CN103045500B - 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 be separated from locust tree root nodule, have acc deaminase can efficient dross promote Autoinducer KDRM295 and the application thereof of Growth of Blaek Locust.This bacterial strain has been preserved in China typical 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, by the N in air ₂be reduced into NH ₃.The nitrogen fixed by nitrogen-fixing microorganism accounts for 65% – 70% of earth's surface combined nitrogen, wherein the strongest with root nodule bacterium and legume symbiosis system nitrogen fixing capacity, accounts for more than 65% of biological nitrogen fixation amount.

Root nodule bacterium and legume symbiosis fixed nitrogen is utilized to increase soil fertility and crop yield is the classical experience of world agriculture.As made green manure with leguminous plants, allow pulse family and Non-legume plants crop rotation, intercropping and interplanting.Improve existing more than the 100 year history of the symbiotic eutectic transformation of leguminous crop and output and soil fertility by Rhizobium Inoculation, being extensively 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 expands, 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 root nodule bacterium nodulation and nitrogen fixation, the nodulation and nitrogen fixation effect of Rhizobium Inoculation sharply declines in the farmland of using 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, the legume inoculation technology application that cultivated area and reinforcement for expansion Leguminosae tree, herbage match with it provides opportunity.

Locust tree ( robinia pseudoacacial.) also known as acacia, be deciduous tree, high 10 – 25 meters, wooden hard, flexible, resistance to wearing, is good ore pillar, sleeper, construction timber.Locust tree originates in eastern united states, after introducing a fine variety Europe, introduces China again in late nineteenth century from Europe.Growth of Blaek Locust is fast, and distribution is more and more wider, has benefited from locust tree strong adaptability on the one hand, can grow, have certain drought-resistance ability in the saline-alkali soil of sour earth, neutral soil and saltiness less than 0.3%; On the other hand locust tree is leguminous plants, can with root nodule bacterium symbiotic nitrogen fixation and obtain and grow necessary nitrogen, thus be suitable for growing in barren soil.The ecologic effect of plantation locust tree, as conserved water and soil, improveing soil etc., also highly significant.Therefore, locust tree becomes one of the whole world most important fast-growing afforestation vanguard tree seed, the vanguard tree seed of Ye Shi China project of conceding the land to forestry.

Along with the exhaustion day by day of the fossil energies such as Global Oil, coal and Sweet natural gas, develop reproducible biomass energy to replace fossil energy more and more for countries in the world government is paid close attention to.At present, China's economic increases fast, the sharp increase of fossil energy consumption, and energy shortage problem is very outstanding, and the development and utilization of the renewable energy sources such as biomass energy has become the key realizing 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 resistance to lean feature such as degeneration-resistant, become important Tree Species as Bio-energy.Barren on the ground waste, be the bottleneck problem that current China locust tree energy forest is produced with the biomass of becoming a useful person that the input-output of Shaoshi fertilizer, low cost are high.With the legume inoculation locust tree seedling of efficient nodulation and nitrogen fixation, make sapling obtain nitrogen on barren soil by symbiotic nitrogen fixation and become a useful person, forestation of locust efficiency may be improved and reduce raw materials cost.At present, on the one hand, technically, modern forestation of locust improves afforestation efficiency, for locust tree the Miaos work Rhizobium Inoculation creates convenience by extensive seedling nursery.On the other hand, be limited to technology and managerial restriction, the large-scale afforestation of China does not also implement the extensive fertilising based on nitrogen to forestry developed country like that, but do not apply fertilizer on the contrary for the symbiotic nitrogen fixation of locust tree and root nodule bacterium creates the condition not having nitrogen to check, be conducive to the efficiency improving symbiotic nitrogen fixation.

Locust tree adapt to a strong factor of lean soil ability be it can with multiple root nodule bacterium symbiotic nitrogen fixation belonging to the genetic diversity of kind.Found to belong to the mainly Autoinducer of locust tree dross ( mesorhizobium) root nodule bacterium, also have Sinorhizobium Pseudomonas ( sinorhizobium), rhizobium ( rhizobium) and Bradyrhizobium ( bradyrhizobium) etc. belong to root nodule bacterium.The ability height of root nodule bacterium and locust tree nodulation and nitrogen fixation differs, and Growth of Blaek Locust needs to select efficient rhizobium strains to utilize Rhizobium Inoculation to promote.The excellent rhizobium strains of usual screening is by being separated multiple rhizobium strains from multiple large and full locust tree root nodule, inoculates locust tree seedling one by one, cultivates 2 months or after the longer time, detects the dross number in locust tree shoot root portion 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, screening process wastes time and energy.

Root nodule bacterium infect fabaceous can cause the 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-one butyric acid and NH ₃.When root nodule bacterium infect root, root cells synthesis ACC and release portion ACC to extracellular.There are the root nodule bacterium of acc deaminase to absorb and the ACC of degrading plant cell release, make the ACC in root cells constantly discharge and reduce, the amount just corresponding minimizing of root cells synthesizing ethylene, thus reduce ethene infects dross restraining effect to root nodule bacterium.If the acc deaminase structure gene of root nodule bacterium ( acdS) knock out, the Noduling ability of root nodule bacterium significantly can reduce (Uchiumi etc., Journal of Bacteriology 2004,186:2439-2448); On the contrary, if do not had 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 to have the root nodule bacterium of acc deaminase to have strong invasiveness, can efficient dross.The machine-processed more complicated of root nodule bacterium regulation and control acc deaminase.Research has found much have acdSautoinducer belong to root nodule bacterium do not express under free cultivation conditions acdS, do not have acc deaminase active, but can express at a high level in root nodule acdS, play effect (Ma etc., the Antonie van Leeuwenhoek 2003,83:285-291 of 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 root nodule bacterium of acc deaminase, if the acc deaminase of root nodule bacterium is active under detecting free cultivation conditions, can lose efficacy to a lot of root nodule bacterium especially Autoinducer, and with polymerase chain reaction (PCR) detect root nodule bacterium with or without 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 be separated from locust tree root nodule, have acc deaminase can efficient dross promote Autoinducer KDRM295 and the application thereof of Growth of Blaek Locust.

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

First use ordinary method abstraction and purification root nodule bacterium from locust tree root nodule, then increase from root nodule bacterium genome acdSgene, to amplify target fragment order-checking determine bacterial strain with or without acdSgene and acc deaminase, again by the rhizobium strains inoculation BLACK LOCUST SEEDLINGS having acc deaminase, detect dross number, the plant height of plant, leading thread and dry weight after 3 months in inoculation, select can efficient dross, significantly promote the growth of locust tree seedling and improve biomass rhizobium strains for breeding and afforestation.On the other hand, amplification has the 16S rRNA gene of acc deaminase root nodule bacterium and checks order, by the category attribution of sequence alignment and Phylogenetic Analysis 16S rRNA gene order determination bacterial strain; Acc deaminase bacterial strain be there will be a known with belonging to together in root nodule bacterium to the root nodule bacterium of acc deaminase that have filtered out simultaneously acdSgene order carries 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 having acc deaminase.

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

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

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

16S rRNA gene order (see SEQ ID NO:1) and the Autoinducer generitype Root or stem of Littleleaf Indianmulberry Autoinducer typical strain of described Autoinducer KDRM295 mesorhizobium lotithe 16S rRNA gene order consistence of ATCC 700743 is 99.7%, with 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%, comparatively near 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, KDRM295 bacterial strain belongs to Autoinducer and belongs to, and can not determine which belongs to plants.

Described Autoinducer KDRM295 has acc deaminase.It acdSgene Partial sequence (see SEQ ID NO:2) and Autoinducer belong to and there will be a known acc deaminase bacterial strain 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, is only 78.8%; Status in phylogeny is also different from other Autoinducers acdS(Fig. 2).This shows that bacterial strain KDRM295 is different from during Autoinducer belongs to the bacterial strain that there will be a known acc deaminase.

The 16S rRNA gene of above-mentioned Autoinducer KDRM295 and acdSthe analytical results of gene combines and shows that the genotype of KDRM295 is different from known Autoinducer.

Described Autoinducer KDRM295 can on locust tree root efficient nodulation and nitrogen fixation, promote the growth of locust tree seedling and become a useful person.

Beneficial effect of the present invention:

Autoinducer KDRM295 of the present invention has acc deaminase, ACC can be resolved into α-one 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 the barren waste aerial that locust tree is not being applied fertilizer provides high-caliber nitrogen, promote the growth of locust tree seedling, increase the biomass that locust tree becomes a useful person, thus drop into the inoculation of low cost and allow locust tree become a useful person high yield, locust tree breeding and afforestation plays a role.

Accompanying drawing explanation

Fig. 1 is Autoinducer bacterial strain KDRM295(●) belong to Autoinducer ( mesorhizobium) phylogenetic tree of 16S rRNA gene of typical strain of existing 25 kinds.In figure, ■ indicates Autoinducer generitype Root or stem of Littleleaf Indianmulberry Autoinducer typical strain mesorhizobium lotiaTCC 700743, ▲ instruction 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 GenBank database in bacterial strain name unquote.

Fig. 2 is Autoinducer bacterial strain KDRM295(●) and Autoinducer genus ( mesorhizobium) in have acc deaminase bacterial strain acdSthe phylogenetic tree of gene.In figure, ■ indicates camel thorn Autoinducer mesorhizobium alhagicCNWXJ12-2, in bacterial strain name unquote is acdSthe accession number of gene nucleotide series in GenBank database.

Embodiment

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

Root nodule large and full on healthy and strong locust tree root is selected from the locust tree at Guishan Mountain, Danjiangkou, Hubei Province artificial growth, carefully divide root to cut root nodule company headquarters with scissors, 5 –, 15 root nodules that same locust tree root obtains are put into and dry discolour silica gel is housed and the tubule being covered with absorbent cotton.Then after the root nodule sterilized water of collection fully being soaked imbibition, with alcohol-pickled 30 s of 95%, then mercuric chloride surface sterilization 5 min of 0.1% is used, use aseptic water washing again 6 times, an aseptic 2-ml centrifuge tube will be put into respectively after each root nodule numbering, with aseptic grinding rod, root nodule is ground, 1 ml sterilized water is added and suction 3 suspension homogenates with pipettor, by suspension serial dilution 10 times, 100 times and 1000 times, then (often liter containing 1 g yeast powder at YMA solid medium to draw 100 μ l suspension respectively, 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 powder) on, culture plate is placed on 28oC light culture.Cultivate 3 – after 5 days picking have typical root nodule bacterium colonial morphology (circle, oyster white, protuberance, neat in edge do not spread, smooth surface and because there being abundant exocellular polysaccharide thickness, more moistening, slightly transparent) bacterium colony, streak culture on YMA flat board, repeat 3 line purifying bacterium colonies.Single bacterium colony of purifying is suspended in sterilized water, microscopy after gramstaining, select Gram-negative, cell be shaft-like and the consistent bacterium of form as the rhizobium strains of purifying.The rhizobium strains of described purifying can be seeded in TY nutrient solution (often liter containing 5 g Tryptoness, 3 g yeast powders, 0.33 g CaCl ₂, pH 6.8) in be cultured to late log phase or stationary phase, with 30%(v/v) glycerine solution equal-volume mix, be frozen in-80oC and preserve for a long time.

2. root nodule bacterium acdSthe amplification of gene and qualification

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

SEQ ID NO:2 is seen from the bacterial strain KDRM295 DNA fragmentation that the obtains sequence after removing primer that increases.Sequence SEQ ID NO:2 is carried out BLAST retrieval in ncbi database, and result display 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 between the two is 78.8%.This sequence obtained that shows to increase is acdSsequence, bacterial strain KDRM295 has acc deaminase.

3. with having the legume inoculation locust tree seedling of acc deaminase and detecting Inoculating efficiency

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

Table 1 root nodule bacterium KDRM295 inoculates the effect of locust tree seedling

	Dross number	Plant height (cm)	Leading thread (mm)	Overground part dry weight (g)	Dry weight increases (%)
						The contrast locust tree seedling do not inoculated	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: after inoculation locust tree, root nodule bacterium KDRM295 and locust tree symbiosis can form more root nodule, by the N in reducing atmosphere ₂for Growth of Blaek Locust provides nitrogen, significantly can promote that locust tree seedling grows, increase biomass.

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

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

SEQ ID NO:1 is seen from the bacterial strain KDRM295 DNA fragmentation that the obtains sequence after removing primer that increases.Sequence SEQ ID NO:1 is carried out BLAST retrieval in ncbi database, result display SEQ ID NO:1 is similar to the 16S rRNA gene order that Autoinducer belongs to 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 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 sequence obtained that shows to increase 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 16S rRNA gene order SEQ ID NO:1 of bacterial strain KDRM295 and Autoinducer being belonged to the various typical strains (see the List of Prokaryotic names with Standing in Nomenclature, http://www.bacterio.cict.fr) of existing 25 kinds carries out Phylogenetic Analysis together.With rhizobium type species beans root nodule bacterium typical strain during analysis rhizobium leguminosarumthe 16S rRNA gene order of USDA 2370 is outer group.With MEGA 5.0 software, Phylogenetic Analysis is carried out to 16S rRNA gene order, the MUSCLE program integrated with MEGA 5.0 software is joined, by Neighbor-joining method with Kimura 2-parameter model construction phylogenetic tree with default parameters running process connection; Tree branch node expanding value Bootstrap method repeats 1000 times and calculates.

The described phylogenetic tree built of analyzing is shown in 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 comparatively near, is in a branch adjacent with these four kinds.Also can determine that bacterial strain KDRM295 belongs to Autoinducer and belongs to thus, but can not determine which KDRM295 belongs to and plant.

6. root nodule bacterium acdSthe Phylogenetic Analysis of gene

By bacterial strain KDRM295's acdSfind that there is during gene order SEQ ID NO:2 and Autoinducer belong to acdS7 bacterial strains acdSgene order carries out Phylogenetic Analysis together.With beans root nodule bacterium during analysis rhizobium leguminosarumbv. viciae 3841 acdSgene order is outer group.With MEGA 5.0 software pair acdSgene order carries out Phylogenetic Analysis, and the MUSCLE program integrated with MEGA 5.0 software is joined, by Neighbor-joining method with Kimura 2-parameter model construction phylogenetic tree with default parameters running process connection; Tree branch node expanding value Bootstrap method repeats 1000 times and calculates.

The described phylogenetic tree built of analyzing is shown in Fig. 2.Fig. 2 shows bacterial strain KDRM295's acdSwith the Autoinducer that there will be a known acc deaminase in phylogeny acdSbe in different branches, show that bacterial strain KDRM295 is different from during Autoinducer belongs to the bacterial strain that there will be a known acc deaminase.

Sequence table

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

 

< 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 ( mesorhizobiumsp.) KDRM295, be preserved in China typical culture collection center, deposit number is CCTCC NO:M 2012331, it is characterized in that: the 16S rRNA gene nucleotide series of described Autoinducer KDRM295 as shown in SEQ NO:1, described Autoinducer KDRM295's acdSgene nucleotide series is as shown in SEQ NO:2.

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