Construction Of Soybean Rhizobia Engineering Strain With NodD Gene And Its Effect On Nodulation

Rhizobia japonicum, a class of symbiotic nitrogen fixation microorganism, fix biological nitrogen after formation of symbiotic nitrogen-fixing system, which provide essential nitrogen element for growth of host plant. Nodulation genes of rhizobia play important role in the process of forming mutualism symbiotic relationship. According to their functions, nodulation genes can be divided into two parts:regulatory gene and structural gene, of which nodD gene acts as important regulatory gene in the process of symbiotic. NodD protein regulates other nod structural genes’expression and synthesis of nodulation factor.As nodD gene is regulatory gene of coding nodulation factor and has important function in the process of nodulation, nodD gene is transfered into rhizobia in order to increase copy number, which will lay a foundation for further studying effects of nodD gene on nodulation ability of rhizobia.Homology-based cloning was used to clone nodulation gene nodD with template of genomic DNA from Sinorhizobia fredii 15067 in this study. The results showed that its sequence had 98% similarity with nodDl gene of Sinorhizobia fredii, R.japonicum as well as that of Rhizobia fredii by the method of bioinformatic analysis. It was presumed that the gene was the nodD gene exsiting in Sinorhizobia fredii 15067.The longest ORF contained 321 amino acids, ranged from the sxth to nine hundred and eventy first nt. Utilization of DNA recombination technique, nodD gene was joined into downstream of lac promoter and kan gene promoter, respectively. And expression vector pTR102-Piac-nodD and pTR-Pkan-nodD were constructed succesfully by using plasmid pTR102 marked by luxAB gene. Both of the expression vectors pTR102-Plac-nodD and pTR-Pkan-nodD were transform into Sinorhizobia fredii 15067 and native Rhizobia japonicum with method of triparental hybridization. Four genetic engineering strains were succesfully constructed succesfully.By means of pot culture nodulation test, significant test results showed that there were differences in different degree in soybeen plant height, fresh weight, dry weight, nodule number and yield comparing with control group after soybean seedlings were infected by four genetic engineering strains, in which the plant height or fresh weight of SF (pTR-Plac-nodD) and SF (pTR-Pkan-nodD) was significantly different from Sinorhizobia fredii 15067 or SF (pTR102). And nodule number was extremely significantly different. Compared to soybean plants infected by Sinorhizobia fredii 15067, nodules of the soybean plants infected by SF (pTR-Plac-nodD) or SF (pTR-Pkan-nodD) increased by about 23.73%. The fresh weight of SFH (pTR-Plac-nodD) and SFH (pTR-Pkan-nodD) was significantly different from Sinorhizobia fredii 15067 or SFH(pTR102). And plant height, nodule number and yield was extremely significantly different, respectively. Compared to soybean plants infected by Sinorhizobia fredii 15067, nodules of the soybean plants infected by SFH (pTR-Plac-nodD) increased by about 23.73% as well as that of SF (pTR-Pkan-nodD) increased by about 22.03%.The detection results of plasmids stability showed that retention of plasmid pTR-Pkan-nodD and pTR-Plac-nodD was 100% on authigenic conditions, and these two vectors were found to be fairly stable in rhizobia. The results of fluorecent detection of nodules from soybean plants infected by engineering strains were 100% under symbiotic condition. Recombinant vectors pTR-Pkan-nodD and pTR-Plac-nodD were able to inherit steadily in both Sinorhizobia fredii 15067 and Rhizobia japonicum.