Construction Of Soybean Hemoglobin Gene - Transformed Rhizobia Engineering Strain

Leghemoglobin in the soybean nodules is a class of plant symbiotic hemoglobin, which is closely related to nitrogen fixation and consists of four main components:Lba, Lbc1, Lbc2, Lbc3. Four leghemoglobins are separately encoded by four corresponding Glycine max leghemoglobin genes (lba, lbc1, Ibc2, Ibc3) which are recessive genes in the soybean plants. These genes specific express in root only after soybean root is invaded by rhizobium. The positive correlation is found between content of Glycine max leghemoglobin in the nodule and nitrogenase activity, so only Glycine max leghemoglobin existing, soybean nodule could be in progress normally and could continuously offer growing demanding nitrogen source.The Glycine max leghemoglobin gene was transferred into rhizobia by means of molecular biology and genetic engineering in this study in order to ensure the normal nitrogen fixation, to enhance nitrogen fixation efficiency of nodules, to provide soybean plants sufficient nitrogen. Meanwhile, it lay an experimental foundation for the further research of symbiotic nitrogen fixation of rhizobia and non-legumes.In this study, the total RNA of soybean nodules was extracted and reversed transcript into cDNA. The coding sequence of Glycine max leghemoglobin gene of lba, lbc1, lbc2and lbc3were respectively cloned using PCR with the template of cDNA by homology-based cloning. Bioinformatics analysis showed that the sequences had100%similarity with previous reported leghemoglobin gene and amplified sequences could be identified as the Glycine max leghemoglobin gene. By DNA recombination technique, the genes of lba, lbc1, Ibc2, lbc3and the tandem genes cluster lbX(lbc3-lbc2-lbc1-lba) were separately joined into rearward of lac promoter to obtain target fragment of Plac-lba, Plac-lbc1, Plac-lbc2, Plac-lbc3and Plac-lbX, which were respectively connected into vector pTR102with marker gene luxAB to successfully construct five prokaryotic expression vectors of pTR-Plac-lba, pTR-Plac-lbc1, pTR-Plac-lbc2, pTR-Plac-lbc3and pTR-Plac-1bX. Furthermore, the five prokaryotic expression vectors were separately transformed into Sinorhizobium fredii15067(SF) and native Sinorhizobium (SFH) with method of triparental hybridization, and ten transgenic engineering strains were successfully constructed.The detection results of the genetic stability of plasmid indicated that the inheritance retention of recombinant plasmid pTR-Plac-lba, pTR-Plac-lbc1, pTR-Plac-lbc2, pTR-Plac-lbc3, pTR-Plac-1bX was100%under authigenic conditions; The results of fluorecent detection of soybean nodules that were infected by transgenic engineering strains were100%under symbiotic condition. Five recombinant vectors could stably inherit in both Sinorhizobium fredii15067and native Sinorhizobium.By means of pot experiment, the result showed that comparing with control group of soybean plants infected by Sinorhizobium fredii15067, soybean seedlings which were respectively infected by SF(pTR-Plac-lba), SF(pTR-Plac-lbc1), SF(pTR-Plac-lbc2), SF(pTR-Plac-lbc3), SF(pTR-Plac-1bX) were significantly increased in different degree in soybeen plant height, fresh weight, dry weight, nodule number, nodule nitrogenase activity and yield. Comparing with another control group of soybean plants infected by native Sinorhizobium, soybean seedlings which were respectively infected by SFH(pTR-Plac-lba), SFH(pTR-Plac-lbc1), SFH(pTR-Plac-lbc2), SFH(pTR-Plac-lbc3), SFH(pTR-Plac-lbX) were significantly increased in different degree in soybeen plant height, fresh weight, dry weight, nodule number, nodule nitrogenase activity and yield. According to the comparison of the physiological indexes of soybean plants infected by different transgenic engineering strains, the result showed that the synergism of five recombinant vectors was pTR-Plac-lba>pTR-Plac-lbc3>pTR-Plac-lbX>pTR-Plac-lbc2> pTR-Plac-lbc1.The results of pot experiment also showed that by comparing the physiological indexes of soybean plants respectively infected by Sinorhizobium fredii15067and native Sinorhizobium, we found that synergism of native Sinorhizobium was better than that of Sinorhizobium fredii15067. And comparing genetic engineering strains which came from the same recombinant vectors were respectively transformed into the two original strains, synergism of engineering strains from native Sinorhizobium was better than that from Sinorhizobium fredii15067.This study gained Financial Aid of ’Harbin Sci-tech Innovation Talent study special capital projects’ and ’Heilongjiang province science and technology key projects’.