Burkholderia BK-1 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and provides Burkholderia sp. BK-1 which is preserved in the China Center for Type Culture Collection on September 30, 2024, the preservation number of the Burkholderia sp. BK-1 is CCTCC NO: M 20242135, the preservation number of the Burkholderia sp. BK-1 is CCTCC NO: M 20242135, and the preservation number of the Burkholderia sp. BK-1 is CCTCC NO: M 20242135. The invention also provides application of the burkholderia BK-1 in reduction of accumulation of heavy metals in crops. The Burkholderia BK-1 provided by the invention not only has mercury reducing capacity, but also has the capacity of dissolving phosphorus and producing siderophores, so that the accumulation of heavy metals in crops can be reduced, for example, the accumulation of mercury in rice can be reduced.

Description

Technical Field

[0001] The present disclosure relates to the field of microbial technology, for example, to a Burkholderia BK-1 and applications thereof. Background Art

[0002] Mercury is one of the most toxic heavy metal pollutants. It can be transmitted over long distances through the atmosphere and settle into terrestrial and aquatic ecosystems, posing a serious threat to the ecological environment and human health. With the rapid development of industrialization in my country, the problem of mercury pollution has become increasingly serious. Every year, 500-600 tons of mercury are discharged into the atmosphere through human activities, accounting for about 30% of the global anthropogenic emissions. A survey of heavy metal pollution in China's cultivated land over the past three decades shows that mercury is the second largest heavy metal pollutant, second only to cadmium. Mercury pollution in farmland soil has led to a decline in soil quality, productivity and food safety. Due to the toxic characteristics of mercury, once absorbed by plants, it will accumulate permanently in the body and eventually enter the human body through the food chain, causing harm to human health.

[0003] Rice is one of the main food crops in my country. In 2022, the rice planting area was 29.4501 million hectares, with a total output of 208.195 million tons. The glutinous rice planting area has been stable at around 789,300 hectares throughout the year, with a total output of about 6 million tons. Due to the topography, soil parent material and the development and utilization of mineral resources, the heavy metal point exceedance rate in farmland soil remains high. A large amount of mercury enters the paddy field soil through inorganic mercury, causing mercury pollution in the paddy field soil. Eating rice from mercury-contaminated areas is one of the main ways for human exposure to methylmercury.

[0004] There are a wide variety of microbial communities in the soil, which are large in number and have vigorous metabolism. Among them, many soil microorganisms are tolerant to heavy metals and play an important role in the absorption, transport and degradation of heavy metals. Studies in recent years have shown that microorganisms can alleviate the toxic effects of heavy metals on crops, and at the same time regulate the absorption and transport of heavy metals by crops, playing an important role in reducing the accumulation of heavy metals in the edible part of crops. Heavy metal-resistant growth-promoting bacteria are a type of bacteria that can grow stably and promote plant growth under heavy metal stress. Studies have found that heavy metal-resistant growth-promoting bacteria have a variety of heavy metal resistance mechanisms, including redox, intracellular transformation and extracellular precipitation, which can reduce the effectiveness of heavy metals in the environment and reduce the toxic effects of heavy metals on plants. In addition, it can also directly or indirectly promote plant growth through a variety of mechanisms such as the production of indoleacetic acid, siderophores, and phosphorus solubilization and nitrogen fixation, thereby improving the tolerance of plants to heavy metals. Studies have shown that in a heavy metal polluted environment, inoculation of heavy metal-resistant growth-promoting bacteria can effectively promote the growth of crops such as wheat, rice, and soybeans, improve the tolerance of crops to cadmium, reduce the effectiveness of heavy metals in the rhizosphere, and reduce the absorption of cadmium by crops.

[0005] However, current research on the use of microorganisms to reduce heavy metals in crops is mostly focused on the heavy metal cadmium, while similar research on mercury is relatively rare. Summary of the invention

[0006] The purpose of the present disclosure is to overcome the deficiencies in the prior art and to provide a Burkholderia BK-1 and its application. The Burkholderia BK-1 not only has the ability to reduce mercury, but also has the ability to solubilize phosphate and produce siderophores, thereby being able to reduce the accumulation of heavy metals in crops, for example, being able to reduce the accumulation of mercury in rice.

[0007] The above purpose is achieved through the following technical solutions:

[0008] In one aspect, a Burkholderia sp. BK-1 is provided. The Burkholderia sp. was deposited in China Center for Type Culture Collection on September 30, 2024, with a deposit number of CCTCC NO: M 20242135.

[0009] In another aspect, a bacterial agent comprising the Burkholderia BK-1 is provided.

[0010] On the other hand, there is provided a use of the Burkholderia BK-1 or the bacterial agent in reducing the accumulation of heavy metals in crops.

[0011] In some embodiments, the crop comprises rice.

[0012] In some embodiments, the heavy metal comprises mercury.

[0013] The beneficial effects of the present disclosure are:

[0014] The Burkholderia BK-1 provided by the present disclosure not only has the ability to reduce mercury, but also has the ability to solubilize phosphate and produce siderophores, and thus can reduce the accumulation of heavy metals in crops, for example, can reduce the accumulation of mercury in rice.

[0015] Biological Deposit

[0016] A Burkholderia sp. BK-1 provided in the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on September 30, 2024, with a deposit number of CCTCC NO:M 20242135. The deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the postal code is 430072. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1This is a graph verifying the mercury resistance of strain BK-1 in Example 2; wherein, Figure 1 A is the result of the validation of mercury resistance of strain BK-1 at mercury concentrations of 0-20 mg / L. Figure 1 B is a graph showing the growth effect of strain BK-1 on LB solid medium at mercury concentrations of 0-20 mg / L;

[0018] Figure 2 is the phylogenetic tree of strain BK-1 in Example 2;

[0019] Figure 3 The result diagram of the growth ability of strain BK-1 in Example 3 at different mercury concentrations, pH values ​​and inoculation amounts; wherein, Figure 3 A is the result of the growth ability of strain BK-1 at different mercury concentrations. Figure 3 B is the result of the growth ability of strain BK-1 at different pH values. Figure 3 C is the result graph of the growth ability of strain BK-1 at different inoculation amounts;

[0020] Figure 4 The figure is a result diagram of the strain characteristic study of the strain BK-1 of Example 4, that is, a result diagram of the comparison of the characteristics of the strain BK-1 on CAS medium, medium for nitrogen-fixing bacteria, inorganic phosphorus medium, organic phosphorus medium and cellulose red medium;

[0021] Figure 5 The mercury removal rate of the strain BK-1 in Example 5 and the result of scanning electron microscopy observation are shown; wherein, Figure 5 A is the result graph of mercury removal rate of strain BK-1. Figure 5 B is the result of bacterial morphology of strain BK-1 at 0 mg / L mercury concentration (scale bar = 500 nm), Figure 5 C is the result of bacterial morphology of strain BK-1 at a mercury concentration of 10 mg / L (scale bar = 500 nm);

[0022] Figure 6 This is a graph showing the results of electrophoresis detection of the merA gene of strain BK-1 in Example 6;

[0023] Figure 7 The result diagram of the real-time fluorescence quantitative analysis of each gene of the mer operon of the strain BK-1 in Example 6;

[0024] Figure 8 The effect of strain BK-1 in Example 7 on mercury accumulation in various organs of indica rice variety N1311r and japonica rice variety WN; wherein, Figure 8 A shows the effect of strain BK-1 on mercury accumulation in various organs of indica rice variety N1311r. Figure 8 B shows the effect of strain BK-1 on mercury accumulation in various organs of japonica rice variety WN. DETAILED DESCRIPTION

[0025] The technical solutions in some embodiments of the present disclosure are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present disclosure.

[0026] Example 1 Formulation of culture medium

[0027] 1.LB liquid medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, add water to 1 L.

[0028] 2.LB solid medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar, add water to 1 L.

[0029] 3. Axubei culture medium for nitrogen-fixing bacteria: 0.2g potassium dihydrogen sulfate, 0.2g magnesium sulfate, 0.2g calcium chloride, 5.0g calcium carbonate, 10.0g mannitol, 0.1g calcium sulfate, 15.0g agar, adjust pH to 7.0±0.1, add water to 1L, sterilize at 121℃ high pressure for 15min, and set aside.

[0030] 4. Organophosphorus bacterial culture medium: 10.0g glucose, 0.5g ammonium sulfate, 0.5g yeast extract powder, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate, 0.03g ferrous sulfate, 0.03g manganese sulfate, 1.0g lecithin, 15g agar, adjust pH to 7.0-7.5, add water to 1L, autoclave at 121℃ for 15min, and set aside.

[0031] 5. Inorganic phosphorus bacterial culture medium: 10g glucose, 0.5g ammonium sulfate, 0.5g yeast extract powder, 0.3g sodium chloride, 0.3g potassium chloride, 0.3g magnesium sulfate, 0.03g ferrous sulfate, 0.03g manganese sulfate, 5.0g calcium sulfate, 15.0g agar, adjust the pH to 7.0-7.5, add water to 1L, autoclave at 121℃ for 15min, and set aside.

[0032] 6.CAS detection medium: chrome azuro (CAS) 0.0605g, hexadecyltrimethylammonium bromide (HDTMA) 0.0729g, ferric chloride hexahydrate 0.002645g, sodium dihydrogen phosphate dihydrate 0.29525g, disodium hydrogen phosphate dodecahydrate 1.2135g, ammonium chloride 0.125g, potassium dihydrogen phosphate 0.0375g, sodium chloride 0.0625g, agar 9.0g, adjust pH to 6.8±0.1, add water to 1L, autoclave at 116℃ for 30min, and set aside.

[0033] 7. Cellulose Congo red medium: 1.0g sodium nitrate, 1.2g disodium hydrogen phosphate, 0.9g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.5g potassium chloride, 0.5g yeast extract powder, 0.5g acid hydrolyzed casein, 0.2g Congo red, 5.0g cellulose powder, 15.0g agar, adjust pH to 7.0±0.1, add water to 1L, autoclave at 121℃ for 15min, and set aside.

[0034] Example 2 Screening and species identification of mercury-resistant strains

[0035] 1. Screening of mercury-resistant strains

[0036] Four rice soil samples from the mercury mines around Xiushan Tujia and Miao Autonomous County, Chongqing were used as materials. In a sterile environment, the four soil samples were mixed evenly, and 5 g of soil sample was taken into a conical flask containing 50 mL of sterile water to make a bacterial suspension. After shaking for 20 minutes, it was allowed to stand, and 1 mL of the supernatant was taken with a pipette and inoculated into a 5 mg / L Hg 2+ LB liquid medium was incubated at 28°C and 150 rpm in a shaker. When the medium became turbid, 1 mL of the culture medium was inoculated into a 10 mg / L Hg 2+ LB liquid medium, and so on, culture for multiple times until the medium does not become turbid, take 1mL of the turbid bacterial liquid and rinse with sterile water for 10 -1 -10 -6 Dilute and take 10 -4 -10 -6 0.1 mL of each dilution was inoculated on LB solid culture medium and cultured in a 28°C incubator for 24 h. A single colony with good growth was streaked 2-3 times to obtain a single colony, which was stored in a 4°C refrigerator.

[0037] 2. Species Identification of Mercury-Resistant Strains

[0038] The strain to be tested was cultured in LB liquid medium (28°C, 150rpm) for 24h, then 2mL of bacterial solution was centrifuged at 4°C, 8000rpm for 10min, the supernatant was discarded to collect the bacteria, 100μL TE Buffer was added to mix, and 10μL lysozyme was added to treat it in a constant temperature water bath at 37°C overnight, and the single bacterial DNA was extracted according to the steps of Shanghai Shenggong Bacterial DNA Extraction Kit. The extracted DNA sample was stored at -20°C for later use. The conserved nucleic acid sequence of the mercury-resistant strain was amplified by PCR using the universal primers 27F and 1492R of the 16S rRNA gene sequence. The PCR amplification product was sequenced by Beijing Qingke Biotechnology Co., Ltd. The amplified sequence obtained was compared on the NCBI official website, and a phylogenetic tree was constructed on the MEGA software.

[0039] 3. Experimental results

[0040] Thirty-eight strains with possible mercury tolerance were screened by selective medium enrichment screening. The 38 strains were inoculated in a medium containing Hg 2+ A mercury-resistant strain BK-1 was screened out based on the growth of the strain in LB liquid culture medium with concentrations of 0 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, and 20 mg / L.

[0041] Among them, the results of the mercury resistance verification of strain BK-1 at 0-20 mg / L mercury concentration are as follows Figure 1 As shown in A; the growth effect of strain BK-1 on LB solid medium at 0-20 mg / L mercury concentration is as follows Figure 1 B; the sequence of the PCR amplified product of strain BK-1 is shown in SEQ ID No. 1; the phylogenetic tree of strain BK-1 is shown in Figure 2 As shown, the analysis showed that strain BK-1 was in the same smallest branch as Burkholderia sp. strain (MK583567.1) and Burkholderia sp. strain (MK459497.1), with the closest evolutionary distance, and strain BK-1 was identified as Burkholderia sp.

[0042] 4. Preservation of mercury-resistant strains

[0043] The screened mercury-resistant strain was identified as Burkholderia sp. BK-1 and preserved in the China Center for Type Culture Collection with the accession number CCTCC NO: M 20242135; the preservation address is China Center for Type Culture Collection (CCTCC), Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the preservation date is September 30, 2024.

[0044] Example 3 Optimization of growth conditions for mercury-resistant strains

[0045] 1. Experimental Methods

[0046] In order to determine the most suitable growth conditions for the strain, a single factor experimental method was used to analyze the growth of the strain under different mercury concentrations, pH and inoculation amounts. The mercury concentration gradient was selected as 0 mg / L, 5 mg / L, 10 mg / L, 15 mg / L and 20 mg / L, the pH was selected as 5, 6, 7, 8 and 9, and the inoculation amount was selected as 5%, 10%, 15%, 20% and 25%. The strain BK-1 was inoculated into 50 mL of LB liquid culture medium according to different mercury concentrations, pH and inoculation amounts. Three replicates were used in each group, and the absorbance value (OD 600 ).

[0047] 2. Experimental results

[0048] like Figure 3 As shown in A, strain BK-1 was exposed to 15 mg / L Hg 2+ The growth of strains was the best at 20 mg / L Hg. The stable period was reached at 48-60 h. 2+ In the medium containing 10 mg / L mercury, the effect of different pH on the growth of strain BK-1 was further determined to determine the optimal growth conditions for strain BK-1 in mercury-contaminated soil, such as Figure 3 As shown in B, strain BK-1 grows best at a pH of 7, so strain BK-1 is suitable for neutral soil. Figure 3 As shown in C, when the inoculum amount was 10%, the growth condition of strain BK-1 was the best.

[0049] Example 4 Study on the characteristics of mercury-resistant strains

[0050] 1. Experimental Methods

[0051] The mercury-resistant strain BK-1 was streaked multiple times on LB solid culture medium to obtain single colonies. A small amount of bacteria was taken with an inoculation loop and inoculated into Ashubei medium for nitrogen-fixing bacteria, organophosphorus bacteria medium, inorganic phosphorus bacteria medium, CAS detection medium and cellulose Congo red medium. The culture was inverted in a constant temperature incubator at 28°C for 72 hours. The colonies were observed according to the characteristics of each culture medium to determine the functional characteristics of the mercury-resistant strain.

[0052] 2. Experimental results

[0053] We conducted a qualitative analysis of other characteristics of strain BK-1, and found that strain BK-1 exhibited different characteristics under different culture conditions. Figure 4 As shown, a yellow halo appeared in the CAS medium of strain BK-1, indicating that it has the ability to produce siderophores; a transparent circle appeared around the bacteria of strain BK-1 in the organic phosphorus medium, indicating that it has the ability to dissolve organic phosphorus; a transparent circle also appeared around the bacteria of strain BK-1 in the inorganic phosphorus medium, indicating that it also has the ability to dissolve inorganic phosphorus; strain BK-1 did not show a specific reaction in the cellulose Congo red medium.

[0054] Example 5 Determination of mercury removal rate of mercury-resistant strains and electron microscopic observation

[0055] 1. Experimental Methods

[0056] Take 1 mL of the bacterial suspension of strain BK-1 cultured under the optimal growth conditions and inoculate it into LB liquid culture medium containing different mercury concentrations. The control group is a medium without Hg. 2+After 48 hours of culture, the bacterial culture was collected and the Hg in the culture medium with different mercury concentrations was detected using an atomic fluorescence mercury analyzer. 2+ The residual amount of Hg is calculated using the formula 2+ removal rate.

[0057]

[0058] Where C represents the mercury removal rate, C0 represents the initial Hg in the sample at 0h 2+ concentration, C1 represents the remaining Hg in the sample after 48 h of strain treatment 2+ concentration.

[0059] Collect the bacterial solution under 10mg / L and centrifuge at 10000rpm for 5min, discard the supernatant and rinse the sample 2-3 times with 1×PBS (pH7.0), 15min each time, centrifuge at 5000rpm for 3min after rinsing, remove the supernatant, add 1mL of 2.5% glutaraldehyde solution to fix, shake and fully mix, resuspend the bacterial solution, send the sample after fixing at 4℃ for 4h, and then wash it with phosphate buffer for 3 times. Dehydrate step by step with ethanol solution of different concentrations, 30%-50%-70% (30%-50%-70% dehydrated at 4℃), 80%-90% (dehydrated at room temperature), dehydrate for 15min per level, 100% ethanol solution needs to be dehydrated twice, and then replaced with isoamyl acetate solution 2 times, 20min each time. Dry the bacterial cells with a critical drying instrument, spray gold plating on the bacterial cells with a gold plating instrument, and finally observe and take pictures with a field emission scanning electron microscope.

[0060] 2. Experimental results

[0061] like Figure 5 As shown in A, the mercury removal rate of strain BK-1 on LB solid medium containing mercury ion concentrations of 5 mg / L, 10 mg / L, 15 mg / L, and 20 mg / L was compared. 2+ When the concentration was 5 mg / L, the mercury removal rate of strain BK-1 was the highest; 2+ When the concentration is 5 mg / L, it can still be maintained above 80%, and then gradually decreases with the increase of ion concentration. Figure 5 As shown in B-5C, in Hg 2+ When the concentration was 10 mg / L, the bacteria were collected and observed under an electron microscope. It was found that in the presence of mercury ions, the bacteria would shrink and become concave.

[0062] Example 6 Extraction of bacterial RNA from strain BK-1 and real-time fluorescence quantitative PCR

[0063] 1. Experimental Methods

[0064] Bacterial RNA was extracted using a column-type bacterial total RNA extraction kit from Sangon Biotech Co., Ltd., catalog number: B518655-0050. For detailed operation steps, see the kit manual. cDNA preparation: A reverse transcription kit from Takara Biotech Co., Ltd. was used. RTMasterMix (TaKaRa, Japan) was used to reverse transcribe bacterial RNA. The specific method is as follows:

[0065] 1) gDNA removal reaction

[0066] Among them, the reaction system of the removal reaction is shown in the following table:

[0067]

[0068] After reacting at 37°C for 20 min, 2.5 μL of 0.5 mmol / L EDTA was added and reacted at 80°C for 2 min, and then the volume was fixed to 100 μL with RNase-free ddH2O.

[0069] 2) RT reaction

[0070] Among them, the reaction system of RT reaction is shown in the following table:

[0071]

[0072] The reaction conditions of RT reaction are shown in the following table:

[0073]

[0074] Real-time quantitative PCR:

[0075] Real-time fluorescence quantification was performed using the SYBR Green dye method. The total reaction system was 25 μL, cDNA 2 μL, PrimerF and PrimerR 1 μL each, 2×SYBR Premix Ex TaqTMⅡ 12.5 μL, and ddH2O 2.5 μL. The reaction program was: 95°C / 30 sec, 95°C / 5 sec, 60°C / 30 sec, 40 cycles. The housekeeping gene of 16S rRNA was used as the internal reference gene, with 3 biological replicates and 3 or more technical replicates.

[0076] 2. Mercury reductase gene analysis

[0077] The mer operon includes genes such as merA, merB, merC, merD, merP, merR and merT. The proteins encoded by these genes are 2+ The mercury reductase encoded by the merA gene plays a leading role in the conversion of Hg 2+ Reduction to Hg 0The other genes encode auxiliary activation proteins and transport proteins, etc. Therefore, this study mainly determines whether the mercury-resistant strains have Hg by detecting the merA gene. 2+ Restore process.

[0078] like Figure 6 As shown in Figure 2, the merA gene was detected in strain BK-1, which indicates that it has the ability to transform Hg 2+ process.

[0079] 3. Response of key genes for mercury reduction in mercury-resistant strains

[0080] like Figure 7 As shown, in strain BK-1, the expression level of merR gene was upregulated at 2h, indicating that merR gene started the expression of mer operator in this time period, and the expression level was downregulated at 6-12h, and the upregulation increased again at 18h, showing the phenomenon of secondary expression; the expression level of merC gene continued to increase within 2-12h, and began to decrease at 18h, indicating that merC gene can transport Hg(Ⅱ) for a long time and is the key gene for transporting Hg(Ⅱ); the expression level of merT gene was the highest at 2h, and the expression level was upregulated again at 6-18h, but the increase was reduced; the expression level of merA gene began to increase at 12h, and decreased after 18h.

[0081] It can be seen that the mercury reductase gene merA was detected in strain BK-1, indicating that strain BK-1 has the ability to reduce mercury; in addition, the results of fluorescent real-time quantitative PCR showed that the genes of the mer operon 2+ The mer operon was expressed successively during the reduction process of Hg 2+ played an important role in the process.

[0082] Example 7 Effect of inoculation with strain BK-1 on mercury accumulation in various organs of rice varieties with different mercury tolerance

[0083] Indica rice variety N1311r and japonica rice variety WN were selected as materials for potted experiments. The strain BK-1 was inoculated after 5 mg / L exogenous mercury was added to study the effect of strain BK-1 on the mercury accumulation in various organs of different mercury-tolerant varieties.

[0084] The results are as follows Figure 8As shown in A-8B, in the rice roots, the total mercury contents of N1311R1 and WN were 31.97 mg / L and 28.52 mg / L when not inoculated, and decreased to 27.92 mg / L and 19.80 mg / L respectively after inoculation with strain BK-1; in the first stem node, the total mercury contents of N1311R1 and WN were 7.46 mg / L and 5.52 mg / L when not inoculated, and decreased to 19.80 mg / L after inoculation with strain BK-1. In the leaves, the total mercury content of WN was 13.03 mg / L when not inoculated, and it decreased to 12.07 mg / L after inoculation with strain BK-1; in the grains, the total mercury contents of N1311R1 and WN were 0.04 mg / L and 0.03 mg / L when not inoculated, and they decreased to 0.03 mg / L and 0.02 mg / L respectively after inoculation with strain BK-1.

[0085] It can be seen that the inoculated strain BK-1 can reduce the accumulation of mercury in various organs of rice varieties with different mercury tolerance.

[0086] In summary, the strain BK-1 provided by the present disclosure not only has the ability to reduce mercury, but also has the ability to solubilize phosphate and produce siderophores, and thus can reduce the accumulation of heavy metals (eg, mercury) in crops (eg, rice).

Claims

1. A Burkholderia sp. BK-1, characterized in that The Burkholderia was deposited in the China Center for Type Culture Collection on September 30, 2024, and its preservation number is CCTCC NO:M 20242135.

2. A bacterial agent comprising the Burkholderia BK-1 as claimed in claim 1.

3. Use of the Burkholderia BK-1 as claimed in claim 1 or the bacterial agent as claimed in claim 2 in reducing the accumulation of heavy metals in crops.

4. The use according to claim 3, characterized in that: The crops include rice.

5. The use according to claim 3, characterized in that: The heavy metals include mercury.

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