CN103045500A - Mesorhizobium KDRM295 and application thereof - Google Patents

Abstract

The invention relates to mesorhizobium KDRM295 which is separated from acacia root nodule, contains ACC deaminase, and can efficiently nodulate and promote growth of the acacia, and an application thereof. The mesorhizobium KDRM295 is now preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCCNO: M2012331. The mesorhizobium KDRM295 contains ACC deaminase which can decompose ACC into alpha-ketobutyrate and NH3, reduce the level of plant cell for synthesizing ethylene, lighten the inhibition effect of ethylene to infection of the root nodule, improve the nodulation rate of root nodule and acacia and the level of symbiotic nitrogen fixation, provide high-level nitrogen for the growth of the acacia on the barren wasteland without fertilization, promote the growth of acacia seeding, and increase mature biomass for the acacia, thereby realizing high yield of the acacia mature with low inoculation investment, and playing a role of raising seeding and afforestation for acacia.

Description

Autoinducer KDRM295 and application thereof

Technical field

The invention belongs to applied microbiology field, be specifically related to one that from the locust tree root nodule, separate, have the acc deaminase can efficient dross and promote Autoinducer KDRM295 and the application thereof of Growth of Blaek Locust.This bacterial strain has been preserved in Chinese Typical Representative culture collection center (CCTCC), and preserving number is CCTCC NO:M 2012331, and bacterial strain is called Autoinducer KDRM295.

Background technology

Some prokaryotic micro-organisms of occurring in nature can synthesize nitrogenase, at normal temperatures and pressures, and with airborne N ₂Be reduced into NH ₃Accounted for 65% – 70% of earth's surface combined nitrogen by the fixing nitrogen of nitrogen-fixing microorganism, wherein the strongest with root nodule bacterium and leguminous plants syntaxial system nitrogen fixing capacity, account for more than 65% of biological nitrogen fixation amount.

Utilizing root nodule bacterium and leguminous plants symbiotic nitrogen fixation to increase soil fertility with crop yield is the classical experience of world agriculture.As making green manure with leguminous plants, allow pulse family and non-leguminous crop crop rotation, intercropping and interplanting.Improving symbiotic nitrogen fixation efficient and output and existing more than the 100 year history of soil fertility of leguminous crop by Rhizobium Inoculation, extensively is that each large agricultural country is used, is one of important measures of Developing sustainable agriculture.Since the eighties in 20th century, the cultivated area of China food crop constantly enlarges, and the cultivated area of leguminous crop and leguminous green manure constantly reduces, and chemical fertilizer especially nitrogen fertilizer amount constantly increases.Because high-level combined nitrogen checks the root nodule bacterium nodulation and nitrogen fixation, the nodulation and nitrogen fixation effect of Rhizobium Inoculation is being used sharply decline in the farmland of a large amount of nitrogenous fertilizer, and the development of legume inoculation cause is contained thereupon.Enter 21 century, implementation of China strategy to develop western regions and Grain for Green Project, using for the cultivated area that enlarges Leguminosae tree, herbage and the legume inoculation technology of strengthening being complementary with it provides opportunity.

Locust tree ( Robinia pseudoacaciaL.) claiming again acacia, is deciduous tree, and 25 meters of high 10 – are wooden hard, flexible, resistance to wear, and be good ore pillar, sleeper, construction timber.Locust tree originates in the eastern united states, introduce a fine variety Europe after, introduce again China from Europe in the 19th-century end.Growth of Blaek Locust is fast, distributes more and more wider, has benefited from the locust tree strong adaptability on the one hand, can grow in sour earth, neutral soil and the saltiness saline-alkali soil below 0.3%, and certain drought-resistance ability is arranged; Locust tree is leguminous plants on the other hand, can obtain with the root nodule bacterium symbiotic nitrogen fixation the necessary nitrogen of growth, thereby is suitable in barren soil growth.The ecologic effect of plantation locust tree, as conserve water and soil, improve soil etc., also highly significant.Therefore, locust tree becomes one of the whole world most important fast-growing afforestation vanguard tree seed, also is the vanguard tree seed of China's project of conceding the land to forestry.

Along with the day by day exhaustion of the fossil energies such as Global Oil, coal and Sweet natural gas, develop reproducible biomass energy to replace fossil energy more and more as the concern of countries in the world government.At present, the China's economic rapid growth, the sharp increase of fossil energy consumption, the energy shortage problem is very outstanding, and the development and utilization of the renewable energy sources such as biomass energy has become the key that realizes Sustainable development.The basic point of the development and utilization of biomass energy is raw material production, and raw materials cost accounts for 60% – 80% of total cost, and raw materials cost determines the competitiveness of product in market and profit.

Forest is the important source material of biomass energy.Locust tree relies on its calorific value high, and fast growth and the anti-lean characteristics such as degeneration-resistant become important Tree Species as Bio-energy.Barren on the ground waste,, cheaply input-output high become a useful person biomass fertile with Shaoshi is the bottleneck problem that present China locust tree energy forest is produced.With the legume inoculation locust tree seedling of efficient nodulation and nitrogen fixation, make sapling obtain nitrogen in barren soil by symbiotic nitrogen fixation and become a useful person, may improve forestation of locust efficient and reduce raw materials cost.At present, on the one hand, technically, modern forestation of locust improves afforestation efficient by extensive seedling nursery, for locust tree the Miaos worker Rhizobium Inoculation has been created convenience.On the other hand, be limited to technology and managerial restriction, the large-scale afforestation of China is not also implemented take nitrogen as main extensive fertilising to forestry developed country like that, do not create the condition that does not have nitrogen to check for the symbiotic nitrogen fixation of locust tree and root nodule bacterium on the contrary but do not apply fertilizer, be conducive to improve the efficient of symbiotic nitrogen fixation.

Locust tree adapt to a strong factor of lean soil ability be it can with the root nodule bacterium symbiotic nitrogen fixation of the genetic diversity of a plurality of genus kinds.Found can with the locust tree dross mainly be Autoinducer belong to ( Mesorhizobium) root nodule bacterium, also have Sinorhizobium belong to ( Sinorhizobium), rhizobium ( Rhizobium) and Bradyrhizobium ( Bradyrhizobium) etc. the root nodule bacterium that belong to.The ability of root nodule bacterium and locust tree nodulation and nitrogen fixation just differs, and utilizes Rhizobium Inoculation to promote Growth of Blaek Locust need to select efficient rhizobium strains.Usually the good rhizobium strains of screening is by separating a plurality of rhizobium strainss from a plurality of large and full locust tree root nodules, inoculate one by one the locust tree seedling, cultivates 2 months or after the longer time dross number of detection locust tree shoot root section and the biomass of seedling; And the rhizobium strains that will filter out efficient nodulation and nitrogen fixation from a fairly large number of bacterial strain will play at some game of chance, and screening process wastes time and energy.

Root nodule bacterium are infected fabaceous can cause precursor 1-amino-cyclopropane-1 carboxylic acid (ACC) that plant produces plant hormone ethylene and synthesizing ethylene, and ethene can suppress root nodule bacterium dross and fixed nitrogen.Research in recent years finds that some root nodule bacterium has acc deaminase.Acc deaminase energy catalysis ACC desamination reaction generates α-ketone butyric acid and NH ₃When root nodule bacterium were infected root, the synthetic ACC of root cells and release portion ACC were to the extracellular.Have the root nodule bacterium of acc deaminase to absorb and ACC that the degrading plant cell discharges, ACC in the root cells is constantly discharged and reduce, the amount of root cells synthesizing ethylene is with regard to corresponding minimizing, thereby reduced ethene infects dross to root nodule bacterium restraining effect.If the acc deaminase structure gene of root nodule bacterium ( AcdS) knock out, significantly reduction of the Noduling ability of root nodule bacterium (Uchiumi etc., Journal of Bacteriology 2004,186:2439-2448); On the contrary, if do not have to script AcdSRoot nodule bacterium import AcdS, the dross rate of that root nodule bacterium engineering bacteria, account for ratio of outflow and nitrogen-fixing efficiency is significantly higher than wild type strain (Ma etc., Applied and Environmental Microbiology 2004,70:5891-5897; Conforte etc., Journal of General and Applied Microbiology 2010,56:331-338; Tittabutr etc., Systematic and Applied Microbiology 2008,31:141-150; Nascimento etc., Letters in Applied Microbiology 2012,55:15-21).This shows that the root nodule bacterium that acc deaminase is arranged have strong invasiveness, can efficient dross.The machine-processed more complicated of root nodule bacterium regulation and control acc deaminase.Research has been found much to have AcdSThe root nodule bacterium that belong to of Autoinducer under free cultivation conditions, do not express AcdS, do not have acc deaminase active, but in root nodule, can express high-levelly AcdS, effect (Ma etc., Antonie van Leeuwenhoek 2003, the 83:285-291 of performance acc deaminase; Nukui etc., Applied and Environmental Microbiology 2006,72:4964-4969; Nascimento etc., FEMS Microbiology Letters 2012,336:26-37).Therefore, when screening has the root nodule bacterium of acc deaminase, if it is active to detect the acc deaminase of root nodule bacterium under the free cultivation conditions, can lose efficacy to a lot of root nodule bacterium especially Autoinducer, have or not and detect root nodule bacterium with polymerase chain reaction (PCR) AcdSGene can be more effective.

Summary of the invention

Technical problem to be solved by this invention be to provide for the deficiencies in the prior art one that from the locust tree root nodule, separate, have the acc deaminase can efficient dross and promote Autoinducer KDRM295 and the application thereof of Growth of Blaek Locust.

The present invention realizes that the technical scheme that above-mentioned technical purpose adopts is as follows:

Separate and the purifying root nodule bacterium from the locust tree root nodule with ordinary method first, from the root nodule bacterium genome, increase again AcdSGene determines that to the target fragment order-checking that amplifies bacterial strain has or not AcdSGene and acc deaminase, again with the rhizobium strains inoculation BLACK LOCUST SEEDLINGS that acc deaminase is arranged, after inoculating 3 months, detect plant height, leading thread and the dry weight of dross number, plant, select the efficient dross of energy, significantly promote the rhizobium strains of the growth of locust tree seedling and raising biomass to be used for breeding and afforestation.On the other hand, amplification has 16S rRNA gene and the order-checking of acc deaminase root nodule bacterium, determines the category attribution of bacterial strain by sequence alignment and Phylogenetic Analysis 16S rRNA gene order; Simultaneously the root nodule bacterium that acc deaminase is arranged that filter out knownly in the root nodule bacterium there is an acc deaminase bacterial strain with belonging to together AcdSGene order is carried out Phylogenetic Analysis, in conjunction with bacterial strain 16S rRNA gene and AcdSThe Phylogenetic of gene finally determines to obtain the new root nodule bacterium strain that acc deaminase is arranged.

Autoinducer KDRM295 provided by the invention is preserved in Chinese Typical Representative culture collection center on September 7th, 2012, deposit number is CCTCC NO:M 2012331, and the preservation address is China, Wuhan, Wuhan University, Classification And Nomenclature is Autoinducer KDRM295 MesorhizobiumSp. KDRM295.

Described Autoinducer KDRM295 is from being separated to the root nodule of Guishan Mountain, Danjiangkou, Hubei Province artificial growth locust tree.

The cell of described Autoinducer KDRM295 is shaft-like, Gram-negative forms typical root nodule bacterium bacterium colony in YMA medium growth: circle, oyster white, protuberance, neat in edge do not spread, smooth surface and because abundant exocellular polysaccharide thickness, more moistening, slightly transparent is arranged; The growth of cell is aerobic, grows under 28oC and condition of neutral pH very fast, and the diameter that formed bacterium colony at YMA medium growth 3 – in 5 days can reach 1 –, 2 mm.

The 16S rRNA gene order of described Autoinducer KDRM295 (seeing SEQ ID NO:1) and Autoinducer generitype Root or stem of Littleleaf Indianmulberry Autoinducer typical strain Mesorhizobium lotiThe 16S rRNA gene order consistence of ATCC 700743 is 99.7%, with the Mesorhizobium ciceri typical strain Mesorhizobium ciceriThe consistence of the 16S rRNA gene order of ATCC 51585 is 99.4%, with Shangri-la Autoinducer typical strain Mesorhizobium shangrilenseThe consistence of the 16S rRNA gene order of CCBAU 65327 is 99.4%, with Australian Autoinducer typical strain Mesorhizobium australicumThe consistence of the 16S rRNA gene order of WSM2073 is 99.1%, and is nearer with the sibship of above-mentioned four kinds of bacterium in phylogeny, is in a branch (Fig. 1) adjacent with these four kinds of bacterium.Therefore, the KDRM295 bacterial strain belongs to Autoinducer and belongs to, and can not determine to belong to which kind.

Described Autoinducer KDRM295 has acc deaminase.It AcdSGene Partial sequence (seeing SEQ ID NO:2) and Autoinducer belong to the known acc deaminase bacterial strain that has AcdSSequence similarity, but differ greatly; With camel thorn Autoinducer typical strain Mesorhizobium alhagiCCNWXJ12-2's AcdSThe similarity of (GenBank accession number AHAM01000292) is the highest, only is 78.8%; Status in phylogeny also is different from other Autoinducers AcdS(Fig. 2).This shows that bacterial strain KDRM295 is different from the known bacterial strain that acc deaminase is arranged in the Autoinducer genus.

The 16S rRNA gene of above-mentioned Autoinducer KDRM295 and AcdSThe analytical results of gene is in conjunction with showing that the genotype of KDRM295 is different from known Autoinducer.

Can be on the locust tree root efficient nodulation and nitrogen fixation of described Autoinducer KDRM295 promotes the growth of locust tree seedling and becomes a useful person.

Beneficial effect of the present invention:

Autoinducer KDRM295 of the present invention has acc deaminase, ACC can be resolved into α-ketone butyric acid and NH ₃Reduce the level of vegetable cell synthesizing ethylene, alleviate the restraining effect that ethene infects root nodule bacterium, improve root nodule bacterium and the dross rate of locust tree and the level of symbiotic nitrogen fixation, for locust tree provides high-caliber nitrogen in the barren waste growth on the ground of not applying fertilizer, promote the growth of locust tree seedling, increase the biomass that locust tree becomes a useful person, thereby drop into cheaply inoculation and to allow the locust tree high yield of becoming a useful person, play a role at the locust tree breeding and afforestation.

Description of drawings

Fig. 1 is Autoinducer bacterial strain KDRM295(●) belong to Autoinducer ( Mesorhizobium) phylogenetic tree of 16S rRNA gene of typical strain of existing 25 kinds.■ indication Autoinducer generitype Root or stem of Littleleaf Indianmulberry Autoinducer typical strain among the figure Mesorhizobium lotiATCC 700743, ▲ indication Mesorhizobium ciceri typical strain Mesorhizobium ciceriATCC 51585, Shangri-la Autoinducer typical strain Mesorhizobium shangrilenseCCBAU 65327 and Australian Autoinducer typical strain Mesorhizobium australicumWSM2073 is the accession number of 16S rRNA gene nucleotide series in the GenBank database in the bacterial strain name unquote.

Fig. 2 is Autoinducer bacterial strain KDRM295(●) and the Autoinducer genus ( Mesorhizobium) in the acc deaminase bacterial strain is arranged AcdSThe phylogenetic tree of gene.■ indication camel thorn Autoinducer among the figure Mesorhizobium alhagiCCNWXJ12-2 in the bacterial strain name unquote is AcdSThe accession number of gene nucleotide series in the GenBank database.

Embodiment

1. the separation of root nodule bacterium, purifying and preservation

From the locust tree of Guishan Mountain, Danjiangkou, Hubei Province artificial growth, selecting large and full root nodule on the healthy and strong locust tree root, carefully root nodule company headquarters root division is cut with scissors, 15 root nodules of 5 – that same locust tree root obtains are put into the tubule that dry discolour silica gel is housed and is covered with absorbent cotton.Then after the root nodule that gathers fully being soaked imbibition with sterilized water, alcohol-pickled 30 s with 95%, follow mercuric chloride surface sterilization 5 min with 0.1%, use again aseptic water washing 6 times, to put into respectively an aseptic 2-ml centrifuge tube after each root nodule numbering, with aseptic grinding rod root nodule is ground, add 1 ml sterilized water and 3 suspension homogenates of suction with pipettor, with 10 times of suspension serial dilutions, 100 times and 1000 times, then drawing respectively 100 μ l suspension is coated on the YMA solid medium (every liter contains 1 g yeast powder, 10 g N.F,USP MANNITOL, 0.5 g K ₂HPO ₄, 0.2 g MgSO ₄, 0.1 g NaCl, 1.0 g CaCO ₃, pH 6.8,15 g agar powders) on, culture plate is placed on 28oC secretly cultivates.Cultivate 3 – after 5 days picking the bacterium colony of typical root nodule bacterium colonial morphology (circle, oyster white, protuberance, neat in edge do not spread, smooth surface and because abundant exocellular polysaccharide thickness, more moistening, slightly transparent is arranged) is arranged, streak culture on the YMA flat board, repeat line purifying bacterium colony 3 times.Single bacterium colony of purifying is suspended in sterilized water, and microscopy behind gramstaining is selected Gram-negative, cell is shaft-like and form is consistent bacterium as the rhizobium strains of purifying.The rhizobium strains of described purifying can be seeded in the TY nutrient solution, and (every liter contains 5 g Tryptoness, 3 g yeast powders, 0.33 g CaCl ₂, pH 6.8) in be cultured to logarithm later stage or stationary phase, with 30%(v/v) glycerine solution equal-volume mixing, be frozen in-the 80oC prolonged preservation.

2. root nodule bacterium AcdSThe amplification of gene and evaluation

Rhizobium strains is inoculated into the 100 μ l bacterium liquid that are cultured to logarithm later stage or stationary phase in the TY nutrient solution to be moved in the 1.5-ml centrifuge tube, centrifugal 5 min of 8000 rpm, suck supernatant liquor, clean 2 times with 0.5 ml sterilization distilled water, with 100 μ l sterilization distilled water Eddy diffusion thalline, get 1 μ l bacteria suspension and carry out pcr amplification; Template is the DNA that thalline discharges behind PCR denaturation reaction heating pyrolyze; Primer is acdSf3:5 '-ATCGGCGGCATCCAGWSNAAYCANAC-3 ' and acdSr3:5 '-GTGCATCGACTTGCCCTCRTANACNGGRT-3 ', and working concentration is 0.4 μ M.2 * Taq PCR MasterMix that reaction system is produced with TIANGEN Biotech (Beijing) Co., Ltd..Pcr amplification carries out at Bio-Rad S1000 type PCR instrument.Amplification program is 94oC denaturation 4 min; 94oC sex change 45 s, 53oC 45 s that anneal, 72oC extends 1 min, 35 circulations; 72oC extends 7 min.Amplified production 1%(w/v) agarose gel electrophoresis detects, and then delivers to the English Weihe River prompt base (Shanghai) Bioisystech Co., Ltd and checks order with primer acdSf3 and acdSr3.

See SEQ ID NO:2 from the sequence of dna fragmentation after removing primer that bacterial strain KDRM295 amplification obtains.Sequence SEQ ID NO:2 carried out BLAST retrieval in ncbi database, the result shows that SEQ ID NO:2 and Autoinducer belong to root nodule bacterium AcdSSimilar, wherein: what similarity was the highest is camel thorn Autoinducer typical strain Mesorhizobium alhagiCCNWXJ12-2's AcdS(GenBank accession number AHAM01000292), the consistence of the two is 78.8%.This shows that the sequence that amplification obtains is AcdSSequence, bacterial strain KDRM295 has acc deaminase.

3. with the legume inoculation locust tree seedling that acc deaminase is arranged and detection inoculation effect

Select the locust tree root of diameter 0.8 – 1 cm, be cut into the root segment of 8 –, 10 cm, root segment is inserted in the seedling medium in seedbed, at 25 – 28oC, cultivate in the greenhouse of relative humidity 75 – 85%, water weekly once, inoculate when locust trees emerge approximately 5 –, 8 cm after about 5 weeks.Be specially: the fresh colony inoculation that the root nodule bacterium that acc deaminase will be arranged first grow at the YMA solid medium is cultivated 48 –, 72 h to stationary phase at 28oC and 200 rpm to the TY nutrient solution; The bottled 100 ml nutrient solutions of each 500-ml taper during cultivation, every milliliter approximately contains 5 * 10 after cultivating ⁹Individual bacterial cell.Then water the seedbed after bacterium liquid being diluted 500 times with tap water.The contrast seedling replaces watering with the tap water of equivalent.Inoculate the dross number, plant height, leading thread and the dry weight that detect the locust tree seedling after 3 months.Wherein: the result of bacterial strain KDRM295 is as shown in table 1.

The effect of table 1 root nodule bacterium KDRM295 inoculation locust tree seedling

	The dross number	Plant height (cm)	Leading thread (mm)	Overground part dry weight (g)	Dry weight increases (%)
						The contrast locust tree seedling of not inoculating	3	65	3.40	6.35	0
The locust tree seedling of inoculation	57	110	5.53	15.14	138%

As can be seen from Table 1: behind the inoculation locust tree, root nodule bacterium KDRM295 and locust tree symbiosis can form more root nodule, by the N in the reducing atmosphere ₂For Growth of Blaek Locust provides nitrogen, can significantly promote the growth of locust tree seedling, increase biomass.

4. the amplification of bacterial 16 S rRNA gene and evaluation

Inoculation is cultured to the 100 μ l bacterium liquid of logarithm later stage or stationary phase in the TY nutrient solution to be moved in the 1.5-ml centrifuge tube, centrifugal 5 min of 8000 rpm, suck supernatant liquor, clean 2 times with 0.5 ml sterilization distilled water, with 100 μ l sterilization distilled water Eddy diffusion thalline, get 1 μ l bacteria suspension and carry out PCR; Template is the DNA that thalline discharges behind PCR denaturation reaction heating pyrolyze; Primer is 27F:5'-AGAGTTTGATCMTGGCTCAG-3' and 1492R:5'-GGTTACCTTGTTACGACTT-3', and working concentration is 0.25 μ M.2 * Taq PCR MasterMix that reaction system is produced with TIANGEN Biotech (Beijing) Co., Ltd..Pcr amplification carries out at Bio-Rad S1000 type PCR instrument.Amplification program is 94oC denaturation 3 min; 94oC sex change 55 s, 50oC 50 s that anneal, 72oC extends 1 min, 35 circulations; 72oC extends 10 min.Amplified production 1%(w/v) agarose gel electrophoresis detects, and then delivers to the English Weihe River prompt base (Shanghai) Bioisystech Co., Ltd and checks order with corresponding amplimer.

See SEQ ID NO:1 from the sequence of dna fragmentation after removing primer that bacterial strain KDRM295 amplification obtains.Sequence SEQ ID NO:1 is carried out the BLAST retrieval in ncbi database, it is similar that the result shows that SEQ ID NO:1 and Autoinducer belong to the 16S rRNA gene orders of root nodule bacterium, wherein: with Autoinducer generitype Root or stem of Littleleaf Indianmulberry Autoinducer typical strain Mesorhizobium lotiThe 16S rRNA gene order consistence of ATCC 700743 is 99.7%, with the Mesorhizobium ciceri typical strain Mesorhizobium ciceriThe consistence of the 16S rRNA gene order of ATCC 51585 is 99.4%, with Shangri-la Autoinducer typical strain Mesorhizobium shangrilenseThe consistence of the 16S rRNA gene order of CCBAU 65327 is 99.4%, with Australian Autoinducer typical strain Mesorhizobium australicumThe consistence of the 16S rRNA gene order of WSM2073 is 99.1%.This shows that the sequence that amplification obtains is 16S rRNA gene order, and bacterial strain KDRM295 belongs to Autoinducer and belongs to.

5. the Phylogenetic Analysis of root nodule bacterium 16S rRNA gene

The 16S rRNA gene order that the 16S rRNA gene order SEQ ID NO:1 of bacterial strain KDRM295 and Autoinducer is belonged to the various typical strains (seeing the List of Prokaryotic names with Standing in Nomenclature, http://www.bacterio.cict.fr) of existing 25 kinds is carried out Phylogenetic Analysis together.During analysis with rhizobium type species beans root nodule bacterium typical strain Rhizobium leguminosarumThe 16S rRNA gene order of USDA 2370 is outer group.With MEGA 5.0 softwares 16S rRNA gene order is carried out Phylogenetic Analysis, join with default parameters running process connection with the MUSCLE program that MEGA 5.0 softwares are integrated, use the Neighbor-joining method with Kimura 2-parameter model construction phylogenetic tree; Tree branch node expanding value repeats 1000 times with the Bootstrap method and calculates.

The phylogenetic tree that described analysis makes up is seen Fig. 1.The 16S rRNA gene that Fig. 1 shows bacterial strain KDRM295 with Mesorhizobium loti, Mesorhizobium ciceri, Mesorhizobium shangrilenseWith Mesorhizobium australicum16S rRNA gene sibship Deng four kinds is nearer, is in a branch adjacent with these four kinds.Can determine also that thus bacterial strain KDRM295 belongs to Autoinducer and belongs to, but can not determine which kind KDRM295 belongs to.

6. root nodule bacterium AcdSThe Phylogenetic Analysis of gene

With bacterial strain KDRM295's AcdSGene order SEQ ID NO:2 and Autoinducer have been found in belonging to AcdS7 bacterial strains AcdSGene order is carried out Phylogenetic Analysis together.During analysis with the beans root nodule bacterium Rhizobium leguminosarumBv. viciae 3841 AcdSGene order is outer group.With MEGA 5.0 softwares pair AcdSGene order is carried out Phylogenetic Analysis, joins with default parameters running process connection with the MUSCLE program that MEGA 5.0 softwares are integrated, and uses the Neighbor-joining method with Kimura 2-parameter model construction phylogenetic tree; Tree branch node expanding value repeats 1000 times with the Bootstrap method and calculates.

The phylogenetic tree that described analysis makes up is seen Fig. 2.Fig. 2 shows bacterial strain KDRM295's AcdSWith the known Autoinducer that acc deaminase arranged in phylogeny AcdSBe in different branches, show that bacterial strain KDRM295 is different from the known bacterial strain that acc deaminase is arranged in the Autoinducer genus.

Sequence table

＜110＞in be full of the Changjiang river international new forms of energy Investment Co., Ltd

＜120＞Autoinducer KDRM295 and application thereof

＜160＞ 2

＜210＞ 1

＜211＞ 1309 bp

＜212＞ DNA

＜213＞Autoinducer ( Mesorhizobium)

＜400＞ 1

gcagacgggt gagtaacgcg tgggaatcta cccatctcta cggaacaact ccgggaaact 60

ggagctaata ccgtatacgt ccttcgggag aaagatttat cggagatgga tgagcccgcg 120

ttggattagc tagttggtgg ggtaatggcc taccaaggcg acgatccata gctggtctga 180

gaggatgatc agccacattg ggactgagac acggcccaaa ctcctacggg aggcagcagt 240

ggggaatatt ggacaatggg cgaaagcctg atccagccat gccgcgtgag tgatgaaggc 300

cctagggttg taaagctctt tcaacggtga agataatgac ggtaaccgta gaagaagccc 360

cggctaactt cgtgccagca gccgcggtaa tacgaagggg gctagcgttg ttcggaatta 420

ctgggcgtaa agcgcacgta ggcggattgt taagttaggg gtgaaatccc ggggctcaac 480

cccggaactg cctttaatac tggcaatctc gagtccgaga gaggtgagtg gaattccgag 540

tgtagaggtg aaattcgtag atattcggag gaacaccagt ggcgaaggcg gctcactggc 600

tcggtactga cgctgaggtg cgaaagcgtg gggagcaaac aggattagat accctggtag 660

tccacgccgt aaactatgag agctagccgt cggcaagttt acttgtcggt ggcgcagcta 720

acgcattaag ctctccgcct ggggagtacg gtcgcaagat taaaactcaa aggaattgac 780

gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcgc agaaccttac 840

cagcccttga catcccggtc gcggtttcca gagatggatc ccttcagttc ggctggaccg 900

gtgacaggtg ctgcatggct gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc 960

aacgagcgca accctcgccc ttagttgcca gcattcagtt gggcactcta aggggactgc 1020

cggtgataag ccgagaggaa ggtggggatg acgtcaagtc ctcatggccc ttacgggctg 1080

ggctacacac gtgctacaat ggtggtgaca gtgggcagcg agaccgcgag gtcgagctaa 1140

tctccaaaag ccatctcagt tcggattgca ctctgcaact cgagtgcatg aagttggaat 1200

cgctagtaat cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg 1260

cccgtcacac catgggagtt ggttttaccc gaaggcgctg tgctaaccg 1309

＜210＞ 1

＜211＞ 629 bp

＜212＞ DNA

＜213＞Autoinducer ( Mesorhizobium)

＜400＞ 2

gcgcatggtg gcagccgtgg cggcgaaaat cggcatgaaa tgccgcctcg tgcaggaaag 60

ctgggtgccc cacgaggacg ccgtctatga tcgcgtcggc aacatccttt tgagccggat 120

catgggcgcc gatatccaga tggtcgatga gggcttcgac atcggaatca gggaaagctg 180

ggaacaggcg atcgccgatg tgaaagccaa aggcggcaaa ccttatccga ttccggccgg 240

ggcttcggtg cataaatatg gcggcctggg ctacgtcggc ttcgccgaag aggtgcgcgc 300

gcaggagaag caactcggcc ttgccttcga ctacatcgtc gtctgtaccg tcaccggctc 360

tacccatgcc ggcatggtgg tcggcttcgc caaggacggt cgtgagcgca aggtgatcgg 420

catcgacgca tcctgcaccc cggcgcagac caaggcgcaa gtgctggaca tcgtgcggac 480

cacggcgacg ctggtcgaac tcggcaagga cctcggtgaa gacgatgtga tcctcatcga 540

ggattatgcc tacccggtct acggcgtacc ctcgcaggag acgaaggaag cgatccgtct 600

ctgcgcgcgc cttgaaggca tgatcaccg 629

Claims (2)

1. Autoinducer KDRM295, it is characterized in that: it has been preserved in Chinese Typical Representative culture collection center, and deposit number is CCTCC NO:M 2012331.

2. the application of Autoinducer KDRM295 according to claim 1 in the locust tree breeding and afforestation.

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