Breeding And Function Analysis Of Green Fluorescent Protein Tagged Pea Rhizobia

Symbiotic nitrogen fixation plays a very important role in biological nitrogen fixation.Legume plant,an important crop in agriculture,is the major nitrogen-fixing symbiont.Rhizobium is a kind of rod-shaped bacterium.Through its bymbiosis with legume,Rhizobium forms root nodules as well as fixing nitrogen in the air for plant nutrition.Such symbiotic system has strong ability of nitrogen fixation.Resulting from Rhizobium-legume interaction,most legumes form root nodules with nitrogen-fixing ability.Rhizobium has certain host specificity when infecting legumes:some have a narrow host range but some wide.the main way for Rhizobium's entering is to form invasive lines on the root hairs. At first,Rhizobium and legumes recognize each other in the soil;Then,Rhizobium attaches to the root hairs of host,and curls the root hairs,so that Rhizobium is parceled by root hairs;cell wall's invagination and elongation cause Rhizobium to form a tube-shaped invasive lines;Through continuous elongation,invasive lines reach the cortical cells and form branches;Rhizobium in the invasive lines propagates endlessly,and is released into the cortex cells;continuous mitosis makes nodule tissue come into being.Rhizobium differentiates to bacteroid in the inner cells of nodule,and then is surrounded by membrane derive from the host(also known as peribacteroid membrane); bacteroid reduces N₂ to NH₃ which be secreted to the nodule cells as precursor of acyl urine compounds which be exported from nodule and then transported by the conductive tissue of root to the overground part for utilization.But the exact mechanism for the infection still remains unclear.GFP has been proven to be a useful tool in the study of diverse biological processes in vivo. Since Chalfie et al(1994) first cloned and expressed GFP in E.coli,GFP quickly had aroused scholars throughout the world great interests as a new type of reporter gene.It has a number of advantages:First of all,it has a smaller molecular weight,only encoding a polypeptide with 238 amino acids,and does not toxic to the cells,and does not interfere with the function and location of target protein.In addition,GFP is the only protein being able to express in different cells and spontaneously emit fluorescence.Since it does not need any cofactors for function,GFP can be used in vivo measurement directly.In another word,GFP is one of the best means in the study of gene expression and protein distribution in living cells,and has broad application prospects.This experiment mainly used the DNA fragment used for homologous recombination was cloned from the gDNA of Rhizobium leguminosarum.Obtained engineering pea Rhizobium with eGFP green fluorescent marker.1.The DNA fragment used for homologous recombination was cloned from the gDNA of Rhizobium leguminosarum.2.Construction of prokaryotic expression vector which contains both homologous recombination fragments of pea Rhizobium gDNA and eGFP gene.3.Obtained engineering pea Rhizobium with eGFP green fluorescent marker.In this study,the expression vector pVCT2123 was successfully constructed and was transformed into pea Rhizobium in order to obtain GFP-tagged pea Rhizobium.4.Obtained nodule of engineering pea Rhizobium with eGFP green fluorescent marker.This study introduced eGFP tagged engineering pea Rhizobium into pea radicle,nobtained nodules of eGFP tagged engineering pea Rhizobium.PCR verification results illuminate that eGFP tagged engineering pea Rhizobium formed nodules in pea.