Construction Of Nitrogen-fixing Bacterium DX120E NifH Mutant Strain And Its Effect On Nitrogen Metabolism Of Sugarcane

Sugarcane is an important cash crop and sugar crop in Guangxi,its planting area and sugar production account for more than 60% of the country.Nitrogen is one of the important nutrients in plant growth and plays an important role in the growth process of sugarcane.The high input of nitrogen fertilizer increases the production cost of sugarcane as well as soil and environmental degradation.Biological nitrogen fixation is important to maintain sustainable crop development.Gram-negative bacteria Klebsiella Varicola DX120 E,isolated from sugarcane variety ROC22,which has the growth promoting characteristics,such as nitrogen fixing,phosphorus solubilizing and iron philophilic.DX120 E can be combined with sugarcane for nitrogen fixation and has a promoting effect on sugarcane.However,the effect of DX120 E nitrogenase nifH gene deletion on nitrogen fixation and sugarcane growth has not been reported so far.In this study,the mutant and complement strains of DX120 E nitrogenase gene nifH were constructed,and their physiological and biochemical characteristics as well as gene and protein expression differences were analyzed.Meanwhile,The mutant and complement strains were inoculated with sugarcane to study their growth effects on sugarcane.The main results are as follows:1.The mutant strain ΔnifH and the complementary strain C(ΔnifH)of DX120E(WT)were successfully constructed by deletion and complementation of the nitrogen fixing enzyme nifH gene using CRISPR/Cas9 technology.The nitrogenase activity of the strains showed that nifH mutation reduced the nitrogenase activity of DX120 E.The nitrogen-fixing enzyme activity of the complementary strain was not significantly different from that of wild bacteria.2.Different nitrogen sources had different effects on the growth of wild bacteria(WT),mutant ΔnifH and complement bacteria C(ΔnifH).The growth trends of the three strains were similar in basic LB medium,but the growth of strain ΔnifH was inhibited when cultured in a medium with urea,sodium nitrate and ammonium sulfate as the only nitrogen sources respectively.3.The results of the experiments on the utilization of different carbon sources and chemical sensitivity by the three strains showed that the mutation of the nifH gene did not change the types of carbon sources utilized by the nitrogen-fixing bacterium DX120 E,and the wild type,mutant and backfill strains and they were all able to utilize 36 carbon sources such as dextrin,D-maltose,D-algose,sucrose and cottonseed sugar.And meanwhile,they were sensitive to p H6,1% Na Cl,1% sodium lactate,acedomycin and rifamycin.4.The q PCR analysis of nitrogen fixation-related genes in the three strains showed that the relative expression of nif D and nif K genes in C(ΔnifH)was up-regulated by 1.45-fold and 1.62-fold,respectively,compared with WT under the culture conditions with sodium nitrate as the nitrogen source;The expression of nif D in strain C(ΔnifH)was up-regulated 1.19-fold compared to WT and 2.27-fold compared to ΔnifH when urea was used as the nitrogen source.When ammonium sulfate was added as a nitrogen source culture,the relative expression of nif D gene in C(ΔnifH)was up-regulated by 3.35-fold compared to WT,while the expression of nif K gene was up-regulated by 8.62-fold.Western bolt results showed that the protein expression level of ΔnifH was lower than that of WT,while C(ΔnifH)was higher than that of WT.5.Inoculating wild-type strain could increase plant height and chlorophyll content of two sugarcane varieties under two fertilization levels.The sugarcane plant height of ΔnifH treatment was lower than that of WT treatment,and the sugarcane plant height of C(ΔnifH)treatment was not significantly different from that of WT treatment.Inoculation with wild strains increased nitrate reductase,glutamine synthetase,sucrose synthase and sucrose phosphate synthase activities in sugarcane variety B8 and ROC22 leaves to different degrees,and the nitrate reductase activity of sugarcane variety B8 was generally higher than that of variety ROC22,while treatment with the nifH gene mutant strain reduced the activities of several related enzymes mentioned above.The sucrose synthase activity of both sugarcane leaves in WT treatment was higher than that in CK and ΔnifH treatments at both nitrogen application levels,while the difference in sucrose synthase activity between WT and C(ΔnifH)treatments was not significant.The contents of nitrate nitrogen and ammonia nitrogen of leaves in both sugarcane varieties inoculated with the wild-type strain were higher than that of both the CK and ΔnifH treatments.The nitrogen content of sugarcane in the ΔnifH treatment in ROC22 was lower than that in the WT treatment under both fertilization conditions.