CN116179457A - Method for promoting plant probiotics methylobacterium She Jiding to be bred - Google Patents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
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- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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Abstract
The invention discloses a method for promoting the reproduction of plant probiotics methylobacterium She Jiding. The method utilizes a mutant strain of the methylobacterium to perform colonization on plant leaf boundaries; the mutant strain is prepared by knocking out a ppsR gene sequence of the bacillus or a promoter sequence of the ppsR gene by utilizing a modern molecular biology technology, or reducing the capacity of the ppsR gene sequence to bind to a promoter region of a photosynthetic gene cluster related gene by disrupting the ppsR gene sequence through random insertion mutation or site-directed insertion mutation, or reducing the transcription expression level of the ppsR gene by changing the promoter sequence of the ppsR gene through site-directed mutation. The invention can effectively improve the application capability of the methylobacterium as plant probiotics and phyllosphaeria bacterial manure. Practice proves that the invention obtains a technical strategy for effectively improving photosynthesis and promoting the growth capacity of the methylobacterium on plant phyllospheres, has obvious effect of improving plant phyllosphere colonization, and has good application potential for promoting the growth and production of crops.
Description
Technical Field
The invention relates to a method for promoting plant phyllostachys colonization by improving photosynthesis of plant probiotics methylobacterium, belonging to the fields of microbial technology and modern agriculture.
Background
The microbial agent is widely applied in agricultural production due to the advantages of green, high efficiency, sustainable application, low side effect and the like. Methylobacillus (Methylobacillus and Methylorubrum) uses organic carbon such as methanol released in the growth and metabolism process of plant leaf as the sole carbon source and energy source for growth to form dominant bacterial colony of plant leaf. Many methylobacterium species (e.g., methylobacterium oryzae Methylobacterium oryzae, methylobacterium nidulans Mb. Nodulons, methylobacterium torvum. Extorquens, methylobacterium radiodurans, methylobacterium flocum Mb. Gregans and methylobacterium spanii Mb. Hispidum) are capable of synthesizing growth hormone, mitogen and secondary metabolite required for plant growth, thereby promoting the growth of crops such as corn, soybean, peanut, lettuce, tomato, melon and the like and inhibiting the reproduction of pathogenic bacteria such as fusarium graminearum, rhizoctonia solani and the like, and are important plant phyllospheric probiotics. Various methyl bacillus bacterial manure and biopesticide products have been developed by agricultural biotechnology companies such as the united states.
However, the release of methanol from the plant phyllosphere surface fluctuates along with day and night and has low concentration, so that the phyllosphere is faced with the adversity ecological niche of an oligomethanol carbon source, the difference of colonization effects on different crops is large, the general colonization efficiency is low, and the effect of promoting the growth and production of the crops is not ideal.
Disclosure of Invention
The invention aims to provide a method for promoting the plant probiotic methyl bacillus She Jiding to grow, and the method for promoting the plant probiotic methyl bacillus to grow in the phyllostatic ecological niche of the plant is realized by improving the photosynthesis intensity of the plant probiotic methyl bacillus so as to make up for the defects of the prior art.
Genome sequencing shows that the methylobacterium separated from plant phyllospheres generally contains gene clusters (Photosynthetic gene cluster, PGC) required for photosynthesis, including bch gene clusters for synthesizing bacteriochlorophyll a, puf gene clusters for synthesizing light-trapping protein complex, puh gene clusters for synthesizing light-reactive centrin complex, and regulatory genes lhaA and pucC for promoting self-assembly of protein complex, and has the capability of performing RC-II type photosynthesis. However, the bacteriochlorophyll synthesis amount of the methylobacterium is significantly lower than that of other aerobic non-oxygen producing photosynthetic bacteria under the light and dark cycle culture, which shows that the photosynthetic methyl metabolism nutrition mode of the methylobacterium mainly obtains carbon sources and energy required for growth through methanol oxidation and assimilation, and photosynthesis can only drive light energy to be converted into a small amount of chemical energy.
Therefore, improving the photosynthesis of methylobacterium can enhance the growth competitiveness and fitness in the plant phyllospheric microbial community, improve the colonization under the plant phyllospheric ecology, and enhance the probiotics effect on the plant growth. Based on this, the present invention provides an effective method for promoting plant phyllospheric colonization by increasing the photosynthetic strength of methylobacterium to enhance the ability of methylobacterium to promote the growth and production of crops.
In order to improve the colonization ability of the methylobacterium in plant phyllospheres, a technical strategy is needed to enhance the photosynthetic productivity efficiency of the methylobacterium. The invention discovers that the ppsR gene is knocked out in the methylobacterium, or the ppsR gene cannot be transcribed normally by inserting a stop codon, so that the content of the bacteriochlorophyll a synthesized by the methylobacterium under light culture and the photosynthetic capacity of the methylobacterium are obviously improved, the growth capacity on carbon sources such as methanol is enhanced, and the plant phyllospheric colonization effect is greatly improved.
In combination with the above research, in order to achieve the above purpose, the specific technical scheme adopted by the invention is as follows:
a mutant strain of methyl bacillus, which is characterized in that a ppsR gene sequence of the methyl bacillus or a promoter sequence of the ppsR gene is knocked out by utilizing the modern molecular biology technology, or the ppsR gene sequence is damaged by random insertion mutation or site-directed insertion mutation to reduce the capacity of ppsR binding to a promoter region of a photosynthetic gene cluster related gene, or the promoter sequence of the ppsR gene is changed by site-directed mutation to reduce the transcription expression level of the ppsR gene.
Such modern molecular biology techniques include, but are not limited to, homologous double crossover techniques, transposition techniques, CRISPR-Cas9 techniques, and the like.
Further, the methylobacterium refers to a type of gram-negative bacteria that is pink in color with organic carbon one, including methane, methanol, formaldehyde, formic acid, methylamine, etc., as a carbon source and energy source for growth, including but not limited to methylobacterium oryzae mb.oryzae, methylobacterium nodosum, methylobacterium torvum, radiotolerans, methylobacterium floccocus mb.gregans, and methylobacterium spani mb.hispidum.
A method of promoting the colonization of a plant by methylobacterium She Jiding, which method utilizes a mutant of said methylobacterium to colonize the plant leaf.
Further, the method specifically comprises the following steps: liquid culturing the mutant strain of the methyl bacillus to the middle and later period of the growth index, and adjusting the concentration of the bacterial body to OD 600 About 0.5, soaking plant seeds or drip irrigation plant roots or spraying plant leaf boundaries by using the bacterial suspension.
The mutant strain of the bacillus methylobacterium can be applied to the promotion of plant phyllosphere growth and colonization.
Furthermore, the mutant strain of the methyl bacillus is applied to preparation of plant probiotics or phyllosphaeria bacterial manure.
Further, the plant phyllosphere refers to a part of a plant which appears above the ground surface and comprises flowers, stems, leaves and fruits; the plants mainly comprise, but are not limited to, vegetables and fruits such as corn, wheat, soybean, peanut, tomato, cucumber, melon and the like, ornamental plants such as azalea flower, chrysanthemum, narcissus, hydrangea and the like, and other plants which are bred by methyl bacillus She Jiding.
The mutant strain of the methyl bacillus effectively enhances the transcription of photosynthesis genes and improves the photosynthesis strength under the culture condition of a light-dark circulation laboratory of light-given on methanol serving as a carbon source or propylene glycol carbon source, and the synthesis amount of the bacterial chlorophyll a is obviously higher than that of wild bacteria. The mutant strain of the bacillus methylobacterium is cultured on a methanol carbon source or a propylene glycol carbon source under the culture condition of a light-dark cycle laboratory, and the intracellular ATP content is obviously higher than that of the wild bacteria. The mutant strain of the methyl bacillus has obviously higher growth capacity than wild bacteria on a methanol carbon source or a propylene glycol carbon source under the culture condition of a light-dark cycle laboratory, and comprises the steps of improving the growth rate and improving the biomass yield.
Advantages and beneficial effects of the invention
The invention provides an improved strain of methyl bacillus, which is characterized in that ppsR genes or promoter sequences are knocked out, or the ppsR genes are damaged through insertion and inactivation, so that the transcription level of photosynthesis genes can be enhanced, the synthesis amount of bacteriochlorophyll a is improved, the intracellular ATP content is improved, the growth capacity on carbon sources such as methanol is improved, and finally the colonization effect on the leaf of plants of different families is remarkably improved; through practical verification, the colonization amount of the mutant strain of the plant phyllobacterium after the microbial inoculum is applied for 3 weeks reaches 10 per gram of fresh weight of the phyllobacterium plant 8 -10 9 CFU or more.
The invention can effectively improve the application capability of the methylobacterium as plant probiotics and phyllosphaeria bacterial manure. Practice proves that the invention obtains a technical strategy for effectively improving photosynthesis and promoting the growth capacity of the methylobacterium on plant phyllospheres, has obvious effect of improving plant phyllosphere colonization, and has good application potential for promoting the growth and production of crops.
Drawings
FIG. 1 is a graph (A) showing comparison of transcription levels of genes involved in photosynthesis of a wild strain of Methylobacillus methylobacterium, methylobacterium wrung, under light-dark cycle (LD) and pure darkness (D) culture conditions, and high resolution mass spectrometry (B) of bacteriochlorophyll a produced under light-dark cycle culture conditions of methylobacterium wrung.
FIG. 2 is a graph showing comparison of transcription levels of genes involved in photosynthesis of a mutant strain of the knocked out gene ppsR of Methylobacillus methylobacterium strain in light-dark cycle (LD) and in pure darkness (D) culture conditions.
FIG. 3 is a graph comparing the results of the mutant strain in which the gene ppsR was knocked out in methylobacterium wrenchii, showing that the synthesis amount of bacteriochlorophyll a was significantly higher than that of the wild strain under the carbon source conditions of 1, 2-propanediol (A) and methanol (B). Wherein, the A1_LD is a wild strain of the methylobacterium wrenchii in a light and dark cycle culture mode; AM1 delta ppsR_LD, a strain of a mutant strain of the gene ppsR knocked out by the methylobacterium torvum in a light dark cycle culture mode; AM1_ D, wild strain of methylobacterium torvum in pure dark culture mode; AM1 delta ppsR_D, a strain of the gene ppsR mutant knocked out by the methylobacterium torvum in a pure dark culture mode; AM1 Δbchcxyz and AM1 Δbchf: a mutant strain of the methylobacterium wraparound for knocking out a gene critical to the bacteriochlorophyll a synthesis pathway.
FIG. 4 is a graph comparing the results of the mutant strain growth rate and biomass accumulation of the knocked out gene ppsR in methylobacterium wrenchii significantly higher than that of the wild strain. Wherein AM1 is a wild strain of the methylobacterium wrenchii; AM 1. DELTA. PpsR, a strain mutated to the knockdown gene ppsR of M.sprain; LD: a light-dark cycle culture mode; d: pure darkness culture mode
FIG. 5 is a graph comparing the results of the knockout gene ppsR mutant strain (AM 1. DELTA. PpsR) in methylobacterium wrenchii showing significantly higher ability to colonize Arabidopsis She Jiding than the wild strain (AM 1).
FIG. 6 is a graph comparing the results of the inactivated strain of M.torpedo ppsR (AM 1 ppsR) with significantly higher ability to colonize Arabidopsis She Jiding than the wild strain (AM 1).
FIG. 7 is a graph comparing the results of the knockout gene ppsR mutant strain (RA 01) in M.radiodurans showing significantly higher ability to colonize Arabidopsis She Jiding than the wild strain (RA).
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present application, the present invention will be further described with reference to examples, which are only a part of examples of the present application, but not all, and the present invention is not limited by the following examples.
The invention cultures the methyl bacillus (or mutant strain) under 30 ℃ in laboratory culture conditions; the culture mode is preferably shake flask culture at a rotation speed of 80-200rpm, and the light and dark cycle culture mode selects light intensity of 30-200 μmol photons m -2 s -1 In the range, the illumination rhythm is 8-16 h illumination, and the whole process of the pure dark culture mode keeps light-proof corresponding to 16-8 h dark circulation culture. In the present invention, the preparation method of the hypo medium is preferably as follows: macroelement (2X): macroelement a:5.06g/LK 2 HPO 4 ，2.585g/L NaH 2 PO 4 The method comprises the steps of carrying out a first treatment on the surface of the Macroelement B:0.4095g/L MgSO 4 ·7H 2 O，1g/L(NH4) 2 SO 4 The method comprises the steps of carrying out a first treatment on the surface of the If a solid medium is prepared, 30g/L of agar is added to the macroelement B. Separately sterilizing, and using according to 1: mixing the materials according to the proportion of 1. Trace elements: the trace element A and the trace element B are stored separately, and 100mL 1000X trace elements are prepared. Trace element A:1g Na 2 EDTA，0.1gFeSO 4 ·7H 2 O, adjusting the pH to 4 by using 1M NaOH; trace element B:0.14g CaCl 2 ·2H 2 O，0.1gMnCl 2 ·4H 2 O，0.02g Na 2 MoO 4 ·2H 2 O，0.03g CuSO 4 ·5H 2 O，0.16g CoCl 2 ·6H 2 O，0.44g ZnSO 4 ·7H 2 O, adjust pH to 1-2 with HCl. Filtering and sterilizing with 0.22 μm sterile filter after microelement preparation, refrigerating at 4deg.C, and adding at a ratio of 1000 times of dilution. Laboratory standard culture conditions methanol and 1, 2-propanediol concentrations were 0.5% (v/v).
Specific examples were studied on behalf of methylobacterium torvum and methylobacterium radioduram.
EXAMPLE 1 Synthesis of bacteriochlorophyll by methylobacterium wrung with upregulation of the transcription level of the photosynthesis-related Gene in the light-dark cycle culture mode
This example demonstrates the difference in transcription level of photosynthetic genes in the light dark cycle and pure dark culture modes of the wild-type strain of methylobacterium wrenchii. The photosynthetic bacteria can regulate and express photosynthetic gene clusters under the light and dark circulation culture mode, and can additionally convert light energy into chemical energy, thereby being beneficial to the growth of thalli.
In this example, it was examined whether or not photosynthetic gene clusters of M.torpedo were expressed under light control, and cells were cultured in Hypho medium containing 1, 2-propanediol (0.5%) and irradiated with lightThe irradiation condition is 50-100 mu mol photons m - 2 s -1 The light rhythm is 12h light/12 h darkness (pure darkness culture is used as a control), the culture is carried out under the condition of a shaking table at 80rpm at 30 ℃, the RNA extraction is carried out on thalli grown to the middle and later stages of an index under the two culture conditions, the transcriptional level change of photosynthetic gene cluster partial genes (bchC, bchX, bchY, bchZ, bchF, bchM, pufM, pufC, pufL, puhC, puhA and pucC) is measured by using a RT-qPCR method, the abscissa is the verified gene type, and the ordinate is the log2 logarithmic value of the transcriptional change multiple of the genes under the condition of the darkness culture and the darkness culture. The results showed that all genes tested, including bchC, bchX, bchY, bchZ, bchF, bchM, pufM, pufL, puhC, puhA, pucC, increased transcription levels by more than 2-fold under light and dark cycling culture, with the bacteriochlorophyll synthesis-related genes, including bchC, bchX, bchY, bchZ, bchF, bchM, increased by nearly or more than 4-fold (fig. 1A). To confirm the bacteriochlorophyll synthesis ability and structural subtype of bacteriochlorophyll of the methylobacterium, the intracellular pigment of the thallus under the condition of light and dark cycle culture is extracted, the thallus is extracted by 10 times of methanol, and the thallus is subjected to qualitative analysis by high-resolution mass spectrum after filtration. The quality spectrograms of the bacteriochlorophyll and bacteriochlorophyll a standard substance extracted from the interior of the methylobacterium wrenchii are completely consistent (figure 1B), and the bacteriochlorophyll a can be generated under the condition of light and dark cycle culture.
Example 2 knockout of the Gene ppsR further increases the transcription level of the photosynthetic-related Gene of Methylobacillus torvus
This example obtains a strain of a mutant strain AM1 Δppsr of methylobacterium torvum by homologous double crossover knockout of the ppsR gene of methylobacterium torvum. Cultivation was performed and the transcription level of the photosynthetic cluster part genes (bchC, bchX, bchY, bchZ, bchF, bchM, pufM, pufC, pufL, puhC, puhA, pucC) was analyzed as in example 1, with the abscissa representing the verified gene type and the ordinate representing log2 log of the fold change of transcription of the gene under light-dark and dark culture conditions. According to experimental results, analysis of RT-qPCR showed that the knockout of negative regulatory factor ppsR resulted in a 20-fold increase in transcription level of most photosynthetic genes (bchC, bchX, bchY, bchZ, bchF, bchM, pufM, pufL, puhC, puhA, pucC) including bacteriochlorophyll synthetic genes (bchC, bchX), photoreaction center synthetic genes (pufL, pufM) and light capturing complex genes (puhA) by more than 100-fold in 1, 2-propanediol as carbon source, light dark cycle culture mode, compared to pure dark culture, indicating that the knockout of ppsR gene released the negative regulation of photosynthetic gene clusters of methylobacterium wrenchii, resulting in a significant enhancement of response to light. The nucleotide sequence of the ppsR gene is shown as SEQ ID NO. 1.
Example 3 knockout of the gene ppsR to increase chlorophyll a Synthesis amount in Methylobacillus torvus bacteria
This example compares the changes in intracellular bacteriochlorophyll content of the M.twister knockout ppsR mutant strain AM 1. DELTA. PpsR and the wild-type strain AM1 in both light and dark cycling and pure darkness culture modes with reference to example 1 for the culture conditions of 1, 2-propanediol carbon source, with the use of Hypho medium containing 0.5% methanol in methanol under illumination conditions of 50-100. Mu. Mol photons m -2 s -1 The light rhythms were 12h light/12 h dark (pure dark culture as control), and the culture was performed at 30℃under the shaking conditions of 200 rpm. Bacteriochlorophyll extraction and quantification methods referring to example 1, other genes bchF and bchCXYZ on photosynthetic gene clusters were knocked out as in example 2. According to the analysis result (figure 3A) under the culture condition of taking 1, 2-propylene glycol as a carbon source, strains with different genotypes under different culture conditions are taken as the abscissa, and the strain AM 1-LD is a strain of the wild strain of the methylobacterium wrenchii in a light dark cycle culture mode; AM1 delta ppsR_LD, a strain of a mutant strain of the gene ppsR knocked out by the methylobacterium torvum in a light dark cycle culture mode; AM1_ D, wild strain of methylobacterium torvum in pure dark culture mode; AM1 delta ppsR_D, a strain of the gene ppsR mutant knocked out by the methylobacterium torvum in a pure dark culture mode; AM1 Δbchcxyz_ld and AM1 Δbchf_ld: a mutant strain of the key gene of the chlorophyll a synthesis pathway of the bacterium is knocked out by the methylobacterium wrenchii in a light-dark circulation culture mode. The result shows that the ppsR gene knockout obviously improves the content of intracellular bacteriochlorophyll a of the strain under the light-dark circulation, the content is improved from 0.144mg/g to 0.241mg/g, the improvement range reaches 67%, and the bacteriochlorophyll synthesis is knocked out to be relevantNo bacteriochlorophyll a was detected intracellularly after genes such as bchF and bchCXYZ. Under the culture condition of methanol as a carbon source (figure 3B), the bacteriochlorophyll a can not be detected by AM1 in both light-dark cycle and pure darkness modes, whereas the methylobacterium torvum knocked out ppsR mutant strain AM 1. DELTA. PpsR culturing in light and dark circulation mode and pure darkness mode, the bacteriochlorophyll a content was 0.198mg/g and 0.319mg/g, respectively. The result shows that the knockout of the negative regulatory factor ppsR improves the synthesis capability of the bacteriochlorophyll a of the methylobacterium wraparound under the conditions of different carbon sources of 1, 2-propanediol and methanol and light dark circulation culture.
Example 4 knockout of the Gene ppsR to increase ATP Synthesis in Methylobacillus torvus
This example demonstrates the difference in intracellular energy levels of ppsR knockout mutant and wild-type strain in both light and dark cycle and pure darkness culture modes, the culture method was referred to in example 1, extraction of ATP, ADP and AMP using lyophilized cell samples, addition of 2ml of ethanol extraction buffer (20% ethanol, 70mM HEPES buffer, ph 5.2), ice bath for 3 minutes after water bath at 100 ℃ for 3 minutes, concentration of supernatant after centrifugation followed by HPLC method, chromatographic column Waters Spherisorb ODS2 (4.6 mm×250mm×5 μm), quantitative analysis according to standard curve of standard, and calculation of cell energy load EC value according to ATP, ADP and AMP content. According to the analysis of the measurement of ATP content (Table 1, wherein AM 1D: a wild strain of methylobacterium torvum in a pure dark culture mode; AM1 LD: a wild strain of methylobacterium torvum in a light dark cycle culture mode; AM 1. DELTA. PpsR_D: a mutant strain of gene ppsR knocked out by methylobacterium torvum in a pure dark culture mode; AM 1. DELTA. PpsR_LD: a mutant strain of gene ppsR knocked out by methylobacterium in a light dark cycle culture mode), the intracellular ATP synthesis amount after light dark cycle was significantly increased from 4.13nmol/mg to 5.91nmol/mg, and the EC value was increased from 0.81 to 0.84 according to the analysis of Energy level (Energy charge, EC) under the culture conditions in which 1, 2-propanediol was the carbon source. Under the culture condition of taking methanol as a carbon source, the strain AM1 delta ppsR of the strain with the knocked-out ppsR of the methylobacterium wrenchii is improved from 5.17nmol/mg to 6.71nmol/mg in the light-dark cycle culture, the improvement range is nearly 30 percent, and the EC value is improved from 0.83 to 0.85 according to the analysis of energy charge level (EC) (table 1). The result shows that the knocking-out of the negative regulatory factor ppsR improves the synthesis level of intracellular ATP of the methylobacterium wrenchii under the conditions of two different carbon sources of 1, 2-propanediol and methanol and illumination circulation culture.
Table 1 shows a comparison of the results of the intracellular ATP synthesis and the cellular energy load in the mutant strain from which the ppsR gene was knocked out in M.torpedo
Example 5 knockout of the Gene ppsR to increase growth Rate and biomass yield of Methylobacillus torvus
This example compares the growth curves and the highest biomass accumulation of the strain AM 1. DELTA. PpsR knockout strain of M.sprain and the strain AM1 wild type strain in 1, 2-propanediol and methanol in both light and dark cycle and pure darkness, and the cultivation method is described in example 3. FIGS. 4A and 4C are graphs showing growth of cells (AM 1 and AM 1. DELTA. PpsR) under light-dark cycle (LD) and dark culture (D) conditions, respectively, with 1, 2-propanediol and methanol as carbon sources, and with the abscissa showing the culture time and the ordinate showing the OD 600 Absorbance below. FIGS. 4B and 4D show the maximum biomass of cells (AM 1 and AM 1. DELTA. PpsR) under conditions of light-dark cycle (LD) and dark culture (D) with 1, 2-propanediol and methanol as carbon sources, respectively. Under 1, 2-propanediol culture conditions (FIGS. 4A and 4B), the same initial inoculum size, methylobacterium torvum knockout ppsR mutant strain AM1 DeltappsR under light and dark cycle culture conditions, absorbance OD during growth thereof 600 Is always higher than other control strains, the highest biomass reaches 1.37g/L, and the biomass is improved by 23 to 35 percent compared with other control strains. Light absorbance OD during growth of the M.torpedo knockout ppsR mutant strain AM 1. DELTA. PpsR and the wild strain AM1 under conditions of cultivation with methanol as a carbon source (FIGS. 4C and 4D) 600 Always higher than the control strain under the dark culture condition, methylobacterium wrenchii knockout ppsR mutant strain AM1 delta ppsR in light dark cycle culture barThe highest biomass under the piece reaches 1.05g/L, and is improved by 13-23% compared with other control group strains. The result shows that the knocking-out of the negative regulatory factor ppsR improves the biomass yield of the methylobacterium wrenchii under the conditions of two different carbon sources of 1, 2-propanediol and methanol and illumination circulation culture, so that the biomass accumulation is obviously improved under the condition of the culture of the carbon sources with the same concentration.
Example 6 knockout of the Gene ppsR to increase the ability of M.torpedo-toxidans to colonize Arabidopsis She Jiding
This example shows that the methylobacterium torvum knockout ppsR mutant strain AM1 Δppsr has an enhanced ability to colonize arabidopsis She Jiding. Culturing the strain AM1 delta ppsR of the strain which is knocked out by the methylobacterium wrenchii and the wild strain AM1 to the middle period of the index under the condition that methanol with a certain concentration is used as a carbon source, the cells were collected and treated with 10mM MgCl 2 Aqueous solution adjustment of OD 600 To 0.5. After taking sterilized Arabidopsis seeds and dibbling the seeds on a 1/2MS culture medium containing 1% sucrose, taking 5 μl of bacterial liquid to soak the seeds, and aseptically culturing the seeds for three weeks under the condition of 16 hours illumination and 8 hours darkness light dark rhythm. The interplanetary fraction of Arabidopsis thaliana was taken and placed in 10mM MgCl containing 0.2% v/v silwet L-77 2 The solution was shaken for 1 hour, post-diluted and plated on 123mM methanol carbon source plates, and CFU was counted after 7 days, respectively, and the number of viable cells per gram of fresh weight of the Arabidopsis thaliana colonized by the strain of the M.torpedo knockout ppsR mutant strain AM 1. DELTA. PpsR was found to be about 2.07 times higher than that of the wild strain AM1, as shown in FIG. 5.
EXAMPLE 7 enhancement of the ability of M.toxidans to colonize Arabidopsis She Jiding by insertion of stop codon to prevent the normal transcriptional expression of the gene ppsR
In this example, a termination codon UAA is inserted into the 12 th nucleotide position of the gene ppsR through homologous double exchange to obtain a strain inactivated by the ppsR of the methylobacterium, wherein the ppsR gene in the strain AM1ppsR cannot be normally transcribed and expressed, so that the negative regulation effect of the strain on the photosynthetic gene cluster of the methylobacterium is reduced. The ability of the methylobacterium wraparound ppsR inactivated strain AM1ppsR to colonize the wild-type strain AM1 in arabidopsis She Jiding was examined as described in example 5, and the number of viable cells per gram fresh weight of the arabidopsis colonizing the methylobacterium wraparound inactivated strain AM1ppsR was found to be about 2.37 times higher than the wild-type strain AM1, as shown in fig. 6.
Example 8 knockout of the Gene ppsR to increase the ability of M.radiodurans to colonize Arabidopsis She Jiding
In this example, the gene ppsR was knocked out in M.radiodurans by homologous double crossover to obtain a M.radiodurans knocked out gene ppsR mutant strain, and the ability of the M.radiodurans knocked out gene ppsR mutant strain to colonize with wild strain Arabidopsis thaliana She Jiding was examined in the manner described in example 5, and it was found that the number of viable cells per gram of fresh weight of the M.radiodurans knocked out gene ppsR mutant strain RA01 was about 1.55 times higher than that of the wild strain RA, as shown in FIG. 7. The nucleotide sequence of the ppsR gene is shown as SEQ ID NO. 2.
Claims (8)
1. A mutant strain of a methylobacterium, wherein the mutant strain is obtained by knocking out the ppsR gene sequence or the ppsR gene promoter sequence of the methylobacterium by using modern molecular biology techniques, or by disrupting the ppsR gene sequence by random insertion mutation or site-directed insertion mutation to reduce the ability of ppsR to bind to a promoter region of a gene associated with a photosynthetic gene cluster, or by altering the ppsR gene promoter sequence by site-directed mutation to reduce the transcription expression level of the ppsR gene.
2. The mutant methylobacterium of claim 1, wherein the modern molecular biology techniques comprise homologous double crossover techniques, transposition techniques, CRISPR-Cas9 techniques.
3. The mutant strain of Methylobacillus bacterium of claim 1, wherein said Methylobacillus bacterium is a pink gram-negative bacterium of the type having an organic carbon one, including methane, methanol, formaldehyde, formic acid, methylamine, etc., as a source of growing carbon and energy, including but not limited to Methylobacillus oryzaeMb. oryzaeMethylobacillus nodosusMb. nodulansMethylobacillus torvus (L.) RickenMr. extorquensMethylobacillus radioduransMb. radiotoleransMethylobacillus floccocusMb. gregansAnd Methylobacillus spanishMb. hispanicum。
4. A method for promoting the colonization of a plant by methylobacterium She Jiding, wherein the method utilizes a mutant of the methylobacterium of claim 1 to colonize the plant phyllosphere.
5. The method for promoting the proliferation of plant probiotic methylobacterium She Jiding according to claim 4, which is specifically: and (3) culturing the mutant strain of the methyl bacillus in a liquid way to the middle and later stages of a growth index, adjusting the concentration of thalli to about 0.5 OD600, and soaking plant seeds or drip irrigation plant roots or spraying plant leaf boundaries by using the bacterial suspension.
6. The method of promoting the proliferation of methylobacterium plantarum She Jiding according to claim 4, wherein the plant phyllosphere refers to the portion of a plant that appears above the ground surface and comprises flowers, stems, leaves, fruits; the plant mainly comprises ornamental plants such as corn, wheat, soybean, peanut, tomato, cucumber, melon and vegetable crops, azalea, chrysanthemum, narcissus, hydrangea and other plants cultivated by methyl bacillus She Jiding.
7. A mutant of the Methylobacillus bacterium of claim 1, which is useful for promoting colonization of plant phyllosphere growth.
8. Use of a mutant of a methylobacterium according to claim 1 for the preparation of a plant probiotic or a foliar bacterial fertilizer.
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| US20220073968A1 (en) * | 2019-02-08 | 2022-03-10 | Newleaf Symbiotics, Inc. | Methods for identifying microbial strains having enhanced plant colonization efficiency |
| CN115433694A (en) * | 2022-06-30 | 2022-12-06 | 广东药科大学 | Application of radiation-resistant methylobacterium L321 in degrading phthalate and promoting growth |
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| US20220073968A1 (en) * | 2019-02-08 | 2022-03-10 | Newleaf Symbiotics, Inc. | Methods for identifying microbial strains having enhanced plant colonization efficiency |
| CN115433694A (en) * | 2022-06-30 | 2022-12-06 | 广东药科大学 | Application of radiation-resistant methylobacterium L321 in degrading phthalate and promoting growth |
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| Title |
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| LUO ET AL.: "CRISPR/Cas9‑deaminase enables robust base editing in Rhodobacter sphaeroides 2.4.1", 《MICROBIAL CELL FACTORIES》, pages 1 - 14 * |
| SYLVIE ELSEN ET AL.: "PpsR: a multifaceted regulator of photosynthesis gene expression in purple bacteria", 《MOLECULAR MICROBIOLOGY》, vol. 57, no. 1, pages 17 - 26 * |
| YIN ET AL.: "PpsR, a Regulator of Heme and Bacteriochlorophyll Biosynthesis, Is a Heme-sensing Protein", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》, vol. 287, no. 17, pages 13850 - 13858 * |
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