellular,endoplasmic reticulum,chloroplast,and mitochondria.The main findings are as follows:1)Codon optimization of the thermophilic endoglucanase gene E1 derived from Acidothermus cellulolytics to eliminate rare codons,enhance the stability of enzyme activity and promote the efficiency of recombinant cellulases expression.2)Plant binary expression vector pBINPLUS-ImpactVectors-E1 constructed with five different subcellular(cytoplasmic,extracellular,endoplasmic reticulum,chloroplast,mitochondrial)localization signal peptide genes using codon-optimized E1 gene.3)In combination with the labeling function of green fluorescent protein and the localization function of different subcellular signal peptides,the transient expression system was successfully constructed using E1 gene and transient expression was achieved in tobacco and onion epidermis.Inverted fluorescence microscopy revealed that the target protein with GFP fluorescent protein and different localization signal peptides was fully expressed in its corresponding subcellular organelles.4)The E1 gene was transformed into plants such as Arabidopsis thaliana by Agrobacterium-mediated transformation using the constructed plant expression vector,and positive transgenic plants were identified by PCR.5)The enzymatic properties of the recombinant thermophilic endoglucanase in transgenic Arabidopsis thaliana were characterized and it was found that both recombinant endoglucanases with endoplasmic reticulum and chloroplast-expressed signal peptides have quite good temperature and acid-alkali stability.The enzymatic properties analysis showed that the optimal activity temperatures of the two recombinant cellulases were around 60°C,and the optimum pH were all around 5.5.Compared with recombinant endoglucanases with endoplasmic reticulum signal peptides,recombinant endoglucanases with chloroplast-expressed signal peptides have higher enzymatic activity.