Method for determining colonization of endophyte Bacillus proteolyticus in soybeans

Abstract

The invention relates to the field of microorganisms and molecular biology, in particular to a method for determining the colonization condition of endophytes in soybeans by combining a molecular biological marker technology of the endophytes Bacillus proteolyticus and colonization dynamic detection. The method comprises the following steps: firstly, carrying out electrotransfection on endophyte Bacillus proteolyticus into plasmids with Amp resistance tags, infecting soybean seeds with constructed strains, diluting tissue fluid of each tissue part of a soybean plant, coating the diluted tissue fluid in a solid culture medium with Amp resistance, and judging whether a bacterial colony exists in the culture medium or not and verifying 16S rDNA (ribosomal deoxyribonucleic acid), according to the present invention, with the technology, the colonization condition of the endophyte Bacillus proteolyticus in the soybean can be determined, and the determination of the colonization condition of the endophyte Bacillus proteolyticus in the soybean can be provided, such that the important significance can be provided for the mastering of the colonization condition of the endophyte Bacillus proteolyticus in the soybean.

Description

Technical Field

[0001] The invention relates to the field of microorganisms and molecular biology, and in particular to a method for determining the colonization of endophytic bacteria in soybean by combining molecular biological marker technology of endophytic bacteria Bacillus proteolyticus with colonization dynamic detection. Technical Background

[0002] This technology is mainly used to determine the colonization of endophytes in soybeans, and its background relies on the development of modern microbiology and molecular biology technology. This technology mainly combines molecular biological marker technology with colonization dynamic detection, that is, using antibiotic resistance marker technology to track the colonization of specific endophytes in soybeans, making it possible to specifically detect endophytes in complex plant environments. Through regular sampling, isolation and culture, sequencing analysis and other means, the colonization quantity and distribution of endophytes in different growth stages and different tissue parts of soybeans are monitored. Create conditions for a deep understanding of the interaction between endophytes and soybeans.

[0003] This technology is different from traditional isolation and culture methods and high-throughput sequencing technology. It not only solves the problems that general technologies are limited by culture medium components and culture conditions, cannot fully reflect the diversity of endophytes, and are costly, labor-intensive, and cumbersome, but also solves the problem of molecular biological markers of endophytes Bacillus proteolyticus. Compared with traditional isolation and culture methods, the technology combining molecular biological markers of endophytes Bacillus proteolyticus with colonization dynamic detection can complete the detection in a shorter time, improve the research efficiency, and has the characteristics of fast and high efficiency. Molecular biological technology can accurately identify and locate the genetic markers of endophytes Bacillus proteolyticus, making the detection process highly sensitive and specific, and can accurately reflect the presence of endophytes Bacillus proteolyticus in soybeans. Summary of the invention

[0004] The invention provides a method for determining the colonization of endophyte Bacillus proteolyticus in soybean plants, and establishes a rapid and efficient method for determining the colonization of endophyte Bacillus proteolyticus in soybeans by combining molecular biological marker technology with colonization dynamic detection.

[0005] To achieve the above objectives, a rapid and efficient method for determining the colonization of endophyte Bacillus proteolyticus in soybeans was established by combining sub-molecular biological marker technology with colonization dynamic detection, which is characterized by comprising the following steps:

[0006] The soybean endophyte Bacillus proteolyticus was transformed with an Amp-resistant plasmid to obtain the soybean endophyte Bacillus proteolyticus with biological markers;

[0007] Then, the transformed endophyte Bacillus proteolyticus was used to infect soybean Jiyu 47 seeds and cultured in soil as plant samples, and soybeans infected with endophyte Bacillus proteolyticus without transformation resistance were set as blank controls;

[0008] The roots, stems and leaves of soybean Jiyu 47 in the seedling stage 14 days after sowing were taken as extraction materials and ground to destroy plant cell tissues and release the endophytic bacteria therein for coating observation;

[0009] The extracts from the non-transformed resistant endophyte Bacillus proteolyticus infection and transformed endophyte Bacillus proteolyticus infection were spread on LB medium and LB medium with Amp resistance, respectively, and then cultured for 3 days to detect the colonization of endophyte Bacillus proteolyticus in different parts of soybean seedlings.

[0010] Each experiment was repeated three times to ensure the reliability of the results.

[0011] Wherein, obtaining a plasmid with Amp resistance to transform soybean endophyte Bacillus proteolyticus to obtain soybean endophyte Bacillus proteolyticus with biological markers, the step further includes:

[0012] The strain Bacillus proteolyticus was prepared into competent cells and then transformed with a T vector with Amp resistance by electroporation to obtain an endophytic bacterium Bacillus proteolyticus with Amp resistance. The specific steps are: inoculating soybean endophytic bacterium Bacillus proteolyticus in LB medium, activating it at 37°C and 180rpm for 12 hours. The activated bacterial liquid was inoculated into 100mL LB liquid medium at an inoculum volume of 1% and continued to be cultured, and the OD 600 The bacterial liquid was 0.5-0.6, and centrifuged at 4°C, 8000rpm for 15 minutes to collect the bacteria. After washing three times with pre-cooled 10% glycerol, the supernatant was discarded. The precipitated bacteria were redissolved in 1mL of 10% glycerol to obtain competent cells of endophytic bacteria Bacillus proteolyticus.

[0013] Gently pipette and mix 10 μL of Amp-resistant T vector and 100 μL of endophytic bacteria Bacillus proteolyticus competent cells, then immediately transfer all of them into a pre-cooled 2 mm electrode cup and place on ice for 5 min.

[0014] Run the electroporator, and the parameters of the electroporator are designed as follows: voltage 2400V, 25μF, 200Ω. Take out the electrode cup, wipe off the surface moisture, and put it into the electroporation chamber for electroporation;

[0015] Quickly remove the electrode cup, add 1 mL of SOC medium preheated at 37°C in the clean bench, gently blow the bacterial solution evenly with a pipette, and transfer it to a 1.5 mL centrifuge tube and resuscitate at 30°C, 200 rpm for 2 hours.

[0016] The revived bacterial solution was centrifuged at 4000 rpm for 1 min, the supernatant was discarded, and the bacterial cells were resuspended in 200 μL SOC medium, spread on LB solid medium containing Amp resistance, and cultured at 37° C. for 12 h to obtain soybean endophytic bacteria Bacillus proteolyticus with Amp resistance.

[0017] Wherein, after the constructed endophytes are infected into soybean seeds, the soybean seeds are soil cultured, and the steps further include:

[0018] The endophytic bacteria Bacillus proteolyticus with Amp resistance were inoculated into LB medium with Amp resistance for enrichment, and the OD 600 Soak soybean Jiyu 47 seeds in 0.8% bacterial solution for 1.5h-2h, then sow and culture in soil. The infection method of non-transformation resistant endophyte Bacillus proteolyticus is the same as above.

[0019] The plant sample is ground and its tissue dilution is applied to observe the colonization of endophyte Bacillus proteolyticus, and the step further includes:

[0020] The extracts infected with Bacillus proteolyticus without transformation resistance and those infected with Bacillus proteolyticus after transformation were ground to destroy plant cell tissues, release the endophytic bacteria and spread them on LB medium and LB medium with Amp resistance, respectively, to observe the colonization of endophytic bacteria Bacillus proteolyticus in various parts of soybean plants;

[0021] After cleaning the soil and impurities on the surface of soybean roots, stems and leaves as plant samples with clean water, wash them three times with 99% ethanol, each time for 60 seconds, then disinfect them with 3% NaOH solution for 6 minutes, soak them in 99% ethanol for 30 seconds, and finally rinse them with sterile 0.9% saline for 5 times. The saline after rinsing was applied on solid LB culture medium to verify the sterilization effect.

[0022] The disinfected soybean roots, stems, and leaves were placed in different sterile mortars, 4 mL of sterilized 0.9% saline was added as a diluent, and they were fully ground using a sterile pestle to obtain dilutions containing endophytes from soybean Jiyu 47 roots, stems, and leaves, respectively;

[0023] Dilutions of soybean roots, stems and leaves infected with non-transformed resistant endophyte Bacillus proteolyticus and transformed endophyte Bacillus proteolyticus were spread on LB medium and LB medium with Amp resistance, respectively, and cultured for 3 days. The colonization of endophyte Bacillus proteolyticus on LB medium and LB medium with Amp resistance was detected by 16S rDNA.

[0024] The present technology relates to a determination method combining molecular biological labeling technology and colonization detection of endophytic bacteria Bacillus proteolyticus. First, competent cells of endophytic bacteria Bacillus proteolyticus are prepared, and then plasmids with Amp resistance tags are electrotransfected into competent cells to construct endophytic bacteria Bacillus proteolyticus with Amp resistance. Non-transformation resistant endophytic bacteria Bacillus proteolyticus and transformed endophytic bacteria Bacillus proteolyticus are taken to infect soybean Jiyu 47 seeds respectively, and then the roots, stems and leaves of soybean Jiyu 47 seedlings at 14 days after sowing are taken to prepare dilutions containing soybean roots, stems and leaves infected with non-transformation resistant endophytic bacteria Bacillus proteolyticus and transformed endophytic bacteria Bacillus proteolyticus, and the dilutions are respectively applied to LB medium and LB medium with Amp resistance, and the colonization of endophytic bacteria Bacillus proteolyticus is detected by 16S rDNA. In order to clarify the distribution and colonization of the endophyte Bacillus proteolyticus in the roots, stems and leaves of soybean Jiyu 47 seedlings after infection, this technology determined the colonization of the endophyte Bacillus proteolyticus in soybean, constructed a method for determining endophytes in soybean, and tracked the colonization of specific endophytes in soybean, making it possible to specifically detect endophytes in complex plant environments, laying the foundation for the study of the interaction between endophytes and soybeans, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0026] Figure 1 This is the growth state in which the dilutions of soybean roots, stems and leaves infected with non-transformation resistant endophyte Bacillus proteolyticus are spread on LB medium respectively;

[0027] Figure 2 This is the growth state in which the dilution of soybean roots, stems and leaves infected with non-transformation resistant endophyte Bacillus proteolyticus is spread on LB medium with Amp resistance respectively;

[0028] Figure 3In this technology, the dilution of soybean roots, stems and leaves infected with Amp-resistant endophyte Bacillus proteolyticus is spread on LB medium respectively;

[0029] Figure 4 In this technology, the dilution of soybean roots, stems and leaves infected with Amp-resistant endophyte Bacillus proteolyticus is applied to the growth state of LB medium with Amp resistance respectively; Specific implementation methods

[0030] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0031] Example 1 The colonization of endophyte Bacillus proteolyticus in soybean is determined by combining molecular biological marker technology with colonization dynamic detection, comprising the following steps:

[0032] 1. Prepare the strain Bacillus proteolyticus into competent cells and transform them with the T vector with Amp resistance through an electroporator to obtain the endophytic bacteria Bacillus proteolyticus with Amp resistance;

[0033] The soybean endophyte Bacillus proteolyticus was inoculated into LB medium and activated at 37°C and 180 rpm for 12 hours. The activated bacterial solution was inoculated into 100 mL LB liquid medium at a 1% inoculum and continued to be cultured. The OD 600 The bacterial solution was 0.5-0.6, and centrifuged at 4°C, 8000rpm for 15min to collect the bacteria. After washing with pre-cooled 10% glycerol three times, the supernatant was discarded, and the precipitated bacteria were redissolved in 1mL of 10% glycerol to obtain the competent cells of endophytic bacteria Bacillusproteolyticus. The endophytic bacteria Bacillusproteolyticus with Amp resistance were constructed by electroporation transformation;

[0034] Gently pipette and mix 10 μL of Amp-resistant T vector and 100 μL of Bacillus proteolyticus competent cells, then transfer all of them into a pre-cooled 2 mm electrode cup and place them on ice for 5 min.

[0035] Run the electroporator. The electroporator parameters are designed as follows: voltage 2400V, 25μF, 200Ω. Take out the electrode cup, wipe off the surface moisture, and put it into the electroporator chamber for electroporation:

[0036] Quickly remove the electrode cup, add 1 mL of SOC medium preheated at 37°C in the clean bench, gently blow the bacterial solution with a pipette, and transfer it to a 1.5 mL centrifuge tube, and resuscitate at 30°C and 200 rpm for 2 hours;

[0037] The revived bacterial solution was centrifuged at 4000 rpm for 1 min, the supernatant was discarded, and the bacterial cells were resuspended in 200 μL SOC medium, spread on LB solid medium containing Amp resistance, and cultured at 37° C. for 12 h to obtain soybean endophytic bacteria Bacillus proteolyticus with Amp resistance.

[0038] 2. After the constructed endophytes are infected into soybean seeds, the soybean seeds are planted in soil;

[0039] The endophytic bacteria Bacillus proteolyticus with Amp resistance were inoculated into LB medium with Amp resistance for enrichment, and the OD 600 Soak soybean Jiyu 47 seeds in 0.8% bacterial solution for 1.5h-2h, then sow and culture in soil. The infection method of non-transformation resistant endophyte Bacillus proteolyticus is the same as above.

[0040] 3. Grind the plant samples and spread their tissue dilutions to observe the colonization of the endophyte Bacillus proteolyticus;

[0041] After cleaning the soil and impurities on the surface of soybean roots, stems and leaves as plant samples with clean water, wash them three times with 99% ethanol, each time for 60 seconds, then disinfect them with 3% NaOH solution for 6 minutes, soak them in 99% ethanol for 30 seconds, and finally rinse them with sterile 0.9% saline for 5 times. The saline after rinsing was applied on solid LB culture medium to verify the sterilization effect.

[0042] The disinfected soybean roots, stems, and leaves were placed in different sterile mortars, 4 mL of sterilized 0.9% saline was added as a diluent, and they were fully ground using a sterile pestle to obtain dilutions containing endophytes from soybean Jiyu 47 roots, stems, and leaves, respectively;

[0043] Dilutions of soybean roots, stems and leaves infected with non-transformed resistant endophyte Bacillus proteolyticus and transformed endophyte Bacillus proteolyticus were spread on LB medium and LB medium with Amp resistance, respectively, and cultured for 3 days. The colonization of endophyte Bacillus proteolyticus on LB medium and LB medium with Amp resistance was detected by 16S rDNA.

[0044] Each experiment was repeated three times, which reduced the chance of a single experimental result and made the results more convincing.

[0045] What is disclosed above is only one or more preferred embodiments of the present application, and cannot be used to limit the scope of rights of the present application. Ordinary technicians in this field can understand that all or part of the processes of implementing the above embodiments and equivalent changes made according to the claims of the present application are still within the scope covered by the present application.

Claims

1. A rapid and efficient method for determining the colonization of endophyte Bacillus proteolyticus in soybeans was established by combining sub-biological marker technology with colonization dynamic detection, characterized in that: The following steps are involved: The soybean endophyte Bacillus proteolyticus was transformed with an Amp-resistant plasmid to obtain the soybean endophyte Bacillus proteolyticus with biological markers; Then, the transformed endophyte Bacillus proteolyticus was used to infect soybean Jiyu 47 seeds and cultured in soil as plant samples, and soybeans infected with endophyte Bacillus proteolyticus without transformation resistance were set as blank controls; The roots, stems and leaves of soybean Jiyu 47 in the seedling stage 14 days after sowing were taken as extraction materials and ground to destroy the plant cell tissues and release the endophytic bacteria therein for coating observation; The extracts from the non-transformed resistant endophyte Bacillus proteolyticus infection and transformed endophyte Bacillus proteolyticus infection were spread on LB medium and LB medium with Amp resistance, respectively, and then cultured for 3 days to detect the colonization of endophyte Bacillus proteolyticus in different parts of soybean seedlings. Each experiment was repeated three times to ensure the reliability of the results.

2. The method for determining the colonization of soybean endophytes in soybeans according to claim 1, characterized in that: The soybean endophyte Bacillus proteolyticus is transformed by using a plasmid having Amp resistance to obtain the soybean endophyte Bacillus proteolyticus with a biological marker, and the step further includes: The strain Bacillus proteolyticus was prepared into competent cells and then transformed with a T vector with Amp resistance by electroporation to obtain an endophytic bacterium Bacillus proteolyticus with Amp resistance. The specific steps are: inoculating soybean endophytic bacterium Bacillus proteolyticus in LB medium, activating it at 37°C and 180rpm for 12 hours. The activated bacterial liquid was inoculated into 100mL LB liquid medium at an inoculum volume of 1% and continued to be cultured, and the OD 600 The bacterial solution was 0.5-0.6, and centrifuged at 4°C, 8000rpm for 15min to collect the bacteria. After washing 3 times with pre-cooled 10% glycerol, the supernatant was discarded. The precipitated bacteria were redissolved in 1mL of 10% glycerol to obtain competent cells of endophytic bacteria Bacillus proteolyticus; Gently pipette and mix 10 μL of Amp-resistant T vector and 100 μL of Bacillus proteolyticus competent cells, then transfer all of them into a pre-cooled 2 mm electrode cup and place them on ice for 5 min. Run the electroporator. The electroporator parameters are designed as follows: voltage 2400V, 25μF, 200Ω. Take out the electrode cup, wipe off the surface moisture, and put it into the electroporator chamber for electroporation; Quickly remove the electrode cup, add 1 mL of SOC medium preheated at 37°C in the clean bench, gently blow the bacterial solution with a pipette, and transfer it to a 1.5 mL centrifuge tube, and resuscitate at 30°C and 200 rpm for 2 hours; The revived bacterial solution was centrifuged at 4000 rpm for 1 min, the supernatant was discarded, and the bacterial cells were resuspended in 200 μL SOC medium, spread on LB solid medium containing Amp resistance, and cultured at 37°C for 12 h to obtain soybean endophytic bacteria Bacillus proteolyticus with Amp resistance.

3. The method for determining the colonization of soybean endophytes in soybeans according to claim 2, characterized in that: The endophytic bacteria Bacillus proteolyticus with Amp resistance were inoculated into LB medium with Amp resistance for enrichment, and the OD 600 Soak soybean Jiyu 47 seeds in 0.8% bacterial solution for 1.5h-2h, then sow and culture in soil. The infection method of non-transformation resistant endophyte Bacillus proteolyticus is the same as above.

4. The method for determining the colonization of soybean endophytes in soybeans according to claim 3, characterized in that: Planting soybean seeds with Amp resistance in saline-alkali soil for cultivation and observation and serving as subsequent plant samples, the steps further include: The extract materials infected with non-transformed resistant endophyte Bacillus proteolyticus and transformed endophyte Bacillus proteolyticus were ground to destroy plant cell tissues, release the endophytic bacteria and spread them on LB medium and LB medium with Amp resistance, respectively, to observe the colonization of endophyte Bacillus proteolyticus in various parts of soybean plants.

5. The method for determining the colonization of soybean endophytes in soybeans according to claim 4, characterized in that: The plant samples in the saline-alkali soil are ground to destroy the plant cell tissue, release the endophytic bacteria therein and apply the culture medium for observation, and the steps also include: cleaning the soil and impurities on the surface of the soybean roots, stems and leaves of the plant samples with clean water, washing them three times with 99% ethanol, each time for 60 seconds, and then disinfecting and soaking them with 3% NaOH solution for 6 minutes, soaking them in 99% ethanol for 30 seconds, and finally washing them with sterilized 0.9% saline for 5 times, and applying the washed saline on the solid LB culture medium to verify the sterilization effect; The disinfected soybean roots, stems and leaves were placed in different sterile mortars, 4 mL of sterile 0.9% saline was added as a diluent, and they were fully ground using a sterile pestle to obtain dilutions containing endophytes from soybean Jiyu 47 roots, stems and leaves.

6. The method for determining the colonization of soybean endophytes in soybeans according to claim 5, characterized in that: The plant samples in the saline-alkali soil are ground to destroy the plant cell tissues, release the endophytic bacteria therein, and apply the culture medium for observation. The step also includes: taking the dilution liquid of soybean roots, stems and leaves infected with the endophytic bacteria Bacillus proteolyticus without transformation resistance and the endophytic bacteria Bacillus proteolyticus after transformation, respectively, applying them on LB culture medium and LB culture medium with Amp resistance, culturing for 3 days, and performing 16S rDNA detection on the colonization of the endophytic bacteria Bacillus proteolyticus on the LB culture medium and the LB culture medium with Amp resistance.

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