CN102978136B - Mesorhizobium KDRM024 and application thereof - Google Patents

Abstract

The invention relates to a mesorhizobium KDRM024 capable of being separated from an acacia root nodule, containing ACC deaminase, efficiently nodulating and promoting acacia growth and application thereof. A mesorhizobium KDRM024 strain is collected in CCTCC (China Center for Type Culture Collection), and the collection number is CCTCC No: M2012336. The mesorhizobium KDRM024 disclosed by the invention contains the ACC deaminase and can decompose the ACC into alpha-ketobutyrate and NH3, lower the level of plant cells on synthesizing ethylene, reduce the inhibiting effect of the ethylene on rhizobium infestation, enhance the nodulation rate and symbiotic nitrogen fixation level of rhizobia and acacia, provide high-level nitrogen for the growth of the acacia on unfertilized sterile wastelands, promote the growth of acacia seedlings and increase the biomass of acacia lumbers, thereby achieving the high yield of the acacia lumbers through low-cost inoculation investment and playing a role in acacia seedling culture and forestation.

Description

Autoinducer KDRM024 and application thereof

Technical field

The invention belongs to applied microbiology field, be specifically related to one separated from locust tree root nodule, have the acc deaminase can efficient dross and promote Autoinducer KDRM024 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 2012336, and bacterial strain is called Autoinducer KDRM024.

Background technology

Some prokaryotic micro-organisms of occurring in nature can synthesize nitrogenase, at normal temperatures and pressures, and by airborne N ₂be reduced into NH ₃.The nitrogen of being fixed by nitrogen-fixing microorganism accounts for 65% – 70% of earth's surface combined nitrogen, wherein the strongest with root nodule bacterium and leguminous plants syntaxial system nitrogen fixing capacity, accounts for the 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 made green manure with leguminous plants, allow pulse family and non-leguminous crop crop rotation, intercropping and interplanting.The symbiotic nitrogen fixation efficiency and output and existing more than the 100 year history of soil fertility that by Rhizobium Inoculation, improve leguminous crop, extensively Wei Ge 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 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, for the legume inoculation technology application that expands the cultivated area of Leguminosae tree, herbage and strengthen matching 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 eastern united states, introduces a fine variety behind Europe, in late nineteenth century, introduces China from Europe again.Growth of Blaek Locust is fast, distributes more and more wider, has benefited from locust tree strong adaptability on the one hand, can in the saline-alkali soil below 0.3%, grow in sour earth, neutral soil and saltiness, has certain drought-resistance ability; Locust tree is leguminous plants on the other hand, can obtain with 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, the vanguard tree seed of Ye Shi China project of conceding the land to forestry.

Day by day exhausted along with fossil energies such as Global Oil, coal and Sweet natural gases, develops reproducible biomass energy and take that to replace fossil energy be more and more the concern of countries in the world government.At present, China's economic rapid growth, the sharp increase of fossil energy consumption, 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 resistance to lean feature 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 current China locust tree energy forest is produced.Legume inoculation locust tree seedling with efficient nodulation and nitrogen fixation, makes sapling obtain nitrogen on barren soil by symbiotic nitrogen fixation and becomes a useful person, and may improve forestation of locust efficiency and reduce raw materials cost.At present, on the one hand, technically, modern forestation of locust improves afforestation efficiency by extensive seedling nursery, for locust tree the Miaos work Rhizobium Inoculation has been created convenience.On the other hand, be limited to technology and managerial restriction, the large-scale afforestation of China does not also implement take nitrogen as main extensive fertilising to forestry developed country like that, but do not apply fertilizer, for the symbiotic nitrogen fixation of locust tree and root nodule bacterium, do not create the condition that does not have nitrogen to check on the contrary, be conducive to improve the efficiency 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 locust tree dross be mainly 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.Conventionally the good rhizobium strains of screening is by separated a plurality of rhizobium strainss from a plurality of large and full locust tree root nodules, inoculates one by one locust tree seedling, cultivates 2 months or after the longer time, detects the dross number of 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 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-one butyric acid and NH ₃.When root nodule bacterium are infected root, the synthetic ACC of root cells release portion ACC are to extracellular.The ACC that root nodule bacterium can absorb and degrading plant cell discharges that has acc deaminase, makes the ACC in root cells constantly discharge and reduce, and the amount of root cells synthesizing ethylene, with regard to corresponding minimizing, infects the restraining effect of dross thereby reduced ethene to root nodule bacterium.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 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 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 can express in root nodule 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 free cultivation conditions, to a lot of root nodule bacterium especially Autoinducer, can lose efficacy, and detect root nodule bacterium with polymerase chain reaction (PCR), have or not 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 separated from locust tree root nodule, have the acc deaminase can efficient dross and promote Autoinducer KDRM024 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 separated and purifying root nodule bacterium from locust tree root nodule, then increase from root nodule bacterium genome acdSgene, determines that to the target fragment order-checking amplifying bacterial strain has or not acdSgene and acc deaminase, again by the rhizobium strains inoculation BLACK LOCUST SEEDLINGS that has acc deaminase, in inoculation, after 3 months, detect plant height, leading thread and the dry weight of dross number, plant, the rhizobium strains that select the efficient dross of energy, significantly promotes the growth of locust tree seedling and improve biomass is for breeding and afforestation.On the other hand, amplification has 16S rRNA gene 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; The root nodule bacterium that have acc deaminase that filter out knownly in root nodule bacterium are had to an acc deaminase bacterial strain with belonging to together simultaneously acdSgene order is carried out Phylogenetic Analysis, in conjunction with bacterial strain 16S rRNA gene and acdSthe Phylogenetic of gene, final definite new root nodule bacterium strain that has acc deaminase that obtains.

Autoinducer KDRM024 provided by the invention is preserved in Chinese Typical Representative culture collection center on September 7th, 2012, deposit number is CCTCC NO:M 2012336, and preservation address is China, Wuhan, Wuhan University, Classification And Nomenclature is Autoinducer KDRM024 mesorhizobiumsp. KDRM024.

Described Autoinducer KDRM024 is separated to from the root nodule peace mountain, Jiangxia District, Wuhan City, Hubei Province artificial growth locust tree.

The cell of described Autoinducer KDRM024 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, comparatively fast, the diameter that 3 – that grows in YMA medium forms bacterium colony for 5 days can reach 1 – 2 mm in growth.

The 16S rRNA gene order of described Autoinducer KDRM024 (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 98.1%, with chance Autoinducer typical strain mesorhizobium opportunistumthe 16S rRNA gene order consistence of WSM2075 is 99.8%, also nearest with the latter's sibship on phylogenetic tree, is in the branch that same node separates (Fig. 1).Therefore, bacterial strain KDRM024 belongs to Autoinducer and belongs to, and may belong to chance Autoinducer kind.

Described Autoinducer KDRM024 has acc deaminase.It acdSgene Partial sequence (seeing SEQ ID NO:2) with mesorhizobium opportunistumwSM2075's acdSthe consistence of corresponding sequence be 86.1%, with false indigo Autoinducer separated from be grown in the root nodule of Gansu Province locust tree mesorhizobium amorphaetwo copies of CCNWGS0123 acdSin one acdSthe consistence of corresponding sequence (can retrieve in the sequence of GenBank accession number AGSN01000010) be 98.8%, be on phylogenetic tree from the former in different branches, be in the branch that same node separates (Fig. 2) with the latter.This shows that bacterial strain KDRM024 is different with typical chance Autoinducer, and its ancestors may be that the mode by horizontal transfer has obtained from the root nodule bacterium of other kinds in evolution acdSgene.

The 16S rRNA gene of above-mentioned Autoinducer KDRM024 and acdSthe analytical results of gene is in conjunction with showing that the genotype of KDRM024 is different from known Autoinducer.

Can be on the locust tree root efficient nodulation and nitrogen fixation of described Autoinducer KDRM024, promotes the growth of locust tree seedling and becomes a useful person.

Beneficial effect of the present invention:

Autoinducer KDRM024 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 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 with inoculation cheaply, drop into and to allow the locust tree high yield of becoming a useful person, on locust tree breeding and afforestation, play a role.

Accompanying drawing explanation

Fig. 1 is Autoinducer bacterial strain KDRM024(●) 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 in figure mesorhizobium lotiaTCC 700743, ▲ indication chance Autoinducer typical strain mesorhizobium opportunistumwSM2075 is the accession number of 16S rRNA gene nucleotide series in GenBank database in bacterial strain name unquote.

Fig. 2 is Autoinducer bacterial strain KDRM024(●) and Autoinducer genus ( mesorhizobium) in have acc deaminase bacterial strain acdSthe phylogenetic tree of gene.■ indication false indigo Autoinducer in figure mesorhizobium amorphaecCNWGS0123, ▲ indication chance Autoinducer mesorhizobium opportunistumwSM2075, 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

From the locust tree at peace mountain, Jiangxia District, Wuhan City, Hubei Province artificial growth, select large and full root nodule on healthy and strong locust tree root, with scissors, carefully root nodule company headquarters root division is cut, 15 root nodules of 5 – that same locust tree root is obtained are put into the tubule that dry discolour silica gel is housed and is covered with absorbent cotton.Then the root nodule of collection is fully soaked after 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 after each root nodule numbering, put into respectively an aseptic 2-ml centrifuge tube, with aseptic grinding rod, root nodule is ground, with pipettor, add 1 ml sterilized water 3 suspension homogenates of suction, by 10 times of suspension serial dilutions, 100 times and 1000 times, then drawing respectively 100 μ l suspension is coated on YMA solid medium (every liter containing 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) and upper, culture plate is placed on to 28oC and secretly cultivates.Cultivate 3 – after 5 days picking have the bacterium colony of 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), streak culture on YMA flat board, repeat line purifying bacterium colony 3 times.Single bacterium colony of purifying is suspended in sterilized water, and microscopy after gramstaining, selects 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 TY nutrient solution, and (every liter containing 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 mix, be frozen in-80oC preserves for a long time.

2. root nodule bacterium acdSthe amplification of gene and evaluation

Rhizobium strains is inoculated into and in TY nutrient solution, is cultured to the 100 μ l bacterium liquid of logarithm later stage or stationary phase and moves in 1.5-ml centrifuge tube, centrifugal 5 min of 8000 rpm, suck supernatant liquor, with 0.5 ml sterilizing distilled water, clean 2 times, 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 μ M.2 * Taq PCR MasterMix that reaction system is produced with TIANGEN Biotech (Beijing) Co., Ltd..Pcr amplification carries out on 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.

The sequence of the DNA fragmentation obtaining from bacterial strain KDRM024 amplification removing primer is shown in SEQ ID NO:2.Sequence SEQ ID NO:2 is carried out to BLAST retrieval in ncbi database, and result shows that SEQ ID NO:2 and Autoinducer belong to root nodule bacterium acdSsimilar, wherein: similarity is the highest is separated false indigo Autoinducer from be grown in the root nodule of Gansu Province locust tree mesorhizobium amorphaetwo copies of CCNWGS0123 bacterial strain acdSin one acdScorresponding sequence (GenBank accession number AGSN01000010), the consistence of the two is 98.8%.This shows that the sequence that amplification obtains is acdSsequence, bacterial strain KDRM024 has acc deaminase.

3. with having the legume inoculation locust tree seedling of acc deaminase and detecting 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, in the greenhouse of relative humidity 75 – 85%, cultivate, water weekly once, when locust tree emerges approximately 5 – 8 cm after approximately 5 weeks, inoculate.Be specially: the fresh colony inoculation of first root nodule bacterium that have acc deaminase being grown on YMA solid medium, to TY nutrient solution, is cultivated 48 – 72 h to stationary phase at 28oC and 200 rpm; The bottled 100 ml nutrient solutions of each 500-ml taper during cultivation, after cultivating, every milliliter approximately contains 5 * 10 ⁹individual bacterial cell.Then after bacterium liquid being diluted to 500 times with tap water, water seedbed.Contrast seedling replaces watering with the tap water of equivalent.Inoculate the dross number, plant height, leading thread and the dry weight that after 3 months, detect locust tree seedling.Wherein: the result of bacterial strain KDRM024 is as shown in table 1.

The effect of table 1 root nodule bacterium KDRM024 inoculation locust tree seedling

	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	47	111	5.58	15.24	140%

As can be seen from Table 1: after inoculation locust tree, root nodule bacterium KDRM024 and locust tree symbiosis can form more root nodule, by the N in 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 moved in 1.5-ml centrifuge tube to being cultured to the 100 μ l bacterium liquid of logarithm later stage or stationary phase in TY nutrient solution, centrifugal 5 min of 8000 rpm, suck supernatant liquor, with 0.5 ml sterilizing distilled water, clean 2 times, 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 μ M.2 * Taq PCR MasterMix that reaction system is produced with TIANGEN Biotech (Beijing) Co., Ltd..Pcr amplification carries out on 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.

The sequence of the DNA fragmentation obtaining from bacterial strain KDRM024 amplification removing primer is shown in SEQ ID NO:1.Sequence SEQ ID NO:1 is carried out to BLAST retrieval in ncbi database, result shows that 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 98.1%, with chance Autoinducer typical strain mesorhizobium opportunistumthe 16S rRNA gene order consistence of WSM2075 is 99.8%.This shows that the sequence that amplification obtains is 16S rRNA gene order, and bacterial strain KDRM024 belongs to Autoinducer and belongs to.

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

The 16S rRNA gene order of the various typical strains (seeing the List of Prokaryotic names with Standing in Nomenclature, http://www.bacterio.cict.fr) that the 16S rRNA gene order SEQ ID NO:1 of bacterial strain KDRM024 and Autoinducer are belonged to 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 to Phylogenetic Analysis, the MUSCLE program of integrating with MEGA 5.0 softwares is joined with default parameters running process connection, uses Neighbor-joining method with Kimura 2-parameter model construction phylogenetic tree; Tree branch node expanding value repeats 1000 times by Bootstrap method and calculates.

The phylogenetic tree that described analysis builds is shown in Fig. 1.Fig. 1 shows 16S rRNA gene and the chance Autoinducer typical strain of bacterial strain KDRM024 mesorhizobium opportunistumthe 16S rRNA gene of WSM2075 is in the branch that same node separates, and shows that bacterial strain KDRM024 belongs to Autoinducer and belongs to, and may belong to chance Autoinducer kind.

6. root nodule bacterium acdSthe Phylogenetic Analysis of gene

By bacterial strain KDRM024's acdSgene order SEQ ID NO:2 and Autoinducer find that there is in belonging to acdS7 bacterial strains acdSgene order is carried out Phylogenetic Analysis together.During analysis with 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, and the MUSCLE program of integrating with MEGA 5.0 softwares is joined with default parameters running process connection, uses Neighbor-joining method with Kimura 2-parameter model construction phylogenetic tree; Tree branch node expanding value repeats 1000 times by Bootstrap method and calculates.

The phylogenetic tree that described analysis builds is shown in Fig. 2.Fig. 2 shows bacterial strain KDRM024's acdSwith from leguminous forage biserrula pelecinusthe chance Autoinducer of separation in root nodule mesorhizobium opportunistumwSM2075's acdSbe in the different branches on phylogenetic tree, with false indigo Autoinducer separated from be grown in the root nodule of Gansu Province locust tree mesorhizobium amorphaetwo copies of CCNWGS0123 acdSin one acdS(GenBank accession number AGSN01000010) is in the branch that on phylogenetic tree, same node separates.This shows that KDRM024 is different with typical chance Autoinducer, and its ancestors may be that the mode by horizontal transfer has obtained from the root nodule bacterium of other kinds in evolution acdSgene.

Sequence table

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

< 120 > Autoinducer KDRM024 and application thereof

＜160＞ 2

＜210＞ 1

＜211＞ 1309 bp

＜212＞ DNA

< 213 > Autoinducers ( mesorhizobium)

＜400＞ 1

gcagacgggt gagtaacgcg tgggaatcta cccatctcta cggaacaact ccgggaaact 60

ggagctaata ccgtatacgt ccttttggag aaagatttat cggagatgga tgagcccgcg 120

ttggattagc tagttggtgg ggtaatggcc taccaaggcg acgatccata gctggtctga 180

gaggatgatc agccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt 240

ggggaatatt ggacaatggg cgcaagcctg atccagccat gccgcgtgag tgatgaaggc 300

cctagggttg taaagctctt tcaacggtga agataatgac ggtaaccgta gaagaagccc 360

cggctaactt cgtgccagca gccgcggtaa tacgaagggg gctagcgttg ttcggaatta 420

ctgggcgtaa agcgcacgta ggcggatact taagtcaggg gtgaaatccc ggggctcaac 480

cccggaactg cctttgatac tgggtatctc gagtccggaa gaggtgagtg gaattccgag 540

tgtagaggtg aaattcgtag atattcggag gaacaccagt ggcgaaggcg gctcactggt 600

ccggtactga cgctgaggtg cgaaagcgtg gggagcaaac aggattagat accctggtag 660

tccacgccgt aaacgatgga agctagccgt tggcaagttt acttgtcggt ggcgcagcta 720

acgcattaag cttcccgcct ggggagtacg gtcgcaagat taaaactcaa aggaattgac 780

gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcgc agaaccttac 840

cagcccttga catcccggtc gcggtttcca gagatggata 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 > Autoinducers ( mesorhizobium)

＜400＞ 2

gcggatggtc gccgcggtcg ccgccaagat cggcatgaaa tgcctcctgg tacaggagag 60

ctgtgttccg catgaggatg ccgtctacga ccgggtcggc aacattctct tgagccgcat 120

catgggagca gaggtgcgct tggtcgacga gggctttgac atcggcatcc gccgcagttg 180

ggaaaaagcg ctctatgagg tcaaggcaag gggcggcaca ccctatgcga taccagccgg 240

ggcgtctgtt cacgaaaagg gcggcctcgg ctatgtgggg tttgcggagg aggtgcgcgc 300

ccaagagaaa cagcttgggt ttgccttcga ctacatcatc gtttgcacgg tcacgggctc 360

gacgcatgct ggcatgctgg ttggatttgc caaggacggt cgacagcgca acgtgatcgg 420

tatcgatgct tctgccacgc ccgccagaac caaggcgcag gtgcttagca ttgcccaaca 480

tacagcgacg ctcgtcgatc tcggaacgga acttgtcgag gatgacgtcg tgttgctcga 540

ggagtacgct ggcccgtgtt atggcattcc gtccgagggg acgaaggaag ccatccgcct 600

gtgtgcgcag ctcgagggca tgattaccg 629

Claims (2)

1. Autoinducer ( mesorhizobiumsp.) KDRM024, it has been preserved in Chinese Typical Representative culture collection center, and deposit number is CCTCC NO:M 2012336, it is characterized in that: it has shown in 16S rRNA gene nucleotide series as shown in SEQ NO:1 and SEQ NO:2 acdSgene nucleotide series.
2. Autoinducer according to claim 1 ( mesorhizobiumsp.) application of KDRM024 in locust tree breeding and afforestation.

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