CN102978139B - Mesorhizobium KDRM495 and application thereof - Google Patents

Abstract

The invention relates to mesorhizobium KDRM495 and application thereof. The mesorhizobium KDRM495 is separated from acacia root nodule, contains ACC deaminase, and can efficiently nodulate and promotes acacia growth. A mesorhizobium KDRM495 strain is collected in CCTCC (China Center for Type Culture Collection), and the collection number is CCTCC No: M2012333. The mesorhizobium KDRM495 disclosed by the invention contains the ACC deaminase which can decompose ACC into alpha-ketobutyrate and NH3, lowers the level of synthesizing ethylene from plant cells, reduces the inhibiting effect of ethylene on rhizobium infestation, enhances the nodulation rate and symbiotic nitrogen fixation level of rhizobia and acacia, provides the high-level nitrogen for the growth of the acacia on unfertilized sterile wastelands, promotes the growth of acacia seedlings and increases the biomass of acacia lumbers, thereby achieving the high yield of the acacia lumbers through low-cost inoculation investment and playing a role in the aspect of acacia seedling culture and forestation.

Description

Autoinducer KDRM495 and application thereof

Technical field

The invention belongs to applied microbiology field, be specifically related to one that from locust tree root nodule, separate, have the acc deaminase can efficient dross and promote Autoinducer KDRM495 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 2012333, and bacterial strain is called Autoinducer KDRM495.

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 improve leguminous crop by Rhizobium Inoculation, be 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, 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, introduces China in late nineteenth century 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.

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 as countries in the world government is paid close attention to.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, be the bottleneck problem of current China locust tree energy forest production with Shaoshi fertilizer, the high biomass of becoming a useful person of input-output cheaply.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, 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 is not also implemented take nitrogen as main extensive fertilising to forestry developed country like that, create for the symbiotic nitrogen fixation of locust tree and root nodule bacterium the condition that does not have nitrogen to check on the contrary but do not apply fertilizer, 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 multiple 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. belong to root nodule bacterium.The ability height of root nodule bacterium and locust tree nodulation and nitrogen fixation 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 separating multiple rhizobium strainss from multiple large and full locust tree root nodules, inoculating 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 precursor 1-amino-cyclopropane-1 carboxylic acid (ACC) that can cause plant generation 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.Have the root nodule bacterium of acc deaminase to absorb and ACC that degrading plant cell discharges, make the ACC in root cells constantly discharge and reduce, the amount of root cells synthesizing ethylene is with regard to corresponding minimizing, thereby reduced ethene root nodule bacterium are infected the restraining effect of dross.If the acc deaminase structure gene of root nodule bacterium ( acdS) knock out, the Noduling ability of root nodule bacterium can significantly 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 been found much to have acdSautoinducer belong to root nodule bacterium under free cultivation conditions, do not express acdS, there is no acc deaminase activity, 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 detect the acc deaminase activity of root nodule bacterium under 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 locust tree root nodule, separate, have the acc deaminase can efficient dross and promote Autoinducer KDRM495 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 separate and purifying root nodule bacterium from locust tree root nodule by ordinary method, 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, after 3 months, detect plant height, leading thread and the dry weight of dross number, plant in inoculation, select the efficient dross of energy, significantly promote the rhizobium strains of the growth of locust tree seedling and raising biomass 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 KDRM495 provided by the invention is preserved in Chinese Typical Representative culture collection center on September 7th, 2012, deposit number is CCTCC NO:M 2012333, and preservation address is China, Wuhan, Wuhan University, Classification And Nomenclature is Autoinducer KDRM495 mesorhizobiumsp. KDRM495.

Described Autoinducer KDRM495 is from being separated to the root nodule of Yunxi County, Hubei Province artificial growth locust tree.

The cell of described Autoinducer KDRM495 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 (seeing SEQ ID NO:1) of described Autoinducer KDRM495 and Autoinducer generitype Root or stem of Littleleaf Indianmulberry Autoinducer typical strain mesorhizobium lotithe 16S rRNA gene order consistence of ATCC 700743 is 98.2%, 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, KDRM495 bacterial strain belongs to Autoinducer and belongs to, and may belong to chance Autoinducer kind.

Described Autoinducer KDRM495 has acc deaminase.It acdSgene Partial sequence (seeing SEQ ID NO:2) with mesorhizobium opportunistumwSM2075's acdSthe consistence of corresponding sequence be 86.8%, with the false indigo Autoinducer separating from the root nodule that is grown in 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 91.9%, 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 KDRM495 is different from typical chance Autoinducer, and its ancestors have obtained from the root nodule bacterium of other kinds by the mode of horizontal transfer acdSgene.

The 16S rRNA gene of above-mentioned Autoinducer KDRM495 and acdSthe analytical results of gene is in conjunction with showing that the genotype of KDRM495 is different from known Autoinducer.

Can be on the locust tree root efficient nodulation and nitrogen fixation of described Autoinducer KDRM495, promotes the growth of locust tree seedling and becomes a useful person.

Beneficial effect of the present invention:

Autoinducer KDRM495 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 drop into and allow the locust tree high yield of becoming a useful person with inoculation cheaply, on locust tree breeding and afforestation, play a role.

Accompanying drawing explanation

Fig. 1 is Autoinducer bacterial strain KDRM495(●) 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 KDRM495(●) 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 select large and full root nodule on healthy and strong locust tree root the artificial growth locust tree of Yunxi County, Hubei Province, carefully root nodule company headquarters root division is cut with scissors, 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, with 95% alcohol-pickled 30 s, 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, root nodule is ground with aseptic grinding rod, add 1 ml sterilized water 3 suspension homogenates of suction with pipettor, 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 the is consistent bacterium rhizobium strains as 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 cleaning 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 detect, then deliver to prompt base (Shanghai) the primer acdSf3 of Bioisystech Co., Ltd in the English Weihe River and acdSr3 and check order.

See SEQ ID NO:2 from the increase DNA fragmentation that obtains of bacterial strain KDRM495 in the sequence of removing primer.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: what similarity was the highest is the false indigo Autoinducer separating the root nodule from being grown in 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 91.9%.This shows that the sequence that amplification obtains is acdSsequence, bacterial strain KDRM495 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: first by the fresh colony inoculation that has the root nodule bacterium of acc deaminase to grow on YMA solid medium to TY nutrient solution, cultivate 48 – 72 h to stationary phase at 28oC and 200 rpm; The bottled 100 ml nutrient solutions of each 500-ml taper when 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 and 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 KDRM495 is as shown in table 1.

The effect of table 1 root nodule bacterium KDRM495 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	55	107	5.22	19.73	211%

As can be seen from Table 1: after inoculation locust tree, root nodule bacterium KDRM495 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 cleaning 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 detect, then deliver to the corresponding amplimer of prompt base (Shanghai) Bioisystech Co., Ltd in the English Weihe River and check order.

See SEQ ID NO:1 from the increase DNA fragmentation that obtains of bacterial strain KDRM495 in the sequence of removing primer.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 98.2%, 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 KDRM495 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 KDRM495 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.When analysis with rhizobium type species beans root nodule bacterium typical strain rhizobium leguminosarumthe 16S rRNA gene order of USDA 2370 is outer group.16S rRNA gene order is carried out to Phylogenetic Analysis with MEGA 5.0 softwares, 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 order and the chance Autoinducer typical strain of bacterial strain KDRM495 mesorhizobium opportunistumthe 16S rRNA gene order of WSM2075 is in the branch that same node separates, and shows that bacterial strain KDRM495 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 KDRM495'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.When 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 KDRM495's acdS acdSwith from leguminous forage biserrula pelecinusthe chance Autoinducer separating in root nodule mesorhizobium opportunistumwSM2075's acdSbe in the different branches on phylogenetic tree, with the false indigo Autoinducer separating from the root nodule that is grown in 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 KDRM49 is different from typical chance Autoinducer, and its ancestors have obtained from the root nodule bacterium of other kinds by the mode of horizontal transfer acdSgene.

Sequence table

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

< 120 > Autoinducer KDRM495 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 ccttcgggag 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 ttcaggagag 60

ctgggttcca catgaggatg ccgtctacga tcgggtcggc aacattcttt tgagccgcat 120

catgggagca gagctgcgcc tggtcgacga gggctttgac atcggcatcc gccgcagttg 180

ggaaaaagcg ctctatgagg tcaaggcaag gggcggcaga ccctatgcga tacccgccgg 240

ggcgtctgtt cacgaaaagg gcggcctcgg ctatgtgggg ttcgcggaag aggtgcgcgc 300

ccaggagaaa cagcttggct ttgccttcga ctacatcgtt gtttgcacgg tcacaggctc 360

aacgcatgcc ggcatgctcg tcggattcgc cgaggacggt cgacagtgca acgtgatcgg 420

tgtcgatgcc tctgccaccc ccaccaaaac caaggcgcag gtgctaaaca ttgcccaaca 480

tacagcgaag ctcgtcgatc tcgaaacgga aatcgtcgaa gacgacgtgg tgctgttcga 540

ggagtatgcg tacccgtgtt atggcattcc gtccgaagaa accaaggagg ccatccgcct 600

gtgcgcgcgg ctcgagggga taattaccg 629

Claims (2)

1. Autoinducer KDRM495, it has been preserved in Chinese Typical Representative culture collection center, and deposit number is CCTCC NO:M 2012333, 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. the application of Autoinducer KDRM495 according to claim 1 in locust tree breeding and afforestation.

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