Application Of Green Fluorescent Protein Gene (gfp) In The Symbiosis Between Mesorhizobium Huakuii And Astragalus Sinicus

The symbiosis between Mesorhizobium huakuii and Astragalus sinicus is a Chinese-characteristic symbiotic nitrogen fixation system, while molecular genetic study on its early symbiotic interaction is still at primary stage. A novel reporter gene, green fluorescent protein gene (gfp) has been widely used in study on gene expression and protein localization among living organisms. The chief aim of the present study was to primarily explore the early symbiotic interaction between M. huakuii and A. sinicus, using gfp and its mutants as reporter genes.The feasibility of broad-host-range plasmid pTR102 as a stable transfer vector in molecular biology of M huakuii was analyzed. Stability of pTR102-derived plasmids pHN106 and pHN109 respectively harboring luxAB and gusA cassette was comparatively assayed in M. huakuii 7653R according to the luminescent activity of LuxAB and histological stain of GusA. The results showed that the both two plasmids could stablely maintained in 7653R under both free-living and symbiotic conditions and the stability was up to 100%. Compared to sensitive, stable and visible GUS histological stain method, luminescent activity of LuxAB could not be directly detected in nodules marked with luxAB. High stability of pTR102-derived marking plasmids indicated that novel vector systems based on pTR102 could be constructed for the study on molecular biology and ecology of M. huakuii.1kb gfp cDNA fragment amplified by PCR was cloned into E. coli expression plasmid pET-HC, which was under the control of RNA polymerase gene promoter from phage T7 with its own translation initiation codon. The expression plasmid pHN115 was transferred into E. coli BL21(DE3) and green fluorescence was detected from all transformants after IPTG induction. A new protein band with approximate molecular weight 27KDa was also obtained by SDS-PAGE after IPTG induction. The results showed that wtgfp was successfully expressed in E. coli. The promoter-probe vector pHN117 in E. coli and pHN127 in G" were further constructed by removing promoter while keeping SD sequence from pHN115. A tetA/tetR bidirectional promoter fragment from pBR322 was respectively cloned into pHN117 and pHN127 and the resulted colonies were all fluorescent. A fusion-trascriptional library was then constructed by inserting Sau3AI-partially digested chromosomal DNA from 7653R into BamHI cloning site of pHN127. A group of constitutive-expression fusions with different fluorescent strength were obtained.A new E. coli promoter-probe vector pHN1005 was constructed by using a red-shift enhanced gfpmut3 as reporter gene and showed the following characters: 1, BamHI at the 5' end of gfpmut3 structure gene could be used to clone promoter-active fragment and the strength of promoter could be quantitatively assayed. 2, A rrnBTlT2 terminator from rRNA gene at the 3' end of gfpmut3 could permit clone strong promoter. 3, Another rrnBT1T2 terminator was inserted upstream BamHI to prevent reading through of promoters from pUC19. 4, An intron sequence was also inserted upstream of gfpmut3 and its six reading frame could all be stopped, which could guarantee gfp translation in right reading frame. SmaI, KpnI and SalI flanking BamHI could be used to analyze the cloned fragment. A broad-host-range promoter-probe vector pHN1006 was further constructed by transferring promoter-probe unit from pHN1005 into pTR102. Lots of constitutive-expression fusions containingpromoters of 7653R and one induced fusion by A. sinicus seed extracts were screened by shot-gun method. No physiological, biochemical or genetical changes were found between marked strains 2-33 and 7653R during the growth and symbiotic process with A. sinicus. Formation of infection thread and nodules at early symbiotic stage between 2-33 and A. sinicus were monitored in situ by using laser confocal scanning microscope (LCSM) and fluorescent microscope. An effective vector system and an examination method were established for further molecular genetics study on rhizobium-legume symbiosis.A blue fluorescen...