Cloning And Transformation Carrot Stem Cells Research Of The PgPMEI09 Gene In Panax Ginseng

Pectinesterase?PME?is widely distributed in bacteria,fungi and advanced plant.It can catalyze the deesterification of methyl esterified pectin,Pectinesterase Inhibitor?PMEI?can specifically inhibit the deesterification of pectinesterase in plant.The results of previous research in our laboratory showed that PMEI may be involved in the regulation of ginsenoside synthesis,but the related research has not been reported.The relationship between carrot and ginseng is closer,and the culture pattern is simple.The synthesis of carotenoids and ginsenosides are use isoamyl pyrophosphate?IPP?as precursor,as well as plant stem cells having strong division or higher genetic stability,and plant stem cells could maintain stable proliferative speed.Therefore carrot stem cells can be used as receptor to study the related genes function of ginsenoside.In view of the above basis,this study screened Jilin ginseng PgPMEIs from Jilin ginseng248,993 Unigenes database.On the basis of system analysis,1 PgPMEI was screened according to the results.The target gene was cloned and the expression vector was constructed,using carrot stem cells as receptors,heterologous receptors were transformed to research the function of target genes.The results showed that:1.12 PgPMEIs were screened from 248,993 Unigenes database of 14 tissue and organs in Jilin Ginseng.The result of GO annotation showed that it is annotated to the molecular function regulator and biological regulation under the level of 2.The enrichment analysis showed there is a highly significant difference in biological regulation.2.Phylogenetic analysis of PgPMEIs and extraneous species showed that the evolution of PgPMEIs is later than that of carrot,and it also has good conservatism.3.The results of 12 PgPMEIs amino acid sequence alignment and conservative motif prediction showed that PgPMEIs had 4 conserved Cys and 1 conservative SNAL amino acid segments.4.The interaction between PgPMEIs and the genes of the key enzymes of saponins biosynthesis showed that genes didn't exist independently,but exist as a cluster,they work together to became a complete regulatory network and participate in the process of the growth of plants.And PgPMEI09 has the closest interaction with all genes in 12 PgPMEIs with the number of edges 8.5.The expression pattern analysis showed that the expressesion of PgPMEIs was not be influenced by farm varieties in ginseng.The expression pattern results in 4 different age of ginseng showed that the expression of PgPMEIs increased with the development stage.The expression pattern of PgPMEIs in 14 tissues showed that the most genes expressed in fruit peticle were 11 and the least expressed genes in stems were 6.There are 4 genes of the 12PgPMEIs participate in the expression in 14 tissues,and they are PgPMEI02,PgPMEI09,PgPMEI11 and PgPMEI12 respectively.6.Based on the results above all,PgPMEI09 was selected,the gene was cloned and the expression vector was constrcucted.7.Arabidopsis thaliana was transformed by Agrobacterium-mediated method to observe wild type and T₂ generation of Arabidopsis thaliana.The results showed that the bolting time and flowering time of the transformed Arabidopsis thaliana were delayed.The length of pod and the number of seed in each pod was less than that in the wild type,and the weight of 1000seeds didn't change obviously.The PME activity of transformed Arabidopsis thaliana was lower than that of the wild Arabidopsis thaliana.8.The carrot stem cell line was successfully induced and the regeneration system was also established.The result showed that WPM+IBA4 mg/L medium had the optimum induction effect on carrot stem cells,and the induction rate was 91.33%.MS+2,4-D 2mg/L+IBA 0.2 mg/L+KT 0.5 mg/L medium had the optimum proliferation effect.The stem cells and calli were inoculated on the base medium of MS,16 h light and 8 h dark alternate culture at 25?,the regeneration rate of stem cells was 95.50%,while the regeneration rate of calli was 89.25%.The results of biochemical indexes showed that the contents of soluble sugar,soluble protein,carotenoid and peroxidase?POD?activity of stem cells were higher than those in calli.9.Transformation of carrot stem cells and calli by Agrobacterium-mediated,the results showed that the transformation rate of stem cells was 39.64%,while the transformation rate of calli was 31.29%.The content of soluble sugar,soluble protein,PME activity and peroxidase?POD?activity in transformed carrot stem cells and calli were determined.The results showed that the content of soluble sugar and soluble protein content and PME activity were decreased in varying degrees,while the POD activity increased than the results before transformation.This study provides a reference for further research on functional genes of ginseng.