Effects Of Pseudomonas Putida To 1-amino Cyclopropane-1-carboxylic Acid And Ethylene The Chemotacticity On Its Colonization In Wheat Rhizosphere

As the main microbial flora in microbial fertilize,plant growth-promoting rhizobacteria(PGPR)can not only increase the crop yield,but also significantly improve the ability of stress and disease resistance of plants,thus PGPR is a better substitute for chemical fertilizers.However,in the practical application process,the effect is often unstable,and the mechanism is not yet clear.The first step or the most critical step for PGPR promoting plant growth is the responding and chemotaxis of PGPR to the root exudates of plants and moving to the rhizosphere.Whether the root exudates 1-aminocyclopropane-1-carboxylic acid(ACC)and ethylene are the key substances affecting PGPR colonization in the plant rhizosphere has not been reported.This article has studied this issue and the main results are as follows:(1)Screening of ethylene chemoreceptor gene of UW4.On the basis of previous studies about ethylene chemoreceptor gene(ecp),through Clustal Omega homologous sequencean phylogenetic tree identified the sequence of ecp of UW4,which has high homology rate compare with the ethylene chemoreceptor of Pseudomonas aeruginosa;then an ethylene chemoreceptor gene(Gen Bank: AFY18818.1)were screened out.Meanwhile,we analysis its conservative functional domain,its transmembrane helix structure has been determined by TOPCONS;Through CD-Search analysis,we identified it with HAMP and signaling domains;And analysis by the Phyre2 server confirmed that it had the N-terminal ligand binding domains.(2)Construction of ethylene chemoreceptor gene delete mutant(UW4-ecp-)and functional complemented strain(UW4-ecp-+p BBR-ecp).Firstly,through PCR technology clone the upstream and downstream homologous arms of ethylene chemoreceptor gene.The homologous fragments of the upstream and downstream homologous fragments were fused by overlapping PCR and ligated to p EASY Blunt E1 for sequencing.The sequencing results showed that the fusion fragments were amplified correctly.The homologous recombination plasmid p EX18Gm-F was then obtained by connecting the fusion fragments to p EX18 Gm.Then the p EX18Gm-F was transformed into UW4 by double parental hybridization.The result of PCR and Southern blot showed that the ecp amplification result was negative and the hybridization band was reduced 2.1 kb equal to the size of ecp.Which illustrate that the mutant strain UW4-ecp-was obtained.Then the ecp was inserted into p BBR1MCS2 to construct a functional complement plasmid p BBR-ecp.The p BBR-ecp and p BBR1MCS2 were transformed into UW4-ecp-,and the PCR detect results showed that the p BBR-ecp and p BBR1MCS2 were transferred to UW4-ecp-,and successfully obtained the ecp functional complement strain(UW4-ecp-+p BBR-ecp)and control strain(UW4-ecp-+p BBR1MCS2).(3)Chemotaxis detection.The typical chemotaxis substances arginine,succinic acid,ethylene and ACC were selected and detect the chemotaxis of UW4,UW4-ecp-,UW4-ecp-+p BBR-ecp,UW4-ecp-+p BBR1MCS2 and UW4-acd S-,respectively.The assay results showed that the UW4-ecp-and UW4-ecp-+p BBR1MCS2 had 30% lower ethylene chemotactic ability compared to UW4;UW4-acd S-completely lost chemotactic ability to ACC;There was no significant change in the chemotactic ability of UW4-ecp-+p BBR-ecp to the four chemoattractants.In addition,there was no difference in the chemotactic abilities of each strain to arginine and succinate.(4)The anlysis of specific growth rate of each strain.After comparing the specific growth rate of the following strains,UW4,UW4-ecp-,UW4-ecp-+p BBR-ecp,UW4-acd S-,it was found that there was no significant difference in the specific growth rate between the these four strains(p< 0.05).(5)Effects of each strain on the biomass of wheat root.The wheat respectively co-culture with UW4,UW4-ecp-,UW4-acd S-,UW4-ecp-+p BBR-ecp for 21 days,then measured the fresh and dry weight of wheat roots,We found that compared with the UW4,the UW4-acd S-has significant reduced the promot effect on the growth of wheat(P<0.05).While UW4-ecp-,UW4-ecp-+p BBR-ecp were not present a difference(P < 0.05).(6)Determination of the colonization of each strain on wheat rhizosphere.After respectly co-culture the strains with wheat under sterile conditions for a period of time,the colonization of each strain on wheat roots was determined.The method used in this process was to extract the microbial genome from the root of wheat,analyzed the copies mount of gyr B of the Pseudomonas putida by Taqman.The final results showed that: compared with the colonization amount of UW4,UW4-ecp-and UW4-ecp-+ p BBR-ecp in the rhizosphere of the wheat were not significantly decreased(P <0.05).The colonization of UW4-acd S-in the rhizosphere of wheat was significantly reduced by 10 times,which was significantly different from the colonization amount of UW4(P<0.05).After these comparison,it was found that the chemotaxis to ACC had a more great influence on the colonization of UW4 in wheat rhizosphere.In summary,after loss of the chemotaxis to ACC,UW4 significantly reduced the growth-promoting effect,and the rhizosphere colonization amount of bacteria in wheat significantly decreased,but there was no significant change after the loss of the chemotaxis to ethylene.It is suggested that the chemotaxis to ACC plays a key role in influencing UW4 colonization in the rhizosphere of plants,and ACC may be a key substance affecting PGPR colonization in the rhizosphere of plants.