The Bioinformatics Analysis Of ?-mannosidases PgGH47 Gene Family In Panax Ginseng And Its Genetic Transformation Into Tomato

As the key enzyme for N-glycans modification of proteins in eukaryote,?-Mannosidase??-Man?could remove the mannose reside of glycan,complicate N-glycans of glycoprotein,which played a decisive role in protein folding.In plant cell,?-Man not only affects the growth and development of plants,but also affects the ripening and softening of the fruit.Based on the Jilin ginseng 240 thousand unigenes database of Jilin ginseng,this experiment takes ginseng glycosyl hydrolase 47 family unigenes gene?PgGH47s?from ginseng unigenes database by using plant glycosyl hydrolase 47 family gene found from the NCBI web site as query sequence.PgGH47s was carried out the bioinformatics analysis,according to bioinformatics analysis results,PgGH47-E was selected to clone and construct expression vector.PgGH47-E was transformed into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated method,and the gene function was prelimianrily verified.At the same time,Tomato was successfully Transformed,in order to further verify the relationship between the PgGH47-E gene and the synthesis of terpenoids.The results are as below:1.47 ginseng PgGH47s were obtained in this experiment.The PgGH47s belongs to I type?-Man.It also participates in many metabolic processes and has many biological functions.2.The expression of PgGH47s in ginseng possess tissue specific,the fruit stem is the most up-regulated tissue part of the gene,and the seed is the most down-regulated tissue part of the gene.The expression of PgGH47s in ginseng is influenced by environmental factors,and the trend of gene expression in different regions is obviously changed.The expression of PgGH47s in ginseng has time specificity,and the gene expression trend is different in the root of different years.3.The relationship between PgGH47-E,PgGH47-D02,PgGH47-D01,PgGH47-F01,PgGH47-F05,PgGH47-F07,PgGH47-F12,PgGH47-H,PgGH47-G13 and other key enzyme genes of ginsenoside synthesis was interacted.4.PgGH47-E is a type of Endoplasmic reticulumMan I,which hydrolyzed the terminal 1,2-?-D-mannose residues in the Man9GlcNAc2.The molecular function is to hydrolyze?-1,2manna glycoside bond and Ca²⁺binding.PgGH47-E exercise interaction with the key Enzymes of Saponin Synthesis,and There is a significant correlation between PgGH47-E expression level and ginsenoside content.5.The PgGH47-E gene was successfully cloned and the overexpression vector was constructed in this study.6.The positive Arabidopsis thaliana of T₂ generation were successfully obtained,PCR test products results were certificated transformation was successful.The analysis of the characters of T₂ positive plants in Arabidopsis thaliana found that:the bolting time was earlier,the flowering time was earlier,the seed pods was increased,the 1000-grain weight with increasing,the length of root is longer,and the time of seed preservation is shortened in Transgenic Arabidopsis.7.T₀ positive transgenic tomato plants were successfully obtained in this research,and PCR test results were prelimianrily determined the successful of conversion.Tomato Genetic Transformation System was established,and we found that optimum infection time was 10min,and the optimum concentration of bacterial concentration was OD₆₀₀=0.6.The he induction rate of resistant buds was 14.65%,and the conversion rate was 6.26%.The conversion rate of different tissues of the explants was statistically analyzed,and the conversion rate of cotyledon was significantly higher than that of the hypocotyl.The content of lycopene in PgGH47 is6.74mg/100g,which is more than that in wild type.