CN1254533C - Engineering strain of Brasil diazotrophic spirillum DraT* containing multiple copied nifA gene - Google Patents
Engineering strain of Brasil diazotrophic spirillum DraT* containing multiple copied nifA gene Download PDFInfo
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- CN1254533C CN1254533C CN 03138429 CN03138429A CN1254533C CN 1254533 C CN1254533 C CN 1254533C CN 03138429 CN03138429 CN 03138429 CN 03138429 A CN03138429 A CN 03138429A CN 1254533 C CN1254533 C CN 1254533C
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Abstract
The present invention provides an azospirillum brasilense DraT-engineering strain containing multi-copied nifA genes. The preservation number of the strain is CGMCC No. 0876. The present invention also provides bacterial fertilizer which comprises the azospirillum brasilense DraT-engineering strain containing multi-copied nifA genes. The engineering strain of the present invention can be used as the bacterial fertilizer after cultivated, can be used in field operation for cultivating crops, such as corn, wheat and gramineous crops, and can decrease urea consumption. Meanwhile, the strain can excrete plant hormones, such as IAA, etc. so as to stimulate plants to grow, and therefore, the purposes of increasing yield and saving fertilizer are achieved.
Description
Technical field
The present invention relates to a kind of engineering bacteria of Brasil diazotrophic spirillum, the invention still further relates to a kind of bacterial manure.
Background technology
The raising of most of crop yields such as wheat, corn and paddy rice mainly depends on the increase of chemical nitrogen fertilizer amount of application, to improve output is unique target and the immoderate disastrous effect of using chemical fertilizer in a large number and being brought is a soil compaction, fertility descends, and environment is problem such as water quality severe contamination particularly.
Biological nitrogen fixation is to reduce nitrogenous fertilizer to use, and alleviates environmental pollution, reduces an important channel of agriculture production cost.According to the relation of nitrogen-fixing microorganism and host plant, nitrogen fixation is divided into from growing nitrogen-fixing, association nitrogen fixation and symbiosis circle nitrogen (outstanding Chong Shao chief editor, biological nitrogen fixation, Science Press, 1987).Spontaneous nitrogen-fixing microorganism free living is in soil or waters, its nitrogen-fixing efficiency is subject to the influence of surrounding environment, they are fixed nitrogen in the soil that is rich in energy substance, nitrogen stress and little oxygen (or anaerobic) that can be utilized, thereby can be the growth of forest and steppe plant some nitrogens are provided; But lacking the energy (carbohydrate) that can be utilized or combined nitrogen (NH is being arranged
4 +Or NO
3 -) time, can not the fixed nitrogen growth.Symbiotic nitrogen fixation microorganism and plant symbiosis form root nodule, and they are lived in the root nodule, and the photosynthate that provides with host plant is that the energy carries out fixed nitrogen, for the host plant growth provides a large amount of nitrogens; But its host range is limited, mainly is limited to leguminous plants (rhizobium leguminosarum) and some non-pulse family trees (Fu Shi fixed nitrogen actinomycetes).The association nitrogen fixation microorganism mainly is grown in rhizosphere and the Gen Biao of plant, and some can enter the cortical tissue of root but not form root nodule; The host of a lot of association nitrogen fixation microorganisms is important crops such as paddy rice, wheat, corn etc., and association nitrogen fixation provides some nitrogens for these food crop.Therefore expose at the roots of plants table owing to combination azotobacter can not form root nodule with the host, be subject to Effect of Environmental, add fixed nitrogen and be difficult for being secreted into external, these factors all are the reasons that causes the combination azotobacter nitrogen-fixing efficiency lower.So the high yield of these food crop has to rely on chemical nitrogen fertilizer.Therefore, improving the nitrogen-fixing efficiency of combination azotobacter and improve the nitrogen supply of vinelandii to the host, is the main path that reduces nitrogen fertilizer amount in food crop produce.
In these three kinds of fixed nitrogen types of growing nitrogen-fixing, association nitrogen fixation and symbiotic nitrogen fixation, comparatively deep to theoretical investigation from growing nitrogen-fixing, but be worth little in application in agriculture.Association nitrogen fixation than symbiotic nitrogen fixation have fixed nitrogen mechanism simple, be easy to carry out advantages such as genetic engineering modified.
In combination azotobacter, studying more both at home and abroad is Brasil diazotrophic spirillum (Azospirillumbrasilense).The mid-1970s Brazil scholar Dobereier finds that Brasil diazotrophic spirillum is present in the graminous pasture rhizosphere in a large number, and isolates many bacterial strains.The Brasil diazotrophic spirillum that studies show that afterwards extensively is present in many in the world areas (comprising the torrid zone, temperate zone even frigid zone), can not only with the graminous pasture united symbiosis, but also with many monocotyledons united symbiosis, as (Dobereiner J such as corn, paddy rice, wheat, Chinese sorghum, sugarcanes, Day J M.Proceeding of the1st International Symposium on Nitrogen Fixation, Newton WeandNyman CJ (eds.), Washington State University Press, Pullman, 1976,518 ~ 538).Because Brasil diazotrophic spirillum can provide certain nitrogen with a lot of important unifacial leaf food crop association nitrogen fixations and for these crops, therefore from that time, scientists has all over the world just been poured into a large amount of energy, and each side such as the form of this bacterium, Physiology and biochemistry, heredity, field application have been carried out omnibearing research.Wherein A.brasilense Sp7 bacterial strain is external at maximum reference cultures of aspect research such as heredity, field application, and this bacterial strain is exactly to separate from Brazilian graminous pasture rhizosphere the seventies to obtain, and derives from the torrid areas.
Since the seventies, people have carried out the inoculation test of a lot of fields with A.brasilense as bacterial manure, wherein great majority with wheat as the fertilising object.From statistics all over the world in 1994, as bacterial manure, the success ratio of inoculation was 60% with this bacterium, and its amount of increase in production is 5～30%.And, factor such as effective bacterium number of effect of inoculation and inoculation, soil fertility relevant (Colnaghi R.et al.Plant and soil, 1997,194:145-150).Generally amount of increase in production is big in poor soil, amount of increase in production is little in rich soil, therefore think that effect of increasing production mainly is owing to A.brasilense excretory plant hormone has promoted plant-growth and improved plant the absorption of mineral substance and moisture is caused, and be limited (Okon Y.et al.SoilBiology and Biochemistry by the effect of increasing production that nitrogen fixation provides nitrogen to cause, 1994,26:1591-1596).The wild bacterium of A.brasilense is only limitting ammonium (less than 5mM NH
4 +) environment in fixed nitrogen, so in the soil high ammonium concentration be the performance this bacterium nitrogen-fixing efficiency main limiting factor.
The U.S. was starting strain with rhizobium melioti (Rhizobium meliloti) PC and 2011 in 1994, made up respectively that 7 strains are cloned additional copy dctABD on karyomit(e) or/and the reorganization bacterium of nifA, show four of Wisconsin, USA field, the sub-district comparison test results that carry out on experimental field: the alfalfa plants biomass of clone bacterial strain RMPBC-2 that dctABD and nifA arranged on the lower Hancock test point of the soil organism and the combined nitrogen cellulose content bacterium PC that sets out is high by 12.9% simultaneously, it is high by 17.9% more not inoculate contrast, its difference has reached statistical conspicuous level (BosworthA.H.et al.Applied Environmental Microbiology, 1994,60 (10): 3815-3832).1996, the U.S. entered the land for growing field crops application stage with the inoculation engineering strain experiment of the rhizobium melioti of the nifA gene recombination of external source dctABD gene and modification.So far, both at home and abroad the Brasil diazotrophic spirillum used of inoculation experiments all is a wild type strain.Foreign literature and foreign patent are looked into new capital and are not seen that the relevant Brasil diazotrophic spirillum engineering bacteria of anti-the ammonium carries out the report of field test, the domestic report of not seeing the usefulness Brasil diazotrophic spirillum engineering bacteria of anti-the ammonium in the field inoculation.
I was separated to Brasil diazotrophic spirillum Yu62 bacterial strain in 1984 from the Beijing suburb corn root in the chamber, under the subsidy of national 863 projects, this seminar is an original strain with Brasil diazotrophic spirillum Yu62, pass through for over ten years of the further investigation of this bacterium from each side such as heredity, regulation and control, physiology, the fixed nitrogen regulatory mechanism of this bacterium is tended to become clear, and experiment has confirmed NH
+ 4Regulation and control to this bacterium nitrogen fixation are worked by two approach---check the synthetic of nitrogenase and suppress the activity of existing nitrogenase.In Brasil diazotrophic spirillum, the nitrogenase activity after the translation is subjected to the regulation and control of DraT-DraG protein system.When ammonium concentration is high in the cell, under the effect of DraT enzyme, make the arginine residues on subunit of nitrogenase ferritin be completely lost activity by an ADP-ribose groups covalent modification; When ammonium concentration in the cell is low, under the effect of DraG enzyme, ADP-ribose is dissociated from the ferritin subunit, it is active that ferritin recovers.After the draT transgenation, its nitrogenase activity then no longer is subjected to the inhibition of ammonium.Therefore, Brasil diazotrophic spirillum DraT
-The structure of mutant strain will be a rebuilding approach breaking through high ammonium restriction nitrogen-fixing efficiency.In the synthetic regulation and control of nitrogenase, NifA is confirmed as the transcription activating protein of nitrogenase structure gene (nifHDK).The copy number that increases nifA just can improve nitrogenase activity.
Summary of the invention
The objective of the invention is to increase the copy number of nifA gene in the Brasil diazotrophic spirillum and the draT gene is inserted inactivation, the anti-ammonium ability of the engineering strain that is built into strengthens and nitrogen-fixing efficiency improves.A further object of the present invention provides a kind of bacterial manure.
The invention provides a kind of Brasil diazotrophic spirillum engineering strain, bacterial strain number is YuMA.This bacterial strain on January 22nd, 2003 by China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation.Preservation centre address: China, Beijing, Zhong Guan-cun.Preserving number is: CGMCCNo.0876.
The present invention also provides a kind of bacterial manure, and it contains the Brasil diazotrophic spirillum DraT that contains multiple copied nifA gene of the present invention
-Engineering strain.
Brasil diazotrophic spirillum engineering strain YuMA of the present invention carries out genetic modification by the Brasil diazotrophic spirillum Yu62 that 1984 are separated to obtain by China Agricultural University's department of microbiology to obtain from Beijing suburb corn rhizosphere soil, its morphological specificity is still identical with wild-type starting strain Brasil diazotrophic spirillum Yu62 with classification position.Brasil diazotrophic spirillum engineering strain YuMA belongs to false monospore bacillus order (Pseudomonadales), spiral Cordycepps (Spirillaceae), Azospirillum (Azospirillum), Brasil diazotrophic spirillum kind (Azospirillum brasilense) in classification.This bacterium form is shaft-like, slightly crooked, thalline is 0.5-0.9 * 2-3mm, what have is longer, Gram-negative is moved about by a polar flagella in the liquid medium within, can produce several adnation flagellums on solid medium, cell contains PHB (poly-) particle, and DNA G+C content is 70%.Optimum growth temperature is 30 ℃, can accumulate carotenoid, old culture pinkiness.To penbritin with how the pyridine ketone acid has the resistance of nature.Its growth does not need vitamin H, belongs to aerobic chemoorganotrophic bacterium, does not produce acid in the minimum medium that contains glucose or ribose.Can utilize organic acid as sole carbon source, as oxysuccinic acid, lactic acid, pyruvic acid and the acid of coral amber etc.Can utilize plant straw (containing a large amount of hemicelluloses and xylan) to carry out fixed nitrogen, under little aerobic condition, carry out fixed nitrogen.Brasil diazotrophic spirillum engineering strain YuMA holds nontoxic to the people, no pathogenicity.100ml engineering strain YuMA bacterium liquid (OD
600) hormone that produces, promptly IAA and GA3 are respectively 3.3574mg and 0.487.
Wild-type Brasil diazotrophic spirillum Yu62 bacterial strain can be grown on nitrogen-free agar, and anti-penbritin and pyridine ketone acid how.Recombinant strain is compared with wild type strain, has increased foreign gene kalamycin resistance and tetracyclin resistance.Kalamycin resistance and tetracyclin resistance are particularly comparatively general in the soil microorganisms in physical environment, so the kalamycin resistance that engineering bacteria increased and tetracyclin resistance can not constitute new harm to human health and ecotope.
2000 and continuous 2 years of calendar year 2001, carry out sub-district inoculation engineering bacteria YuMA between corn field in Inst. of Soil ﹠ Fertilizer, Shanxi Prov. Academy of Agricultural Sciences, promoted corn yield increasing significantly and saved nitrogenous fertilizer that than inoculation wild-type bacteria and sterile carrier using for further land for growing field crops provides foundation.
The field test results of stating culture inoculation corn is shown engineering strain YuMA shows the fertile effect of increasing production of good joint, amount of increase in production is saved nitrogen fertilizer amount between 15-20% between 11.27-44.7%.Therefore, this bacterium has potential and promotes market in the application of Gramineae food crop, be expected to obtain good economic benefit.
Description of drawings
Fig. 1 is the stereoscan photograph that the Brasil diazotrophic spirillum engineering bacteria distributes at the Zea mays root table.
Embodiment
Below the invention will be further described for structure example by engineering strain YuMA and application example.
1. the structure of engineering strain YuMA
(1) Brasil diazotrophic spirillum DraT
-The structure of mutant strain
The present invention is the total chromosomal DNA of extracting from the genomic library of Brasil diazotrophic spirillum Yu62 at first, cut with the Sau3A enzyme then, reclaim the dna fragmentation of 8-20kb, be connected with phage vector EMBL3A (Huamei Bio-Engrg Co.) and pack, be built into the gene library of Brasil diazotrophic spirillum Yu62 external.Part draT gene fragment with Brasil diazotrophic spirillum Sp7 bacterial strain is made probe, screens a positive colony from Brasil diazotrophic spirillum Yu62 gene library, and the length of this positive colony is 8.0kb.With SalI positive colony is carried out enzyme and cut, and reclaim the external source fragment of the 8.0kb that contains the draT gene, and be cloned on the pUC19 (available from Huamei Bio-Engrg Co.), obtain recombinant plasmid pLYM106.Plasmid pLYM106 (containing the draT gene) cuts with the EcoRI+KpnI enzyme, reclaims the draTG gene fragment of 3.0kb, cuts with the BamHI enzyme after the flat suicide type plasmid pSUP202 (Promega company) of Klenow benefit is connected, and obtains recombinant plasmid pSUTG.To cut 1.4kb kantlex (Km) fragment that reclaim the back with the PstI enzyme from the pUC4K plasmid, be inserted into the PstI site of the draT gene of recombinant plasmid pSUTG, and cause the draT gene inactivation, thereby obtained plasmid pSUTG-1 (draT::Km).Plasmid pSUTG-1 (draT::Km) is imported among the intestinal bacteria S17-1 by transformation.
The intestinal bacteria S17-1 that will contain plasmid pSUTG-1 (draT::Km) is inoculated in the liquid LB substratum that contains kantlex, 37 ℃ of shaking culture 16 hours; Simultaneously, Brasil diazotrophic spirillum Yu62 is inoculated into contains penbritin (Amp) and naphthalene is stung in the liquid LD substratum of ketone acid, 30 ℃ of shaking culture 20 hours.Get each 500 μ l of intestinal bacteria S17-1 bacterium liquid and Brasil diazotrophic spirillum Yu62 bacterium liquid, join, in the 1.5ml Eppendorf pipe, centrifugal 30 seconds of 1000rpm, abandon supernatant, bacterium mud mixture is moved into LD solid plate central authorities, just putting overnight incubation for 30 ℃ with pipettor.Then, scrape a little bacterium mud with transfering loop, sting on the LB flat board of ketone acid and to draw single bacterium colony containing kantlex, penbritin and naphthalene, the single bacterium colony that grows after 30 ℃ of cultivations, separation and purification 2-3 time is DraT
-Mutant strain.Because plasmid pSUTG-1 belongs to suicide type plasmid, so DraT
-Do not contain exogenous plasmid in the mutant strain.
(2) clone of nifA gene and nifA gene are to Brasil diazotrophic spirillum DraT
-The importing of mutant strain
Increasing from Brasil diazotrophic spirillum Yu62 with the PCR method obtains containing the 2.4kb of nifA gene, and is cloned on pGEM-T (Promega company) carrier, forms the pT-nifA recombinant plasmid.With XhoI and HindIII carrier pVK100 (available from magnificent engineering corporation) is carried out double digestion, with XhoI and HindIII the pT-nifA recombinant plasmid is carried out double digestion simultaneously, reclaim 2.4kb nifA fragment, obtain the pKC11 recombinant plasmid with being connected.In the pKC11 recombinant plasmid, the nifA gene is constitutive expression under the promotor of kalamycin resistance gene, and owing to the insertion of nifA gene makes the kanamycin gene inactivation, but this plasmid still carries tetracycline resistance gene.Then the pKC11 recombinant plasmid is transformed by electricity and import intestinal bacteria S-17.The intestinal bacteria S-17 (Huamei Bio-Engrg Co.) and Brasil diazotrophic spirillum DraT that will contain the pKC11 recombinant plasmid then
-Mutant strain carries out biparent cross, and screening contains the zygote of tetracyclin resistance and kalamycin resistance, and this zygote is exactly engineering strain YuMA.
Wild-type Brasil diazotrophic spirillum Yu62 bacterial strain can be grown on nitrogen-free agar, and anti-penbritin and nalidixic acid.Recombinant strain is compared with wild type strain, has increased foreign gene kalamycin resistance and tetracyclin resistance.Kalamycin resistance and tetracyclin resistance are particularly comparatively general in the soil microorganisms in physical environment, so the kalamycin resistance that engineering bacteria increased and tetracyclin resistance can not constitute new harm to human health and ecotope.
2. the nitrogenase activity of engineering strain
Engineering strain YuMA of the present invention and wild-type Yu62 bacterial strain are together carried out the mensuration of nitrogenase activity, then their nitrogenase activity relatively.The mensuration of nitrogenase activity is measured simultaneously by semi-solid method nitrogenase measuring method and two kinds of methods of liquid method nitrogenase measuring method.
(i) mensuration of semi-solid method nitrogenase activity
(1) engineering strain YuMA to be measured and wild type strain Yu62 are activated simultaneously, keep its growth conditions unanimity;
(2) engineering strain YuMA after will activating and wild type strain Yu62 insert respectively and contain corresponding antibiotic liquid LD substratum (LD liquid nutrient medium: 10g Tryptones, 2.5g sodium-chlor, 5g yeast powder, 1000ml distilled water.15 pounds, 121 ℃ of sterilizations sterilization in 15 minutes) in, 30 ℃ of shaking culture 20 hours.Need to add kantlex (concentration is 10 μ g/ml), tsiklomitsin (12.5 μ g/ml) and penbritin (25 μ g/ml) in the LD substratum of culturing engineering bacterial strain YuMA; Need to add penbritin (25 μ g/ml) and nalidixic acid (5 μ g/ml) in the LD substratum of cultivation wild-type Yu62 bacterial strain;
(3) measure the OD of engineering strain YuMA and wild type strain Yu62 respectively
600Value, and with OD
600Value is adjusted to 1.Get 1ml OD
600Value is 1 bacterium liquid, 12, and the centrifugal 1min of 000rpm collects thalline; (the K-Lac minimum medium: every liter contains K with liquid K-lac substratum
2HPO
41.67g, KH
2PO
40.87g, MgSO
47H
2O 0.29g, NaCl 0.48g, 60% sodium lactate solution 6.3ml, 10 pounds of sterilization 30min.Every 100ml adds 200 * CaCl before using
2Solution 1ml, 100 * trace element solution 1ml, 1000 * FeCl
3Solution 0.1ml, 1000 * NaMoO
4Solution 0.1ml adds 5M NH as required
4Cl solution is to required final concentration.Semisolid K-Lac adds 0.2% agar) fully be suspended in the 100 μ l liquid K-Lac substratum after washing twice; Then 100 μ l bacterium liquid are inoculated into the tubule of the K-Lac semisolid medium that contains, cultivated 48 hours for 30 ℃.If 3 repetitions are promptly respectively inoculated 3 semi-solid tubules with engineering strain YuMA and wild type strain Yu62;
(4) fill in a little tampon in each semi-solid tubule, all change cap test tube into plug again, each injects the acetylene of 1ml new system, and 30 ℃ were reacted 10-12 hour;
(5) from each semi-solid tubule, take out 100 μ l gas gas chromatography determination ethylene content, come its nitrogenase activity height of comparison according to the height at each sample ethene peak with microsyringe.
(ii) liquid method is measured nitrogenase activity
(1) engineering strain YuMA to be measured and wild type strain Yu62 are activated simultaneously, keep its growth conditions unanimity;
(2) each bacterial strain after will activating inserts and contains in the corresponding antibiotic liquid LD substratum, 30 ℃ of shaking culture 20 hours;
(3) survey the OD of each bacterial strain
600Value, and with OD
600Value is adjusted to 1.Get 1ml OD
600Value is 1 bacterium liquid, 12, and the centrifugal 1min of 000rpm collects thalline; Fully be suspended in the 100 μ l liquid K-lac substratum after washing twice with liquid K-lac substratum; Then 100 μ l bacterium liquid are linked into the little serum bottle of 10ml, 30 ℃ of concuss are cultivated, and coerce 3 ~ 4 hours;
(4) inject the acetylene of 10% volume new system to each little serum bottle, with microsyringe took out 100 μ l gas gas chromatography determination ethylene content from each the little serum bottle every 30 minutes, come its nitrogenase activity height of comparison according to the height at each sample ethene peak.
The (iii) demarcation of nmol ethene:
(1) prepares two of 120ml serum bottles, be designated as 1
#, 2
#Bottle;
(2) serum bottle is filled water, and good with the plug plug that is inserted with syringe needle, prevent that bubble from producing, take off plug, pour out 100ml water (measuring) with volumetric flask, change new plug, the volume of air in the bottle is 100ml like this;
(3) to 1
#Inject 2.24ml (for 2.24ml, under the non-standard situation, calculating injection rate under the mark condition) ethene in the bottle, from the 1# bottle, take out 1ml gas again and inject 2 according to PV=nRT
#In the bottle, from 2
#Take out 100 μ l gases in the bottle and squeeze into gas chromatograph, shown peak height is the amount of 1nmol ethene, can calculate on the recording paper per unit peak height in view of the above and represent how much nmol ethene.
All show by the result who measures with semi-solid nitrogenase activity determination method and two kinds of methods of liquid nitrogenase activity determination method, the nitrogenase activity of the engineering strain YuMA that the present invention relates to doubles above than wild-type Yu62 bacterial strain, the nitrogenase of mensuration sees Table 1.
The nitrogenase activity of table 1. engineering strain YuMA and wild type strain Yu62 relatively
The relative nitrogenase activity of bacterial strain phenotype and/or genotype (%)
Wild-type Brasil diazotrophic spirillum Yu6 2 wild-types 100
Brasil diazotrophic spirillum engineering bacteria YuMA DraT
-Contain additional copy nifA gene 224 in the mutant strain
3. sub-district, the field inoculation test of engineering strain YuMA
Below the invention will be further described for the application example by engineering bacteria YuMA.
Used medium component of Brasil diazotrophic spirillum engineering bacteria and culture condition are as follows:
LD liquid nutrient medium (15 pounds, 121 ℃ of sterilizations sterilization in 15 minutes)
Tryptones 10g NaCl 2.5g
Yeast powder 5g distilled water 1000ml
PH6.8 places 200 rev/mins of constant temperature shaking tables, cultivates 48 hours for 30 ℃.
Add 1.2-1.5% agar in the aforesaid liquid substratum, preparation solid inclined-plane or flat board, pH6.8,30 ℃ left standstill 48 hours.
With the LD liquid nutrient medium culturing engineering bacterium YuMA that contains kantlex, tsiklomitsin and penbritin, bacterium liquid and turfy soil are mixed with microbial inoculum.Microbial inoculum is used in conjunction with seeding operation as seed manure.Every strain spread manuer in holes 5 the gram microbial inoculums, microbial inoculum content is approximately 5 * 10
8Individual/the strain cave.
Calendar year 2001 and 2002 are in the Shanxi Province, experimental result shows in the sub-district, field of lightweight sandy loam, in corn ear length, tassel row number, a row grain number, 100-grain weight, cell production and per mu yield, inoculation engineering bacteria and inoculation wild-type bacteria all have significantly production-increasing function and save the nitrogenous fertilizer effect than aseptic contrast.
The test Treatment Design:
(1) aseptic contrast (not connecing nutrient solution+100% nitrogenous fertilizer of bacterium)
(2) aseptic contrast (not connecing bacteria culture fluid+80% nitrogenous fertilizer)
(3) wild-type Brasil diazotrophic spirillum Yu62+80% nitrogenous fertilizer
(4) Brasil diazotrophic spirillum engineering bacteria YuMA+80% nitrogenous fertilizer
100% nitrogenous fertilizer is that 15kg urea/mu, 80% nitrogenous fertilizer are 12kg urea/mu mu.Totally 4 processing, 4 repetitions, random alignment, sub-district area 33.3m
2Test-results sees Table 2.
Table 2. inoculation engineering bacteria YuMA is to the influence and the yield increasing effect of corn growth
| Handle | (1) | (2) | (3) | (4) |
| Spike length (cm) | 20.8 | 21.9 | 22.7 | 23.7 |
| Tassel row number | 14.9 | 14.1 | 14.4 | 15.4 |
| Row grain number | 44.2 | 46.9 | 48.8 | 50.5 |
| 100-grain weight (g) | 27.5 | 32.88 | 35.23 | 36.3 |
| The bright equal output of fringe (kg) in sub-district | 26.75 | 30.38 | 32.88 | 36.50 |
| Cell production (kg) | 15.25 | 16.71 | 23.02 | 25.92 |
At the lightweight silty loam, be rich under organic matter, the middle fertility soil condition, corn combination azotobacter wild-type and engineering strain have been carried out the field inoculation test of corn sub-district.
Test-results shows, under different levels nitrogenous fertilizer condition, inoculation engineering bacteria ratio wild bacterium of inoculation and the contrast of not inoculating all have volume increase in various degree, be under the condition of 12kg urea/mu (80%) particularly in Fertilization Level, the inoculation engineering bacteria on average increases production 10% than the inoculation wild-type, than the contrast volume increase of not inoculating 32%, under the situation that reduces fertilizer amount 20%, the inoculation engineering bacteria is still compared according to volume increase 32%.
Claims (2)
1. Brasil diazotrophic spirillum (Azospirillumbrasilense) DraT-engineering strain that contains multiple copied nifA gene, preserving number is: CGMCC No.0876.
2. bacterial manure, it contains the described Brasil diazotrophic spirillum DraT-engineering strain that contains multiple copied nifA gene of claim 1.
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| CN100376666C (en) * | 2006-03-01 | 2008-03-26 | 华南农业大学 | Malasses weed nitrogen fixing spirillum and its application |
| ES2378040B1 (en) * | 2010-03-31 | 2013-02-18 | Probelte, S.A | A BIONEMATICIDE BIOLOGICAL PREPARATION AND STIMULATOR OF THE VEGETABLE GROWTH AND PURE CROPS OF THE NAMES N11, SR11 AND ALO1, CONTAINED IN THE SAME. |
| WO2014071182A1 (en) | 2012-11-01 | 2014-05-08 | Massachusetts Institute Of Technology | Directed evolution of synthetic gene cluster |
| CN102965307B (en) * | 2012-11-09 | 2014-02-12 | 广东省生态环境与土壤研究所 | Azospirillum brasilense strain and microbial preparation thereof |
| CN103451130B (en) * | 2013-07-25 | 2014-11-26 | 中国农业大学 | Nitrogen fixing gene cluster and application thereof |
| EP3322679A4 (en) | 2015-07-13 | 2019-07-10 | Pivot Bio, Inc. | Methods and compositions for improving plant traits |
| EP3359664A4 (en) | 2015-10-05 | 2019-03-20 | Massachusetts Institute Of Technology | Nitrogen fixation using refactored nif clusters |
| WO2017195732A1 (en) * | 2016-05-13 | 2017-11-16 | 国立研究開発法人国際農林水産業研究センター | Fertilizer or soil conditioner and plant growth method |
| CN110799474B (en) | 2017-01-12 | 2022-07-26 | 皮沃特生物公司 | Methods and compositions for improving plant traits |
| CN111587287A (en) | 2017-10-25 | 2020-08-25 | 皮沃特生物股份有限公司 | Methods and compositions for improved nitrogen-fixing engineered microorganisms |
| CN111057665B (en) * | 2019-12-11 | 2020-12-25 | 安徽农业大学 | Cellulose degrading bacterium n3 for producing IAA and application thereof |
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