| Symbiotic nitrogen-fixing can support30%～50%of the nitrogen requirement for legumes. Artificial inoculation of Rhizobium can increase soil nitrogen content, reduce fertilizer useing, and increase fresh yield or nitrogen fixation. But indigenous Rhizobia may limit the Rhizobium rhizosphere colonization and nodulation capacity, resulting in failing to achieve the effect, screening a fine strain with high competitive nodulation can solve this problem. The process of symbiotic nitrogen fixation is regulated by a highly coordinated signal exchange between host and Rhizobium genes. Genes expressed in the process help to understand the regulation in the process of nodulation, which can provide a new way to enhance the efficiency of nodulation and nitrogen fixation.In recent years, competitive nodulation received extensive attention, nodulation-related and the legume host restrictive nodulation genes have been studyed. In the first chapter, we use Ningbo Bridge as the host plant to study competitive nodulation between7653R and other seven Mesorhizobium huakuii strains (Z1-Z7) taken from different soil regions of Zhejiang, the eight strains can nodulate with plant when fifteen days but the strains#8～#10can’t. Useing plant inoculated by sterile water as blank control, we adjust density of eight strains to106CFU/mL,107CFU/mL, and inoculate singly or1:1mixed. After30days, record the number and weight of nodules, the rate of nodules containing different strains and colonization ability to study the competitive nodulation between7653R and other seven strains. In the rate of nodules containing different strains,7653R and Z2～Z7are similar far higher than Z1; in the majority nodules containing two kinds of Rhizobia, the two strains have similar colonization.Rhizobium genes expressed in vivo plays an important physiological role in the process of nodulation and nitrogen fixation, affecting the efficiency of nitrogen fixation and competitive nodulation. In the second chaper, using ZN1(Derivative of R.etli CFN42wild-type, Spontaneous SmT) and ZNO (Derivative of M.loti NZP2213wild-type, Spontaneous Smr), we try to construct the system to screen rhizobium genes expressed in VlVO.Early, we attempt to construct a system based on antibiotic resistant label to screen genes expressed in vivo. Using pJZ260(transposon containing the promoterless Km resistant gene)as a starting plasmid, we construct pZNN121(Km resistant gene replaced by Gen resistant gene on the transposon). In vitro, using Km500μg/mL to deal with ZN1, ZNO2hours respectively, the mortality rate is97%,78%; using Gen75μg/mL to deal with ZN1for2hours, the mortality rate is99.9%, and using Gen25μg/mL to deal with ZNO for1hour, the mortality rate is99.9%. Gen resistance label is feasible preliminarily. In vivo, using Gen to deal with ZN1, the mortality rate is not stable, and sometimes even below70%; using Gen100μg/mL to deal with ZNO for2hours, the mortality rate is99.9%stably. The strain ZNO can be used in this study initially.Through conjugation we get the mutants library ZK with Gen resistant gene from ZNO, whose genome are inserted by transposon. We use five mutants as negative controls, they are ZK1, ZK2, ZK3, ZK4and ZK5, inserted by transposon on different sites.In vitro, the five mutants are dealed respectively by Gen100μg/mL,200μg/mL,500μg/mL for2hours, the mortality rate are33.49%,23.74%,18.76%, indicating that when Gen resistance gene are inserted in reverse direction, the mutants ZK still have a certain resistance to Gen. It is bound to produce a large number of false-positive mutants, the screening strategy is not feasible.At the same time, we congstruct a system based on flow cytometry to screen rhizobium genes expressed in vivo. By transposon random insertion, we get the mutants library FK and NK containing gfp-lacZ reporter genes. Select the blue and white colony from FK and NK, and then detect β-galactosidase glycoside enzyme activity and expression of green fluorescent protein (GFP). Both β-galactosidase glycoside enzyme and GFP are expressed synchronized. But in ZN1the expression of reporter genes is too low, which may be associated with gene expression background, choseing ZNO in this study.Use NK1(bule colony) and NK5(white colony) as positive control and negative control, because their expresstion of GFP is about10-fold differences. Through Arbitrary PCR and sequencing, we find that gfp-lacZ is respectively in the same and different direction of promoter in NK1and NK5. Set door as101and adjust the density of cells to106CFU/mL, we can sort the mutants having high expression of GFP, the sorting rate is approximately98%. The methord to prepare fresh cells suspension is fixed too. After crushing nodules, bacteria and nodule tissue can be separated through differential centrifugation by50%PEG6000. About75%of the bacteria can be gathered for sorting, after washed and adjusted the density of cells to106CFU/mL by PBS, then use the flow cytometry to sort cells from the nodule tissue in two hours. Select white colony from TY (Sm Km X-gal) as target strains after the sorted cells grow to colony. So far, the system based on flow cytometry to screen genes in M.loti NZP2213vivo is constructed.Through studying the competitive nodulation between7653R and other seven Mesorhizobium huakuii strains, we find that7653R and Z2～Z7have a similar competitive nodulation, they are far higher than Z1. In majority nodules containing two kinds of strains,7653R has considerable colonization with Z1～Z7. Denying the screening system based on the Km or Gen resistant labels, we initially established the system based on flow cytometry to screen M.loti NZP2213genes expressed in vivo. |
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