Investigate The Mechanisms Of Myo-Inositol Metabolic Regulated Agrobacterium-mediated Transformation Of Arabidopsis Root

In this study,we used the roots of Arabidopsis thaliana ecotype Col-0 cultured on medium with supplied or without myo-inositol(MI)and the roots of A.thaliana mutant atipkl that AtIPK1 gene was disrupted by T-DNA insertion as the receptors in Agrobacterium tumefaciens strain A208 mediated transformation.The phytate(IP6)is a terminal product of MI metabolism catalyzed by AtIPK1 gene encoded enzymes.We tried to explore the mechanism of sensitivity of Arabidopsis root receptors influenced by exogenous MI and AtIPKl gene of inositol phosphate(IPs)metabolic pathway in Agrobacterium-mediated transformation adopted morphological observation,high-throughput sequencing,real-time quantitative PCR,histochemical analysis,high performance ion chromatographic(HPIC)analysis and genetic transformation analysis methods.Our results include the following:Exogenous MI pretreatment significantly affected on the sensitivity of Arabidopsis thaliana root receptors to Agrobacterium-mediated transformation.The additional exogenous MI(MI+,100 mg/L MI)treatment promoted the transformation efficiency of roots.The expression level of genes had some timeliness before or after transformation.Addition of MI was beneficial to the growth and development of calli,and the expression level of plant defense and T-DNA integration genes was significant different in callus of.Compared with MI-treated roots and calluses of MI+ treatment,IP6 content is higher and Pi content is lower.These results suggest that exogenous MI play an important roles in regulation and maintenance of cell growth and development,and affects the material metabolism in plant cell.At least 184 new genes and 183 differentially expressed genes(DEGs)were excavated in two treatments(MI-and MI+)by transcriptome anlysis.Gene function annotations of DEGs are involved in the synthesis of plant cell wall,hormone signal regulation,stress response(plant defense),material transport,chromosome modification and DNA repair,secondary metabolic processes.These results indicated that exogenous MI treatments affected the transcription level of plant endogenous genes,and affected the molecular genetic metabolism of plants.Transformation efficiency of mutant atipkl was significantly different from that of wild type Col-0.The mutant atipkl could restore the wild type phenotype by overexpressed AtIPKI1.Although the mutant atipk2? was able to partially block the synthesis of some IP6,but the phenotype and transformation efficiency of the mutant atipk2? was not significantly different compared with the wild type Col-0.The sensitivity of receptor influenced by AtIPK1 to transformation may be related to Pi and IP6 metabolism and also play a role in genes regulation.Exogenous MI regulated gene expression in T-DNA integration(AtKu70,AtKu80,AtXRCC4),plant defense(AtWRKY62,AtWRKY70,AtPRl,AtPR2),associated with Agrobacterium-mediated transformation of plant root(AtCSLA9,AtAGP17),cell wall polysaccharide precursor synthesis(AtMIOX2)genes,and so on.The expression differences of these genes in the root receptor of atipkl and AtIPK1 overexpressing strains were more significant.These results indicated that the sensitivity of root receptor to Agrobacterium transformed is related to the regulation and expression of endogenous genes.Exogenous MI and improve expression of AtIPK1 are beneficial to Agrobacterium-mediated transformation of Arabidopsis root receptors.Exogenous MI and AtIPK1 can regulate the metabolic balance between IP6 and Pi,and can directly or indirectly regulate the expression of endogenous genes and the metabolism of the receptor,which has a significant effect on the sensitivity of receptor to transformation.The significant effect may be the results of synergistic work or work together with many factors.