Study On The Mechanism Of Promoting Growth Of Enterobacter Cloacae DJ Under Saline-alkali Stress

Plant probiotics（PGPR）are a class of bacteria present in the inter-root environment of plants that can promote plant growth and development and improve plant tolerance to abiotic stresses through a variety of mechanisms.We isolated the polyamine-producing plant probiotic bacterium DJ from the inter-rhizosphere soil of Leymus chinensis（Trin.）Tzvel.in the western grassland region of Jilin Province.The results of probiotic experiments showed that DJ could promote the growth of cucumber seedlings under saline conditions,and the results of high performance liquid chromatography showed that the content of putrescine and spermidine in the supernatant of the bacterium was elevated under saline conditions The results of high performance liquid chromatography（HPLC）showed that the levels of putrescine and spermidine in the supernatant of the bacterium were increased under saline conditions.Through the research of this project,we hope to reveal the adaptation mechanism of the growth-promoting bacterium DJ under saline conditions and to investigate the growth-promoting effect of the bacterium under saline conditions.（1）The DJ bacterium was identified using molecular biology techniques,and the results showed that the bacterium was E.cloacae.The growth of the bacterium was followed under different p H conditions with different substrates as nitrogen sources,and the concentration of the bacterium with arginine as nitrogen source was higher than that with ornithine as nitrogen source in different time periods,which showed that the growth of DJ was better in the medium with arginine as nitrogen source.The results showed that the p H of the culture medium was decreasing in the period of 2-8 h,which proved that DJ bacteria would produce some acidic substances at the beginning of the culture period,and with the extension of the culture time,the final p H of the control group and the saline group was about p H 9 at 24 h,and the biomass of the bacterium can reach 0.9(OD₆₀₀),indicating that DJ bacteria are more adapted to grow in alkaline conditions,the above results reveal that DJ bacteria have strong saline-alkali adaptability.（2）Based on the above findings,the flow plate experiment was used to further verify the tendency of DJ bacteria to saline and alkaline substances.In the saline-alkali medium（30 m M Na Cl,p H 9）,the diameter of DJ colonies was 3.3 cm on average,which was significantly different from that of 1.9 cm in neutral medium.In order to further investigate the molecular mechanism of saline-alkali adaptation of DJ bacteria,proteome sequencing was performed on the bacterial broth in neutral and saline-alkali culture conditions,and the sequencing results revealed a total of 116 up-regulated proteins and 72 down-regulated proteins related to chemotaxis,signal transduction,transmembrane signaling receptor activity,which were mainly involved in amino acid metabolism,carbohydrate metabolism,cell motility These proteins are mainly involved in amino acid metabolism,carbohydrate metabolism,cell motility,energy metabolism,membrane transport processes and other pathways.In this study,we performed validation experiments on the genes of chemotactic proteins Che Y and Che Z,and methyl acceptor chemotactic protein MCP,which are related to salinity adaptation of DJ bacteria.The fluorescence quantitative PCR results showed that the expression of Che Y,Che Z,and MCP genes were significantly higher compared with the control group.Based on the experimental results,we hypothesized that DJ improves the behavioral response of bacteria to adapt to the external environment by up-regulating Che Y,Che Z,and MCP gene products under saline conditions,so that the bacteria can better adapt to the saline environment.（3）High performance liquid chromatography detected the changes of polyamine content in DJ bacterial broth under neutral culture conditions and saline-alkali culture conditions,and the results showed that the culture broth of DJ bacteria was dominated by putrescine and spermidine,and the content of putrescine in the supernatant of the bacteria undersaline-alkali culture conditions was 4.73μg/m L,which was significantly different from that of the control group of 3.02μg/m L;the content of spermidine was46.18 ng/m L,which was significantly different from that of the control group of 0.34ng/m L.Therefore,we examined the expression changes of polyamine-producing genes ADC,ornithine decarboxylase ODC,and S-adenosylmethionine decarboxylase SAMDC under saline conditions,and the results showed that the expression of ADC,ODC,and SAMDC genes were significantly up-regulated compared with the control group,and thus their putrescine and spermidine contents were increased.（4）A preliminary study on the growth-promoting effect of DJ bacteria under saline-alkali conditions showed that DJ bacteria could significantly increase the fresh weight of seedlings of cucumber plants under saline-alkali stress conditions,and the average fresh weight of seedlings in the experimental group was 0.42 g compared with0.28 g in the control group.It was found that the number of lateral roots of cucumber seedlings increased after the DJ bacterium dipping,and the average number of lateral roots of seedlings in the experimental group was 17/plant,while the average number of lateral roots of seedlings in the control group was 14/plant,and all the indexes of the measured seedlings were significantly different compared with the control group.Afterwards,the fluorescence quantitative PCR results showed that the expression of polyamine transporter protein PT was significantly increased in cucumber seedlings under saline conditions,while the changes of their antioxidant enzymes SOD,POD and CAT activities were analyzed by enzyme-linked immunoassay,and the results showed that its SOD content of cucumber seedling leaves under saline-alkali conditions was 13.26 ng/m L,which was significantly different compared with the control group of 6.48 ng/m L;POD content was 5.84 ng/m L,which was significantly different compared with the control group of 1.97 ng/m L;CAT content was 18.53ng/m L,which was significantly different compared with the control group of 7.34ng/m L,indicating that the treatment of DJ bacteria could improve the antioxidant capacity of cucumber seedlings under saline-alkali stress,thus promoting the growth of cucumber seedlings.