Isolation Of LORE1 Insertion Mutants And Interaction Between BAK1/SERK3 And SymRK In Lotus Japonicus

Legume is a kind of important food,feed and biofuel crops.With a few exceptions,they can form a complex symbiotic system with a special soil bacteria-rhizobia,and lead to the formation of a new root organs-nodule.Rhizobia in the nodule can convert the nitrogen from the air into available fixed nitrogen.In recent years,based on the research of the symbiotic nitrogen fixation signaling pathways,researchers isolated and identified a series of symbiotic related genes,such as SymRK,SIP1 and PSK.Using leguminous plants Lotus japonicas as the research object,we got 12 SymRK,SIP1 and PSK related gene LORE1 insertion mutants by searching the Lotus Retrotransposons 1 insertion(LORE1)mutants library(http://users-mb.au.dk/pmgrp).Through identifying the symbiosis phenotype of those mutants,the results of this study provided material for the further reveal of SymRK,SIP1 and PSK function and regulatory mechanism in rhizobium infection and process and development of nodules organs.This research mainly results are as follows:1.Trough PCR amplification to separat and identify the mutants,in addition to pskr(30005459),rop-like2 and rop-like3,the other mutants got homozygotes and collected different homozygote seeds of M2 generation.2.In rop-likel and rop-like4 mutants,rhizobium infection was affected.Through statistical analysis and identification of M2 generation in early symbiotic phenotype,we found that compared with wild type,root nodules primordias numbers of rop-likel declined obviously,both the infection threads numbers per plant and infection threads numbers per centimeter of rop-like4 homozygote were significantly decreased.3.The process of nodules' formation was affected in rop-like1,pskl and psk4.Through identification and statistical analysis of M2 generation in nodulation phenotype,we found that the nodule numbers of pskr,rop-likel and rop-like4 decreased significantly,and the numbers of nodule per centimeter in pskr induced obviously.For the further exploration of the connection between symbiotic signaling pathways and immune system,we took the Arabidopsis SERK3/BAK1 as basis and got L.japonicus SERK3/BAK1,SERK2 through searching and analyzing the sequence of amino acids in model legume L.japonicas database.Using yeast Y2H experiment to identify the interacting proteins with LjSERKs.At the same time,we also introduced a new report protein of split-ub system and 2inl cloning vector system.These two systems were convenient for other subsequent research of protein interaction.The main results are as follows:1.Using of yeast Y2H system,the results showed that LjSERKs can specificly interact with LjSymRK;SERK3/BAK1 and SymRK interactions among different legume species is conservative;Inactivating mutation of BAK1 phosphorylation suppressed its interactions with SymRK;we didn't detect the interaction between LjBAK1 and LjFLS2,BRI1 in yeast.2.Using LexA-VP 16 as reports protein split-ub system proved that TM-SymRK(shortened to the transmembrane domain)interacted with LjBAKl/SERK3 and LjSERK2.3.Using Gateway technology,we constructed 8 different pFRET-SymRK-SERKs restructuring vectors,and transformed pFRETgc-SymRK-SERKs into protoplast for c colocation.