CN117187130A

CN117187130A - Pantoea capable of producing DDP-IV inhibitor and application thereof

Info

Publication number: CN117187130A
Application number: CN202311139093.1A
Authority: CN
Inventors: 吕晓楠; 徐立蒲; 夏邦华; 张文; 王静波; 王姝; 王小亮; 曹欢
Original assignee: Beijing Aquatic Technology Promotion Station Beijing Fish Disease Prevention And Control Station; Northeast Agricultural University
Current assignee: Beijing Aquatic Technology Promotion Station Beijing Fish Disease Prevention And Control Station; Northeast Agricultural University
Priority date: 2023-09-05
Filing date: 2023-09-05
Publication date: 2023-12-08
Anticipated expiration: 2043-09-05
Also published as: CN117187130B

Abstract

The invention relates to a Pantoea (Pantoea deley i) LX1 for producing a DDP-IV inhibitor and application thereof, wherein the Pantoea for producing the DDP-IV inhibitor is preserved in China general microbiological culture collection center (CGMCC) No.28105. The invention adds the Pantoea delei LX1 bacterial liquid producing the DDP-IV inhibitor into a rice field of a rice-shrimp co-cropping mode to promote the growth of rice and procambarus clarkia. Pantoea deley i LX1 can greatly improve the plant height and fresh weight of rice seedlings, and slightly improve the root length and stem thickness of the rice seedlings, thereby being very beneficial to the growth of rice plants.

Description

Pantoea capable of producing DDP-IV inhibitor and application thereof

Technical Field

The invention relates to a strain and application thereof.

Background

In recent years, the integrated planting and breeding of rice and crayfish (procambarus clarkia Procambarus clarkii) has become one of typical planting and breeding modes in the middle and downstream regions of Yangtze river of China. By 2019, the yield of crayfish cultivated in the national paddy field is 177.25 multiplied by 104t, and the cultivation area is 110.54 multiplied by 104hm ² 84.84% and 85.96% of the total crayfish cultivation yield and total area, and 60.46% and 47.71% of the total national rice and fish comprehensive cultivation yield and total area. Compared with the traditional rice single-cropping mode, the mode improves the quality and quantity of rice crops, also harvests animal proteins and improves economic benefits.

The main physiological function of sugar is to provide energy, which is the cheapest feed energy source. It is generally considered that the ability of aquatic animals to utilize sugar is low, and that aquatic animals are considered to have congenital "diabetic constitution". Therefore, an important topic in the field of aquatic animal nutrition research is how to improve the utilization of feed sugar.

Disclosure of Invention

The invention provides a pantoea strain producing a DDP-IV inhibitor and application thereof.

The Pantoea delley i LX1 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.28105.

The invention also provides application of the Pantoea which produces the DDP-IV inhibitor in a rice and shrimp co-cropping mode.

The invention adds the Pantoea delei LX1 bacterial liquid producing the DDP-IV inhibitor into a rice field of a rice-shrimp co-cropping mode to promote the growth of rice and procambarus clarkia.

The bacterial colony of the Pantoea deley I LX1 is light yellow, glossy, smooth in surface, round, flat, sticky, neat in edge and slightly raised on an LB solid medium, and is gram-negative bacteria after gram staining.

The diameter D of the hydrolytic circle of the milk protein of Pantoea deley i LX1 on SKM solid medium is 10.73mm, the colony diameter D is 4.69mm, and the D/D is 2.29; the yield of DDP-IV inhibitor was as high as 38.2% in 37 ℃. Pantoea delley i LX1 can produce DDP-IV inhibitor, can control blood sugar of Eriocheir sinensis, and can absorb saccharide substances in feed, especially some non-starch polysaccharides; further can improve the digestion utilization rate of the feed and enhance the growth performance.

Pantoea delley i LX1 strain of the present invention has hypoglycemic effect. The Pantoea delley i LX1 of the invention is added into a paddy field with a comprehensive rice and crab breeding mode, so that the feed digestion utilization rate of procambarus clarkia can be improved. The Pantoea delley i LX1 can form obvious inorganic phosphorus-soluble rings on a Meng Jinna inorganic phosphorus culture medium, the diameter D of the inorganic phosphorus-soluble rings is 9.86mm, the colony diameter D is 3.51mm, and the D/D is 2.81; the inorganic phosphorus-dissolving agent has a strong function of dissolving inorganic phosphorus, can dissolve inorganic phosphorus in soil, can decompose the inorganic phosphorus which is difficult to convert and utilize in soil into soluble phosphorus and can be utilized by rice plants, and can promote the growth of rice. Pantoea delley i LX1 on Meng Jinna organophosphorus medium, diameter D of organophosphorus circle of 14.58mm, colony diameter D of 7.91mm, D/D of 1.84; has a certain function of decomposing organic phosphorus.

IAA secretion ability is considered to be characteristic of rice growth-promoting bacteria having high colonisation efficiency, and is closely related to whether or not the growth-promoting strain can exert a growth-promoting effect stably in the plant body. The Pantoea delley i LX1 contains 87.62 mug IAA in per milliliter of fermentation liquor, has high IAA secretion amount, is favorable for improving the comprehensive planting and breeding effect of rice and crabs, has the capability of producing auxin and has growth promoting effect on plants.

Pantoea delley i LX1 has strong tolerance to acidity and bile salts at 37 ℃, and Pantoea delley i LX1 can survive and play a role in the intestinal tract of procambarus clarkia; pantoea (Pantoea deley i) LX1 has good stress resistance. The Pantoea (Pantoea delley i) LX1 strain is safe and reliable, does not cause death of procambarus clarkia, compared with a control group, the feed coefficient of the Pantoea (Pantoea delley i) LX1 group is obviously lower than that of the control group, the weight gain rate and the specific growth rate of the treated group are obviously higher than those of the control group, and the feed utilization rate of the Pantoea (Pantoea delley i) LX1 can be obviously improved.

Pantoea deley i LX1 can greatly improve the plant height and fresh weight of rice seedlings, and slightly improve the root length and stem thickness of the rice seedlings, thereby being very beneficial to the growth of rice plants.

The use of Pantoea deley i LX1 can improve the nutrient availability of soil, increase the yield of rice and play a role in improving the fertilizer for the submerged rice soil.

Pantoea delley i LX1 is Pantoea belonging to Pantoea (Pantoea Gavini et al., 1989); the culture medium is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is 1 # 3 of North West Lu of the Korean area of Beijing, the preservation number is 28105 of CGMCC, and the preservation date is 2023, 08 and 04 of China academy of sciences microbiological culture Collection center.

Drawings

FIG. 1 is a graph showing the results of an experiment of decomposing inorganic phosphorus by Pantoea delley i LX1 on Meng Jinna inorganic phosphorus bacteria medium;

FIG. 2 is a graph of experimental results of the lysis of inorganic phosphorus by Pantoea delley i LX1 on Meng Jinna organophosphorus bacteria medium;

FIG. 3 is a graph showing the results of measuring IAA-producing ability of Pantoea (Pantoea deley) LX1 by Salkowski colorimetry;

FIG. 4 is a graph showing the results of a hydrolysis test of milk protein of Pantoea delley i LX1 on SKM medium;

FIG. 5 is a phylogenetic tree constructed from Pantoea deley i LX 1;

FIG. 6 is a graph showing the comparative effect of Pantoea (Pseudomonas glycinae) LX1 strain in example 2 on the effect of rice seedling growth on pot culture.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.

The first embodiment is as follows: the Pantoea (Pantoea deley i) LX1 which is the Pantoea (Pantoea deley i) inhibitor produced by the embodiment is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.28105.

And (3) selecting a procambarus clarkia population in a hula aquatic product test field of the institute of aquatic products of Heilongjiang, china, and the academy of aquatic products, at 5 months 2023, randomly selecting 300 healthy procambarus clarkia (25.0+/-1.87 g), filling air and water, bagging, then transporting to the university of northeast agricultural animal science and technology, selecting 20 procambarus clarkia with normal body color and active reaction, and anesthetizing by using an MS-222 anesthetic (250 mg/L). Wiping the surface of procambarus clarkia with absolute ethanol, dissecting with sterilized scissors and tweezers in an ultra-clean workbench, taking out the whole intestinal tract of procambarus clarkia, gently squeezing out the content, placing into a conical flask containing glass beads and 50mL of sterile water, oscillating at 180r/min for 30min at room temperature, performing gradient dilution, and collecting 10 ^-3 、10 ^-4 、10 ^-5 100 mu L of the gradient is coated on an LB solid culture medium plate, each gradient is repeated for 3 times, and the mixture is placed at 28 ℃ for culturing for 24-48 hours. After 48h of culture, strains with different shapes are selected for separation and purification, wherein the strain LX1 is obtained.

After purification, strain LX1 was inoculated with a sterile toothpick onto a plate of Meng Jinna inorganic phosphorus bacteria medium. Each gradient was repeated 3 times and incubated at 28℃for 24-48 h. Apparent inorganic phosphate rings were formed around colonies of the strain LX1 (as shown in FIG. 1), and the colony diameter D and inorganic phosphate ring diameter D were measured by the crisscross method, and D/D was calculated. The diameter D of the inorganic phosphate ring of the strain LX1 was 9.86mm, the colony diameter D was 3.51mm, and the D/D was 2.81. The strain LX1 has a strong function of decomposing inorganic phosphorus.

After purification of strain LX1, a sterile toothpick was used to inoculate a plate of Meng Jinna organophosphorus bacterial medium. Each gradient was repeated 3 times and incubated at 28℃for 24-48 h. Apparent inorganic phosphate rings were formed around colonies of the strain LX1 (as shown in FIG. 2), and the colony diameter D and inorganic phosphate ring diameter D were measured by the crisscross method, and D/D was calculated. The diameter D of the organophosphorus circle of the strain LX1 is 14.58mm, the colony diameter D is 7.91mm, and the D/D is 1.84. The strain LX1 has a certain function of decomposing organic phosphorus.

Bacterial strain LX1 colony isolate was inoculated into R containing L-tryptophan ₂ And (3) in the liquid culture medium A, placing the culture medium A in a shaking table at a constant temperature of 28 ℃ for 180r/min for shake culture for 4d. mu.L of the bacterial suspension was pipetted into a 2mL glass bottle and 500. Mu.L of Salkowski broth was added. A Salkowski colorimetric solution was added simultaneously with 500mg/L IAA as a positive control. The 2mL glass bottle was kept at room temperature in a dark place for 30min, and the color change was observed. If the color turns red, the strain has the function of producing IAA. After the Salkowski colorimetric solution is dripped into the bacterial suspension of the bacterial strain LX1, the color in a 1mL glass bottle is changed into red under the condition of being protected from light at room temperature (as shown in figure 3); the strain LX1 has the capability of producing auxin and has a certain growth promoting effect on plants.

The IAA 10mg is precisely weighed, firstly, a small amount of absolute ethyl alcohol is used for dissolution, then distilled water is added for volume fixation to 100mL, IAA solution with the concentration of 100 mug/mL is prepared as stock solution, and then the stock solution is diluted to prepare serial standard solutions with the concentrations of 0 (blank), 0.5, 1.0, 5.0, 10.0, 15.0, 20.0 and 25.0 mug/mL respectively as working solutions. Respectively adding 2mL of the working solution into 8 test tubes in sequence, adding Salkowski colorimetric solution with volume of 2 times, maintaining at 40deg.C in dark for 30min, and measuring waveAbsorbance at 530nm long. At OD ₅₃₀ IAA concentration is plotted on the abscissa as the ordinate as the IAA standard curve. Bacterial strain LX1 colony isolate was inoculated into R containing L-tryptophan ₂ And (3) in the liquid culture medium A, placing the liquid culture medium A in a shaking table at a constant temperature of 28 ℃ for 180r/min for shake culture for 4d, then measuring the OD value of the bacterial suspension at the wavelength of 600nm by using a spectrophotometry, taking the bacterial suspension, centrifuging the bacterial suspension at a rotating speed of 10000r/min for 10min, taking the supernatant, adding an equal volume of Salkowski colorimetric solution, placing the supernatant in a dark place at 40 ℃ for 30min for color development, and measuring the OD value at the wavelength of 530 nm. Calculation of OD ₆₀₀ When the value is 1, the IAA concentration per unit volume of the fermentation liquid (when the concentration of the bacterial liquid is too high, dilution is required). The measurement result shows that the strain LX1 contains 87.62 mug IAA in per milliliter of fermentation broth, and the IAA secretion capacity of the strain LX1 is strong.

Strain LX1 was plated onto SKM (2% nonfat dry milk medium) to form transparent circles, and the colonies of strain LX1 formed distinct hydrolysis circles (as shown in fig. 4) around the colonies, with a diameter D of 10.73mm for the hydrolysis circles of strain LX1, a diameter D of 4.69mm for the colonies, and a D/D of 2.29. Centrifuging the strain LX1 bacterial liquid cultured for 2 generations for 15min at 8000r/min, and removing supernatant to leave bacterial mud. The cells were resuspended in PBS and the absorbance of the cell solution was adjusted to 1.0 by washing 3 times with 0.1mol/L sterile phosphate buffer solution (PBS, pH 6.8). Culturing at 37deg.C for 24 hr, centrifuging at 4deg.C and 8000r/min for 15min, filtering supernatant with 0.22pm water-based filter membrane bacterial filter to obtain cell metabolite (CFS), and preserving at-80deg.C. Further test: 25. Mu.L, 1.6mmol/L glycyl-prolyl-p-nitroaniline and 25. Mu.L CFS were accurately added dropwise to a 96-well plate. The reaction was stopped by adding 50. Mu.L of DDP-IV (0.01U/mL) at 37℃for 15min, continuing the reaction at 37℃for 1h, and finally adding 100. Mu.L of 1mol/L sodium acetate buffer (pH=4.0), and the absorbance of the reaction solution was measured at 405nm using an ELISA reader. DDP-IV inhibition rate calculation formula:

A _{sample to be measured} : 25. Mu.L of sample +25. Mu.LGly-pro-phy +50. Mu.LDDP-IV +100. Mu.L of sodium acetate;

A _{sample blank} : 25. Mu.L of sample +50. Mu.L of LTris-HCl +25. Mu.L of LGly-pro-phy +100. Mu.L of sodium acetate;

A _{negative control} :25 mu LTris-HCl+25 mu LGly-pro-phy+50 mu LDDP-IV+100 mu L sodium acetate;

A _{negative blank} :75 mu LTris-HCl+25 mu LGly-pro-phy+100 mu L sodium acetate.

The yield of DDP-IV inhibitor at 37℃was found to be as high as 38.2% for strain LX1, indicating that strain LX1 has a certain ability to produce DDP-IV inhibitor.

Stress resistance test of strain LX 1:

acid resistance detection: bacterial strain LX1 was inoculated in an inoculum size of 2% (v/v) to LB liquid medium having pH values of 2.0, 2.5 and 3.0, and after plating by pipetting for 0, 1, 2 and 3 hours, respectively, and cultured at 37℃for 24 hours, the viable count was measured.

And (3) detecting the bile salt resistance: the activated strain LX1 is diluted by a sterile physiological saline solution in a multiple ratio, a proper dilution gradient is selected, and 1mL of the dilution is sucked and placed in a sterilized plate. Plates were then poured with LB solid medium containing 0.3% and 1.0% sodium taurocholate, while MRS solid medium without sodium taurocholate was used as a control group, cultured at 37℃for 48 hours, colony counts were performed, and the survival rate of the strain was calculated.

As shown in Table 1, the effect on the number of spores of the strain LX1 is small when the pH is 4.0, and the number of spores of the strain LX1 is obviously reduced when the pH is 3.0; and when the concentration of bile salts is increased to 1.5%, the number of spores of the strain LX1 is greatly changed. The strain LX1 has stronger tolerance to acidity and bile salts.

TABLE 1

Physiological and biochemical identification of strain LX 1: the preserved strain LX1 is marked on a solid LB culture medium plate in three areas, single colonies are separated and the morphology of the single colonies is described, and gram staining and physiological and biochemical identification are carried out on the strain according to the general bacterial System identification handbook.

Bacterial colony of the strain LX1 is light yellow, glossy, smooth in surface, round and flat, sticky, neat in edge and slightly raised, and is gram-negative bacteria after gram staining. The physiological and biochemical indexes of the strain LX1 are shown in Table 2. According to the description of physiological characteristics of Enterobacter from the "Berger's Manual of bacteria identification", the strain LX1 has the same characteristics as physiological biochemistry of a species of Pantoea (Pantoea delley i), and it is inferred from each physiological biochemistry that the strain LX1 may be Pantoea (Pantoea delley i).

TABLE 2

Identification of 16S rRNA of Strain LX 1: and (3) selecting a bacterial genome DNA extraction kit of Beijing Soxhaust biological technology company, and extracting, separating and purifying the strain DNA. The bacterial universal primer 27F/1492R is adopted for PCR amplification, and the PCR amplification system is a 25 mu L system: 10 Xbuffer 2.5. Mu.L, taq enzyme 0.5. Mu.L, primer 27F 0.5. Mu.L, primer 142R 0.5. Mu.L, DNA template 1. Mu.L, ddH ₂ O20. Mu.L. The reaction procedure is set to 95 ℃ for 5min of pre-denaturation; denaturation at 94℃for 50s, annealing at 56℃for 30s, extension at 72℃for 1.5min, cycle times for 30 times, extension at 72℃for 10min again, and preservation at 4 ℃. The PCR amplified product was sent to RuiBiotech company for sequencing. Sequencing results of strain 16S rRNA were aligned by NCBI database and phylogenetic tree was constructed (as shown in FIG. 5). A BLAST alignment was performed in NCBI to find that the 16S rRNA gene sequence of strain LX1 has 99% similarity to Pantoea (Pantoea delley i). From the phylogenetic tree of strain LX1, it can be seen that the smallest branch of strain LX1 is located in the same place as Pantoea delley (NR 116114.1), and that the evolution distance is closer, and the comprehensive physiological and biochemical index identifies LX1 strain as Pantoea delley (Pantoea delley).

Example 1

Effect of Pantoea delei LX1 on Procambrus clarkii growth performance.

The procambarus clarkia culture test is carried out in a circulating water tank of a laboratory of the institute of aquatic products of Heilongjiang, china academy of aquatic products, and procambarus clarkia used for the test is all derived from China water obstetricsThe academy of China's aquatic products test field of Heilongjiang aquatic products institute. Selecting 200 tails of procambarus clarkia with initial weight of 25.0+ -1.87 g propagated in the current year, placing in a cement pond with water depth of 4.3m×1.5m×1.0m (10 cm), and temporarily culturing with a large amount of herba Sagittariae as shielding material for 1 week to adapt to test environment, and feeding control group fodder during temporary culturing periodPuffed feed special for shrimp feed). After temporary rearing, selecting 120 tails of procambarus clarkii with similar weight, better activity and no damage on the body surface, randomly dividing the tails into 2 groups (a control group and a treatment group), wherein each group is 3 parallel, each parallel 20 tails are placed in 6 circulating water rearing boxes with the height of water body of 10cm and a large amount of water grass as a shielding object, tap water with aeration time of more than 24 hours is used as test water body, an oxygenation pump is adopted for 24 hours for aeration during the test, and the daily consumption is 08:00 and 16:00 feeding.

The treatment group was fed with 1.5X10 mix ⁶ CFU/mL Pantoea (Pantoea deley i) LX1 bacterial liquidThe special puffed feed for shrimp feed is characterized in that before feeding, bacterial liquid is fully mixed with the feed (the feed-liquid ratio is 1:0.1), and the bacterial liquid is fully adsorbed on the feed. Control group is fed with control group feed (+)>Puffed feed special for shrimp feed); feeding for 2 times a day, apparent feeding, sucking at the bottom of the day for 1 time, changing water amount about 1/2, water temperature (23+ -1) deg.C during test, dissolved oxygen (6+ -0.5) mg/L, pH value of 7.1+ -0.5, and cultivation period of 7d. Observing and recording whether the appendages are complete or not at 09:00 a day, and calculating the mortality and the stump rate; weighing each treated procambarus clarkia after the cultivation is finished, and calculating the average weight, the weight growth rate and the feed coefficient, wherein the specific calculation formula is as follows:

body weight gain = [ (W) ₁ -W ₀ )/W ₀ ]×100％

Mortality = D/Z x 100%

Stump rate = C/Z x 100%

Feed coefficient=w ₂ /(W ₁ -W ₀ )×100％

Wherein W is ₀ For initial weight, W ₁ For final body weight, W ₂ For feed consumption, D is the death number of each treatment in 24 hours, C is the shrimp mantissa with appendage loss in each treatment in 24 hours, and Z is the mantissa of procambarus clarkia put in each treatment.

The experimental results are shown in Table 3, and as can be seen from Table 3, the mortality and the amputation rate of procambarus clarkia are not significantly different among groups, which means that Pantoea (Pantoea delei) LX1 does not affect the mortality and the amputation rate of procambarus clarkia, and the strain is safe and reliable. The feed coefficient of the treatment group is obviously lower than that of the control group, and the weight gain rate of the treatment group is obviously higher than that of the control group, which indicates that the Pantoea (Pantoea delei) LX1 can obviously improve the feed utilization rate.

TABLE 3 Table 3

Example 2

Inoculating Pantoea delley i LX1 strain into 50mL LB liquid medium, shake culturing at 28deg.C and 180r/min overnight, diluting the culture solution with sterile water to thallus concentration of 1×10 ⁵ CFU/mL was used as bacterial suspension for further use. 150 seeds of Longjing 31 rice with plump and consistent grains are selected, soaked in warm water at 55 ℃ for 18 hours, and then are grouped and respectively placed in 1 multiplied by 10 ⁵ CFU·mL ^-1 Soaking Pantoea (Pantoea delley i) LX1 bacterial suspension or clear water for 5h; the seeds after soaking are washed 3 times by distilled water, then are evenly placed in a culture dish (diameter is 10 cm) filled with 3 layers of filter paper, and then are respectively sprayed with 5mL of the bacterial suspension (the pantoea LX1 bacterial suspension is used for soaking seeds before) or clear water (the clear water is used for soaking seeds before) on the surfaces of the seeds. After accelerating germination at 28 ℃, sowing the seedlings in 15kg paddy field soil transplanting pots, taking 30 seedlings from each pot after 28 days, and measuring root length, plant height, stem thickness and fresh weight of the seedlings, wherein the seedlings are repeated for 3 times.

The experimental results are shown in Table 4 and FIG. 6, and Pantoea (Pantoea deley i) LX1 has obvious growth promoting effect on rice under potting conditions. As can be seen from Table 4, the root length, plant height, stem thickness and fresh weight of the rice plant subjected to seed soaking treatment by Pantoea deley i LX1 are respectively increased by 5.23%, 54.01%, 5.83% and 30.58%, so that the Pantoea deley i LX1 can greatly improve the plant height and fresh weight of the rice seedling, and the root length and stem thickness of the rice seedling are slightly improved, thereby being very beneficial to the growth of the rice plant.

TABLE 4 Table 4

In this example, 1000ml of Pantoea (Pantoea deley) LX1 bacterial liquid medium was composed of 10g of chitosan (as a carbon source), 10g of ammonium sulfate (as a nitrogen source), 0.2g of magnesium sulfate, 2.2g of dipotassium hydrogen phosphate (as a source of inorganic salts and metal ions), and the balance of distilled water.

Inoculating Pantoea delley i LX1 seed solution into Pantoea delley i LX1 bacterial solution culture medium at 32deg.C, 200r/min, and pH 8 for 24 hr to obtain viable count of 5.23X10 ⁸ CFU/mL. The number of viable bacteria before the optimization is 3.18×10 ⁷ CFU/mL (non-optimized medium is bean sprout juice medium, bean sprout juice medium: 100mL of bean sprout juice, 10g of sucrose, (NH) ₄ ) ₂ SO ₄ 2g，NaCl 0.4g，ZnSO ₄ 0.08g, distilled water to 1000mL, pH 7, and autoclaved at 115℃for 30 min).

Claims

1. A Pantoea producing DDP-IV inhibitor is Pantoea @Pantoea deleyi) LX1 is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.28105.

2. Use of the ubiquitin bacterium producing an inhibitor of DDP-IV according to claim 1 in a rice shrimp co-mode.