| Microbial resources are valuable strategic biological resources,which is one of the priority developments in the world in the new era.Plant growth-promoting rhizobacteria?PGPR?has aroused general concern of researchers because of their excellent promoting function and safe to environment.The current study has mostly aimed at promoting growth functions of PGPR;however,there was little research about the molecular mechanism of their functions.Therefore,there are still lack of answer about PGPR molecular mechanism of promoting functions,metabolic regulation rule and promoting functions genes.If we can answer these questions,not only the PGPR molecular mechanism will be revealed but also it will contribute to PGPR be better utilized and benefit agricultural development.Hence,this study researched on the characteristics and mechcanism of a PGPR?named Gnyt1?which was isolated in prior of the research group.Taking strain Gnyt1 as research materials,the aim of this study was?i?to investigate the features and biological characteristics of Gnyt1,explore the influence of Gnyt1 on the plant and soil,and re-identify Gnyt1;?ii?after that,to determine the whole genome sequence of Gnyt1 by the second and third generation sequencing technology;?iii?according to whole genome data,to analyze and forecast possible functional genes in nuclear genome and plasmid genomes;and?iv?finally,to analyze deeply the genes related to promoting growth characteristics of Gnyt1.The results were as follows:?1?The results of characteristic of Gnyt1 showed that Gnyt1 had a variety of functions such as dissolved phosphorus,nitrogen fixation,and production plant hormones.The nitrogenase activity reached 3193.07 nmol?C2H4?·h-1·m L-1.The inorganic and organic phosphorus can be dissolved efficiently.The three plant hormones?including IAA,GA3 and t-Z?can be produced at the same time.In contrast to other strains of our previous studies,the functions of Gnyt1 were more outstanding.In addition,compared with strains obtained by other researchers,Gnyt1 also had some advantages.?2?The results of biological characteristics indicated that the growth curve of Gnyt1 was relatively short?about 4 h?in lag phase,and fast multiplication rate in exponential phase.The concentration of live bacteria was higher than the other strains in stationary phase,which indicated that Gnytl was suitable for high density fermentation.Gnyt1 had a good tolerance of temperature and pH.The drug resistance results demenstrated that the five strains including Gnyt1 and four control bacteria were no resistence to four kinds of antibiotics.The Gnyt1 had not pathogenicity to five kinds of crops such as naked barley,tomatoes,rape,angelica and alfalfa with two kinds of treatments including liquid filling root and leaf inoculation.Stability tests showed that with the increase of passage number,the promoting ability of strains decreased under the condition of pure culture.whether the selected bacteria Gnyt1 reaction,or contrast bacteria Jm92,LSH11,Lx191 and Jm170,whether nitrogen-fixing character or dissolved phosphorus or plant growth hormone secreted properties.?3?The results of the effect of PGPR on plant showed that the Gnyt1 promoted the growth and yield of five kinds of crops including naked barley,tomatoes,rape,angelica,and alfalfa;however,it played different roles in different plants.PGPR strains can also improve the quality of the crops in addition to promoting function,such as increasing the content of Chinese angelica naphtha and ferulic acid,improving the content of crude protein and crude fat in alfalfa hay,and decreasing crude fiber content.?4?The results of the influence of PGPR on soil properties showed that with the Gnytl bacteria liquid treatments,the soil respiration increased by 6.3%,the soil induced respiration increased by 19.6%,the soil microbial biomass carbon increased by 9.8%,and the soil metabolism entropy?qCO2?reduced by 11.6%.The 17 kinds of phospholipid fatty acid were detected by PLFA method.The amount of total PLFA and the amount of soil bacteria increased,whereas the number of fungi and microbial pressure index decreased after used PGPR bactericide,which indicated that PGPR bactericide can improve soil micro-ecosystem.The results from azotobacter group showed that the amount of soil aerobic nitrogen-fixing bacteria increased dramatically,by more than 11.2%,and aerobic nitrogen-fixing bacteria number increased by as high as 31%.In addition,the number of soil anaerobic nitrogen-fixing bacteria also increased slightly by 4.2%-7.6% after used PGPR bactericide.The study of PGPR influence on nitrogen-fixing bacteria in the soil from gene level showed that the similar result with the pure culture method.The relative content of nif H genes also increased by as high as 13.4 after used Gnyt1 bactericide.?5?The identification results showed that the physiological and biochemical characteristics of Gnyt1 were 100% coincident with these of Bacillus mycoides.Strain Gnyt1 was determined to Bacillus mycoides through BLAST in Genbank database and combining with the results from the phenotypic characteristics of observation,physiological and biochemical characteristics and 16 S rDNA sequence analysis.Strain Gnyt1 was named Bacillus mycoides Gnyt1.?6?The strain whole genome sequencing analysis of Bacillus mycoides Gnyt1 found as following:1)The DNA quality and concentration was higher and conform to the sequencing requirements of construction library by CTAB method.The whole genome sequence measured with combining the second and third generation sequencing and building sequencing library respectively.The total Reads numbers were 3,025,470 and total base numbers were 742,832,445 based on second generation sequencing.The proportion of fuzzy bases was 0.001%.The GC content of total base percentage was 38.8%.The base recognition accuracy rate in more than 99% of the base percentage was 99%.The base recognition accuracy rate in more than 99.9% of the base percentage was 64.4%.2)The single-base quality was high after sequence filtering.Base content distribution analysis showed that the GC and the AT content fit better.GC content distribution showed that the consistency was better of the theory and the actual distribution.This sequencing peak was around Q37 by sequence base quality inspection.The high quality reads numbers were 2,217,564 and the high quality base numbesr were as high as 471,926,886 bp by second generation sequencing.The length was large of the third generations sequencing.There were no uncertain bases.The sequence total length was 5,597,907 bp after genome sequence splicing.The GC percentage was 35.57%.There were ORF numbers 5,876 by ORF forecast.There were two kinds ncRNA including tRNA and rRNA by ncRNA prediction.There were not exist CRISPRs.The Gnyt1 had prophage by 9 in its genome through prophage prediction.The No.3,5,6 were incomplete prophage,while the rest of prophage were complete.The GIs prediction results showed that total had 23 GIs in the Gnyt1 nuclear genome.There were found 10 virulence genes by VFDB analysis,respectively was clpC,clpP,htpB,galE,nheA,nheB,hblC,inhA,lplA1,icl.Through CARD found that there were 51 genes associated with antibiotic resistance,27 genes associated with antibiotic target and 4 genes related with antibiotics synthesis.The Gnyt1 had produced bacitracin by in-depth analysis of the production of antibiotics genes.There were 30 genes of glycoside hydrolysis enzyme in the genome by CAZy analysis.There had 36 genes of glycosyl transferase enzyme.There had 38 genes of carbohydrate ester enzyme.There had 6 auxiliary active enzymes.There had 19 genes of carbohydrate combination of related enzymes.There had 15 genes of the other associated with carbohydrate,excluding polysaccharide lyase.The Gnyt1 nuclear genome sequence had 348 protein-coding genes exist in the signal peptide sequence by signal peptide forecasts strains.The Gnyt1 nuclear genome sequence total had 1,601 genes of encoding protein by transmembrane helical structure prediction.These genes exist in the transmembrane helical regions,which accounted for 27.25% of total number encoding protein genes.3)There were largest number and kinds genes related to biological process by GOSlim annotation in Bacillus mycoides Gnyt1 nuclear genome.Followed by the related molecular function genes.The linked to cellular component genes number and variety were minimum.All coding protein gene S can be divided into 25 classes by COG classification annotations.Besides Not egg NOG in class,the highest genes number was the general function prediction only,the number were as high as 609.The 2987 genes information were learned by Swiss-Prot annotation.Finally,in this study,the genome graph of Bacillus mycoides Gnyt1 was constructed and the genome size was 5,597,907 bp.?7?The strain plasmid genome research of Bacillus mycoides Gnyt1 found as following:1)Agarose gel electrophoresis had 8 strips of plasmid DNA.The quality and concentration were higher of the extraction DNA.In this study,Bacillus mycoides Gnyt1 total had 8 plasmids according to joining together result.No.1 plasmid length was 460,379 bp.No.2 plasmid length was 114,265 bp.No.3 plasmid length was 102,550 bp.No.4 plasmid length was 82,671 bp.No.5 plasmid length was 73,408 bp.No.6 plasmid length was 64,682 bp.No.7 plasmid length was 51,563 bp.No.8 plasmid length was 10,665 bp.2)Through ncRNA prediction,there was a tRNA gene and 18 other ncRNA genes in plasmid1.In the rest of the plasmid were not found ncRNA.There was no found CRISPRs in Gnyt1 plasmid genome.The 6 plasmid were predicted had prophage genes including Plasmid1,Plasmid2,Plasmid3,Plasmid4,Plasmid6 and Plasmid7.There were two incomplete prophage genes in Plasmid.There was an incomplete prophage gene in Plasmid2,Plasmid3,Plasmid4 respectively.There were 2 prophage genes exist in Plasmid 6,one complete phage gene,the other incomplete.There were total 5 genes island in the Plasmid1 genome by GIs forecasts.,There was only 1 gene island in the Plasmid2 genome.There were two genes island in the Plasmid5 genome.The rest of plasmid were not found gene island.,There were nhe C and nhe B genes in plasmid1 and the rest of the plasmid genome were not detected VFDB.There were two associated with antibiotic resistance genes by CARD analysis in Plasmid1.And the other plasmid were not detected in genes associated with antibiotics.The CAZy analysis results showed that only plasmid1,plasmid3 and plasmid4 had associated with carbohydrates active enzyme genes of all plasmid.Signal peptides forecast showed that different plasmid had different signal peptide sequence number.The across membrane structure prediction showed that there were different numbers transmembrane helical structure in the plasmid.3)In this study,the 8 plasmid gene existed a variety of function genes through GO analysis.There mainly had related plasmid replication genes.And the different plasmid contained different number functional genes.The plasmid1 was highest and plasmid8 was most simple.These 8 plasmids had a common characteristic was genes number of related to the nitrogen metabolism and associated with the DNA binding higher than other functional genes.There was a variety of egg NOG by the egg NOG analysis in 8 plasmid.Mostly genes related plasmid replication and different plasmid contained different number of functional genes.The plasmid1 had most number of eggnog.There were 18 classes.The plasmid8 was most less,only 1 class.This chapter study finally build the genome graph of 8 plasmid.?8?The research related to promoting of Bacillus mycoides Gnyt1 showed that there were many genes related to the performance of dissolved phosphorus.There were related organic acids genes such as pyruvate,malate,acetate,citrate,butyrate,Shikimic and so on.And the metabolic pathways up to 14 kinds related to produced acid.There were total 9 genes related to the iron carrier secretion.And mainly existed in the Porphyrin metabolism and ABC transporters pathway.There were two genes of related to the bacitracin secretion.They had an acitracin transport system-ATP binding protein function in different metabolic pathways.This study filled the blank of reaearch on the PGPR functional genes,and meantime it had an important scientific significance,which provided theoretical instructions for further rational utilization PGPR resources. |
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