Expression Analysis Of GmMIPS Gene And Subcellular Localization Under SCN Stress

Soybean is an important crop in China that subjected to biotic and abiotic stress all year round,several reports have been studied about abiotic stresses such as salinity and srought,but few biotic stress.Soybean Cyst Nematode(SCN)is the main biotic stress diseases.Therefore,preventing and controling the demage of SCN has gradually become the focus of scientists‘ research.The traditional control methods is crop rotation and planted resistant varieties,but the traditional metods is causing the nematodes provide adaptation.With the development of fenetic engineering technology,screening resistant genes and cultivating resistant plants have become the main prevention and control methods,and achieved results.Myo-inositol phosphate synthase catalyzes the rate-limiting step in synthesis of inositol-1-phosphate by glucose-6-phosphate(G-6-P).Inositol is the main storage form phosphorus in plants and inositol can produce polysaccharides involved in cell wall synthesis through auto-oxidative metabolic pathways,mainly xylan and pectin.Therefore,increased the expression of MIPS gene can enhance the resistance of soybean plants to nematodes.The MIPS gene was found in soybean genome that named Gm MIPS.MIPS gene family have four members.In this study,the resistant cultivars Huipizhi Heidou,Harbin Xiao heidou and Xiaoli Heidou and the susceptible cultivar Liaodou 15 were infected by SCN3,RNA was extracted at differernt time.This study aim is to investigate the expression analysis of the candidate genes Gm MIPS1 and Gm MIPS2 under infected by SCN3,construct the transient expression vector and detect the subcellular localization of MIPS protein for the preliminary study.The main research results include:1.The length of Gm MIPS1 and Gm MIPS2 is 1533 bp and encoding 511 amino acids,both of which are stable hydrophilic amino acids,with 38 and 39 phosphorylation sites and strong phosphorylation ability.MIPS belongs to the upstream gene of inositol synthesis,involved in inositol biosynthesis and belongs to the inositol phosphorylation pathway.Among the 15 common plants,the closest relationship with Gm MIPS is the bean and mung bean,and the farthest relationship is corn and wheat.2.The results showed that SCN3 can infect the roots of soybean and colonize in it by acid fuchsin staining and continue to develop in third and forth instar larvae.Meanwhile,it found that the number of SCN3 infected susceptible plants was more than that of resistant varieties.3.The Gm MIPS1 and Gm MIPS2 genes from the the resistant cultivars Huipizhi heidou,Harbin Xiao heidou and Xiaoli Heidou and a susceptible cultivar Liaodou 15 were cloned by RT-PCR that the 1533 bp length.By real-time quantitative PCR method identified Gm MIPS1 and Gm MIPS2 genes‘ regulation under the stress of SCN3.The results showed that the expression of Gm MIPS1 and Gm MIPS2 in three resistant cultivars were increased and higher than the susceptible cultivar Liaodou 15 at 10 dpi,indicating that the Gm MIPS genes exerted resistance at 10 dpi and inhibited the development of SCN.4.Selected the c DNA from the experimental resistant soybean variety,constructed p MD19-Gm MIPS1 and p MD19-Gm MIPS2.After double enzyme digestion and plant binary expression vector p CAMBIA1303 connection,then constructed p CAMBIA 1303-Gm MIPS1 and p CAMBIA130-Gm MIPS2.5.Gm MIPS1 and Gm MIPS2 were expressed in the leaves of tobacco by agrobacterium-mediated tobacco transient expression system.Observed the fluorescent protein GFP by laser confocal microscopy.The subcellular localization result showed that Gm MIPS1 and Gm MIPS2 protein was located on the cytomembrane or cell wall.