Paenibacillus strain and application thereof

Abstract

The invention provides a paenibacillus strain which is named as Paenibacillus sp. And is preserved in the Guangdong Microbial Culture Collection Center, the preservation number is GDMCC No: 65116, and the preservation date is September 12, 2024. The paenibacillus strain is named as Paenibacillus sp. And is preserved in the Guangdong Microbial Culture Collection Center, the preservation number is GDMCC No: 65116. The nucleotide sequence of the 16S rRNA is as shown in SEQ ID NO. 1. The invention also provides an application of the bacillus strain, which is used for promoting the growth of Shanghai green, improving the nutritional quality and enhancing the enrichment of selenium. The strain has the function of producing ACC deaminase, and has obvious effects of promoting the growth of Shanghai green, improving the nutritional quality and strengthening selenium enrichment.

Description

Technical Field

[0001] The invention belongs to the technical field of Paenibacillus, and particularly relates to a Paenibacillus strain and application thereof. Background Art

[0002] Selenium (Se) is one of the essential trace nutrients for humans and animals. It is the active center of selenoenzymes and selenoproteins and plays an important role in energy metabolism and gene expression in organisms. Selenium has many important biological functions, such as antioxidant effect, immunomodulatory function, anti-tumor activity, antagonism of heavy metals, and regulation of protein synthesis. Excessive intake of selenium can lead to health problems such as hair loss, nail loss, skin damage, and nervous system disorders, while insufficient intake of selenium can also pose a greater threat to human health, such as Kashin-Beck disease, Keshan disease, malignant tumors, and decreased immunity. my country has more than 700 million people with varying degrees of insufficient selenium intake, and is internationally recognized as one of the "large countries with selenium deficiency". According to a survey by the Chinese Nutrition Society, the selenium intake of the Chinese population is generally low, at 26-32μg / d, far below 50-250μg / d. Therefore, taking enough selenium every day can maintain human health, prevent diseases, and slow down the aging process.

[0003] The human body mainly ingests selenium through diet, and plant selenium is the main source of selenium intake. In view of the importance of selenium to human health and to make up for the insufficient per capita selenium intake, it is imperative to increase the selenium content of vegetables and crops through the biofortification of selenium. There are many kinds of beneficial microorganisms, which play a vital role in promoting plant growth, nutrient absorption, heavy metal repair and disease control. Studies have shown that inoculation of selenium-fortified microorganisms can effectively improve the absorption of selenium by plants.

[0004] Shanghai green (Brassica rapa var.chinensis) is a variant of the Brassica rapa subspecies of the Brassica family, commonly known as Chinese cabbage or green vegetables. It is highly favored because of its rich nutrition, fast growth, wide adaptability, and low cost. It has become a popular vegetable in the world. It also has a certain ability to absorb selenium and is a secondary selenium-accumulating plant. Therefore, it is feasible to enrich selenium in 'Shanghai green' through functional microorganisms, which can alleviate the current situation of selenium deficiency to a certain extent. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a Bacillus strain and its application in view of the deficiencies of the above-mentioned prior art, which has the function of producing ACC deaminase activity, can promote the growth of 'Shanghai Green', improve the nutritional quality of 'Shanghai Green' and enhance the selenium enrichment of 'Shanghai Green'.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is: a Paenibacillus sp. strain, the Paenibacillus sp. strain is named Paenibacillus sp., deposited in Guangdong Microbiological Culture Collection Center, with a deposit number of GDMCC No: 65116 and a deposit date of September 12, 2024; the nucleotide sequence of 16S rRNA of the Paenibacillus sp. is shown in SEQ ID NO.1.

[0007] The present invention also provides the application of the above-mentioned Paenibacillus strain, wherein the Paenibacillus strain is used to promote the growth of 'Shanghai Green', improve the nutritional quality, and enhance the selenium enrichment.

[0008] Preferably, the Paenibacillus strain is used to increase the aboveground dry weight of 'Shanghai Qing', increase the Vc content, and increase the total protein content.

[0009] Preferably, the growth rate of the aboveground dry weight of the 'Shanghai Qing' is 16.57% to 31.89%, the growth rate of the Vc content is 31.58% to 91.67%, and the growth rate of the total protein content is 37.89% to 45.16%.

[0010] Preferably, when the Bacillus strain is used to enhance selenium enrichment in Shanghai Green, when the Shanghai Green is cultivated in the field, the growth rate of selenium content is 12.41%, and when the Shanghai Green is cultivated in hydroponics, the growth rate of selenium content is 25.44%.

[0011] Compared with the prior art, the present invention has the following advantages:

[0012] The Paenibacillus sp. of the invention has the function of producing ACC deaminase activity, can promote the growth of 'Shanghai Qing', improve the nutritional quality of 'Shanghai Qing' and strengthen the selenium enrichment of 'Shanghai Qing'.

[0013] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a sampling point diagram of the source of strain ES5-4 in Example 1 of the present invention.

[0015] Figure 2 This is a diagram of the dominant plant from which strain ES5-4 in Example 1 of the present invention is derived.

[0016] Figure 3 It is a colony diagram of strain ES5-4 and other selenium-resistant strains in Example 1 of the present invention.

[0017] Figure 4This is a colony morphology diagram of Paenibacillus sp.ES5-4 in Example 1 of the present invention.

[0018] Figure 5 This is the Gram staining picture of Paenibacillus sp.ES5-4 in Example 1 of the present invention.

[0019] Figure 6 It is a scanning electron microscope image of Paenibacillus sp.ES5-4 in Example 1 of the present invention.

[0020] Figure 7 It is a transmission electron micrograph of Paenibacillus sp.ES5-4 in Example 1 of the present invention.

[0021] Figure 8 It is a systematic evolutionary diagram of the Paenibacillus sp.ES5-416S rRNA gene in Example 1 of the present invention.

[0022] Fig. 9 This is a graph showing the polar lipid determination of Paenibacillus sp. ES5-4 in Example 1 of the present invention.

[0023] Fig.10 This is a graph showing the ANI value of Paenibacillus sp.ES5-4 in Example 1 of the present invention.

[0024] Fig.11 This is a cluster diagram of the protein sequence of Paenibacillus sp.ES5-4 bac120 in Example 1 of the present invention.

[0025] Fig.12 The growth conditions of the strain Paenibacillus sp. in Example 2 of the present invention at different temperatures.

[0026] Fig.13 This is the standard curve of ACC deaminase activity produced by the strain Paenibacillus sp. in Example 3 of the present invention.

[0027] Fig.14 This is the effect of the strain Paenibacillus sp. in Example 4 of the present invention on the aboveground biomass of 'Shanghaiqing'. (* indicates a significance of P < 0.05)

[0028] Fig.15 This is the effect of the strain Paenibacillus sp. in Example 5 of the present invention on the nutritional quality of 'Shanghai Qing'. (* indicates a significance of P < 0.05)

[0029] Fig.16 This is the effect of the strain Paenibacillus sp. on selenium enrichment in 'Shanghai Qing' in Example 6 of the present invention. (* indicates significance of P < 0.05) DETAILED DESCRIPTION

[0030] Example 1

[0031] This example is the separation and identification of Paenibacillus sp.ES5-4

[0032] 1. Isolation of Paenibacillus sp. ES5-4

[0033] From five sampling points near the natural selenium mining area of ​​Yutangba, Enshi Prefecture, Hubei Province, namely ES1-ES5, the sampling points are as follows Figure 1 The dominant plants (ES1-ES5 are located at 30°10′56″N 109°47′5″E, 30°10′34″N 109°47′35″E, 30°9′42″N 109°48′9″E, 30°9′16″N 109°48′29″E, 30°8′43″N 109°49′32″E) are shown in Table 1. Figure 2 ) were collected from rhizosphere soil, Figure 2 In the middle, a, b, c, d, and e are Veronica persica, Thlaspi rotundifolium, Artemisia argyi, Brassica campestrisssp.chinensis Makino, and Galinsoga parviflora Cav., respectively. ES5-4 was isolated from the rhizosphere soil of Galinsoga parviflora. The loose soil between the roots and on the surface of the sample plants was removed, and the soil tightly attached to the rhizosphere was taken as the rhizosphere soil. The collected rhizosphere soil was placed in a sterile bag, stored in a low temperature environment, and transported back to the laboratory as soon as possible for subsequent experiments.

[0034] Weigh 10 g of sample soil and place it in a 250 mL conical flask filled with 90 mL of sterile water. Elute the soil bacteria on a magnetic stirrer and shake for 30 min. After the elution stops, let the resulting eluate stand for 20 min, draw the supernatant and dilute it in a 10-fold gradient. The concentrations of the diluted solutions are 10 -2 , 10 -3 , 10 -4 and 10 -5 The concentration was selected as 10 -3 , 10 -4The dilutions were spread on LB solid medium with a selenium concentration of 10 mg / L, and repeated 3 times. After the colonies grew, they were transferred 3 times to obtain pure cultures. The plate streak method was used to streak and inoculate on solid medium with increasing selenium concentrations (20 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 500 mg / L, 1000 mg / L, 2000 mg / L) for cultivation. The growth of rhizosphere bacteria was observed, and colonies with poor selenium tolerance were gradually eliminated. 47 strains with strong selenium tolerance (2000 mg / L) were selected ( Figure 3 ES5-4 is one of the strains, and a single colony of pure cultured ES5-4 strain was obtained, which was isolated from the rhizosphere soil of Galinsoga parviflora Cav. at the fifth sampling point ES5.

[0035] 2. Identification of Paenibacillus sp. ES5-4

[0036] (1) Morphological identification of Paenibacillus sp. ES5-4

[0037] The strain ES5-4 was cultured on LB solid medium, transferred 3 times, cultured at 28°C for 2 days, and the colony morphology was observed. Figure 4 As shown in the figure, the colonies of ES5-4 are round, 2-3 mm in size, with a moist, smooth and neat surface and a white color. The strain is purplish after Gram staining, indicating that ES5-4 is a Gram-positive bacterium. Figure 5 Scanning electron microscopy and transmission electron microscopy analysis showed that ES5-4 is rod-shaped ( Figure 6 ), with flagella ( Figure 7 ), can exercise.

[0038] (2) Systematic identification of Paenibacillus sp.ES5-4

[0039] According to the instructions of the DNA extraction kit of Shanghai Bioengineering Co., Ltd., DNA of ES5-4 strain was extracted and diluted for use. Universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-AAGGAGGTGATCCAGCCGCA-3′) were used for 16S rRNA gene amplification. The reaction system was 25 μL system: Taq PCR Master Mix 12.5 μL, primer 27F 1 μL, primer 1492R 1 μL, DNA template 0.5 μL, ddH2O 10 μL. The amplification conditions were: 95°C pre-denaturation for 4 min, 94°C denaturation for 30 s, 55°C annealing for 40 s, 72°C extension for 30 s, 32 cycles, 72°C extension for 10 min, and storage at 4°C.

[0040] After the PCR product was qualified by 1% agarose gel electrophoresis, it was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The result is shown in SEQ ID NO.1. The sequencing result was submitted to the EzBioCloud database for homology comparison. The results showed that ES5-4 was similar to the model strain Paenibacillus pabuli NBRC 13638. T and Paenibacillus dongdonensis KUDC0114 T The 16S rRNA gene sequences of the two genes have the highest similarity, which are 98.43% and 98.32%, respectively. The phylogenetic tree was constructed using ClustalX8.1 and MEGA5.1. Figure 8 The results showed that the ES5-4 strain was similar to Paenibacillus pabuli NBRC 13638 T and Paenibacillus dongdonensisKUDC0114 T In the phylogenetic tree, they are all located in Paenibacillus sp., but they form a separate branch. Therefore, it is speculated that this strain may be a new species of the genus Paenibacillus.

[0041] 3. Biochemical characteristics of Paenibacillus sp.ES5-4

[0042] The ES5-4 strain and its evolutionarily closely related model reference strain Paenibacillus oceanisediminis JCM17814 were tested according to the instructions of the Biolog GEN III microtest system (Biolog, USA). Tand Paenibacillusdongdonensis KCTC33221 T The carbon source utilization and chemical sensitivity of ES5-4 were tested. The results are shown in Table 1: ES5-4 can use 16 substances such as α-D-glucose, D-mannose, D-fructose, D-galactose, D-fucose, L-glutamic acid, L-serine, D-galacturonic acid, L-galacturonolactone, D-gluconic acid, D-glucuronic acid, glucuronamide, mucic acid, quinic acid, citric acid, and L-malic acid as the sole carbon source; it is acid-resistant (pH=6), salt-resistant (4%), resistant to 1% sodium lactate, fusidic acid, D-serine, troleandomycin, rifamycin SV, lincomycin, sodium tetradecyl sulfate, vancomycin, tetrazolium violet, tetrazolium blue, nalidixic acid, lithium chloride, potassium tellurite, aztreonam and other substances, which provides a basis for the production and application of this strain. Its performance is different from that of the reference strains JCM17814 and KCTC33221.

[0043] Table 1 Biolog GEN III test results

[0044]

[0045]

[0046]

[0047] Note: “+”, positive; “-”, negative

[0048] 4. Chemical classification characteristics of Paenibacillus sp.ES5-4

[0049] Quinone component determination: strain ES5-4 and reference strain KCTC33221 were tested using the FMIC-QO01-008-2015 microbiological detection high performance liquid chromatography (HPLC) method. T The results showed that the respiratory quinone components of ES5-4 and the reference strain were both MK-7.

[0050] Polar lipid determination: The FMIC-QO01-008-2015 microbiological detection of microbial cell wall chemical composition thin layer chromatography (TLC) detection method was used to detect the ES5-4 strain and the reference strain JCM17814. T The polar lipids of strain ES5-4 were determined. The results showed that the polar lipids of strain ES5-4 included diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and two unknown phospholipids; JCM17814 TThe main polar lipids include diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and an unknown phospholipid (PL) ( Fig. 9 ).

[0051] 5. Calculation of ANI and dDDH values ​​of Paenibacillus sp.ES5-4

[0052] The strain ES5-4 was cultured in LB liquid medium at 28°C to the logarithmic phase, centrifuged, and the cells were collected. The collected cells were sent to Shanghai Paisono Biotechnology Co., Ltd. for whole genome sequencing. The reads of each data set were filtered, and the high-quality data sets were assembled using the SPAdes 3.13.0 version in the UGENE software package to obtain the ES5-4 whole genome. The genome sequence was uploaded to the NCBI (https: / / www.ncbi.nlm.nih.gov / ) database, and the relevant information is: BioProject: PRJNA1020054; BioSample ID: SAMN37505765; GenBank accession number: GCA_032334105.2. The results showed that the genome size of strain ES5-4 was 7.2Mb, and the G+C content was 50.5%.

[0053] The online analysis tools https: / / ggdc.dsmz.de / ggdc.php# and https: / / www.ezbiocloud.net / tools / ani were used to calculate the relative abundance of strain ES5-4 and four similar typical strains (Paenibacillusillinoisensis NBRC 15959 T , Paenibacillus pabuli NBRC 13638 T , Paenibacillus xylanivorans A59 T , Paenibacillus taichungensis DSM 19942 T ) digital DNA-DNA hybridization (dDDH) values ​​and average nucleotide identity (ANI) of ES5-4. The results showed that the dDDH values ​​of ES5-4 and the above four typical strains were 21.60%, 21.20%, 20.60% and 20.90%, respectively, all lower than the threshold of 70% for the same species; the ANI values ​​were 76.82%, 76.41%, 76.30% and 76.19%, respectively, all lower than the threshold of 95% ( Fig.10 The low levels of dDDH and ANI values ​​provide more sufficient support for ES504 as a new species.

[0054] 6. Phylogenetic analysis of protein sequences based on bac120

[0055] The bac120 gene set based on multi-site protein sequence analysis can be used to infer a superphylogeny, which includes common single-copy genes in 120 bacterial domains. This tree based on protein sequence analysis is called the "bac120 tree". The gene sequences of each gene cluster were aligned according to Clustal Omega version 1.2.1. After aligning, trimming and splicing the common genes of the bac120 set in all genomes of interest, the maximum likelihood tree was inferred using FastTree 2.1. The results are shown in Fig.11 As shown, systematic analysis of the bac120 protein sequence further confirmed that ES5-4 belonged to the genus Paenibacillus sp.

[0056] In summary, through the phylogenetic tree analysis of the 16S rRNA gene of the strain ES5-4, the whole genome bac120 evolution analysis, the physiological and biochemical and chemical classification analysis, and the calculation of the ANI and dDDH values, it was proved that ES5-4 is a new species of the genus Paenibacillus sp., and was named Paenibacillus sp., which is preserved in the Guangdong Microbial Culture Collection Center, the address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Biology, Guangdong Academy of Sciences, the preservation number is GDMCC No: 65116, the preservation date is September 12, 2024, the 16S rRNA nucleotide sequence is shown in SEQID NO.1, and the GenBank accession number is GCA_032334105.2. The discovery of this strain further enriches the resources of beneficial bacteria.

[0057] Example 2

[0058] This example is an analysis of the growth characteristics of Paenibacillus sp. ES5-4 (i.e., Paenibacillus sp.)

[0059] 1. Effect of temperature conditions on the growth of Paenibacillus sp.ES5-4

[0060] Prepare LB solid medium (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 15g / L), natural pH, sterilize, and make plates. Draw a line on the bottom of the plate and divide the plate into three as replicates. Transfer the strain ES5-4 to the plate and culture the plates at different temperatures (4℃, 10℃, 20℃, 28℃, 37℃ and 42℃). After 3 days of cultivation, take photos to record the growth of ES5-4 and observe the effect of temperature on its growth. The results are as follows Fig.12 As shown, ES5-4 can grow at all the measured temperatures, but its suitable growth temperature is 20℃~37℃, and the optimum temperature is 28℃.

[0061] 2. Effect of NaCl concentration on the growth of Paenibacillus sp.ES5-4

[0062] Prepare LB liquid culture medium (tryptone 10g / L, yeast extract 5g / L, NaCl concentration depends on the experiment) for use in the experiment, and dispense into 50mL conical flasks, 20mL per flask, set 9 NaCl concentration gradients (0%, 0.5%, 1%, 2%, 3%, 5%, 7%, 10% and 12%), sterilize and set aside. Cultivate ES5-4 normally to the logarithmic phase, inoculate 1% of the inoculum (200μL) into conical flasks containing culture medium with different NaCl concentrations, place at 28℃, 200rpm constant temperature shaking culture, and measure OD after 24 hours. 600 The results are shown in Table 2. ES5-4 grew well in the NaCl concentration range of 0.5%-2%, and the optimal NaCl concentration was 0.5%-1%.

[0063] Table 2 Effect of NaCl concentration on the growth of ES5-4

[0064] NaCl concentration (w / v) <![CDATA[OD 600 ]]> 0％ 0.5721±0.04989d 0.5％ 1.2406±0.01355a 1％ 1.2655±0.00403a 2％ 1.1395±0.00863b 3％ 0.9547±0.04963c 5％ 0.4676±0.02021e 7％ 0.1423±0.01816f 10％ 0.0682±0.01561g 12％ 0.0574±0.00710g

[0065] 3. Effect of pH on the growth of Paenibacillus sp.ES5-4

[0066] Prepare LB liquid medium (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L), adjust the pH of the medium to be used to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 with 0.1mol / L HCl or NaCl solution, and then dispense into 50mL conical flasks, 20mL per flask, sterilize, and set aside. Cultivate ES5-4 normally to the logarithmic phase, inoculate 1% of the inoculum (200μL) into conical flasks containing medium with different pH values, place at 28°C, 200rpm under constant temperature shaking, and measure OD after 24 hours. 600The results are shown in Table 3. ES5-4 can grow at pH values ​​between 5.0 and 10.0, but the optimum pH is between 5.0 and 7.0.

[0067] Table 3 Effect of pH on the growth of ES5-4

[0068] pH <![CDATA[OD 600 ]]> 4.0 0.1357±0.01309d 5.0 1.3923±0.03022a 6.0 1.396±0.02301a 7.0 1.448±0.03483a 8.0 1.3200±0.02359b 9.0 1.2868±0.01455b 10.0 1.065±0.0714c

[0069] Example 3

[0070] This example is an analysis of the activity of ACC deaminase produced by Paenibacillus sp. ES5-4 (i.e., Paenibacillus sp.)

[0071] The strain ES5-4 was inoculated into LB liquid medium and shaken for 24 hours. After the culture was completed, the cells were collected by centrifugation at room temperature and 12000×g for 5 minutes, and then washed and centrifuged 2 to 3 times with 0.1mol / L Tris-HCl buffer (pH=7.5), the supernatant was discarded, and the cells were resuspended in a 15mL centrifuge tube containing 3.8mL DF salt minimal medium (Table 4) and 0.2mL 3mmol / L ACC solution, and shaken for 24 hours. After the culture was completed, the cells were collected by centrifugation, washed and centrifuged once with 0.1mol / L Tris-HCl buffer (pH=7.5), the supernatant was discarded, and the cells were resuspended in 600μL 0.1mol / L Tris-HCl buffer (pH=8.5), and 30μL toluene was added and vortexed for 30s to lyse the cells.

[0072] Table 4DF salt minimal medium

[0073]

[0074] Table 4-1 Liquid A

[0075]

[0076] Table 4-2B solution (iron salt)

[0077] Reagents Dosage <![CDATA[FeSO4·7H2O]]> 100mg <![CDATA[ddH2O]]> Make up to 10mL

[0078] Use a pipette to draw 100 μL of cell disruption solution into a 1.5 mL centrifuge tube, add 10 μL of 0.5 mol / L ACC solution and 100 μL of 0.1 mol / L Tris-HCl buffer (pH = 8.5) and mix thoroughly. After heating in a water bath at 30°C for 30 min, add 1 mL of 0.56 mol / L HCl, centrifuge at 12000 × g for 2 min at room temperature, and use a cell-free suspension and a solution without ACC solution as controls. Take 400 μL of the suspension into a 2 ml centrifuge tube, add 320 μL of 0.56 mol / L HCl and 120 μL of 2,4-dinitrophenylhydrazine, and incubate in a water bath at 30°C for 30 min. Then, add 800 μL of 2 mol / L NaOH to each centrifuge tube, and measure the OD after it stabilizes. 540 The value was used to determine the amount of α-ketobutyric acid produced by the strain secreting ACC deaminase to decompose ACC. Three replicates were set.

[0079] Prepare α-ketobutyric acid solutions with concentrations of 1, 0.8, 0.4, 0.2, 0.1 and 0 mmol / L, and measure OD 540 Values ​​and make a standard curve ( Fig.13 ), the linear equation of the standard curve is Y = 0.6476X-0.0047 (R 2 =0.9994). The results showed that the amount of α-ketobutyric acid produced by strain ES5-4 was 1.10 mmol / L (Table 5). This indicated that strain ES5-4 had the ability to secrete ACC deaminase and had the function of promoting plant growth.

[0080] Table 5 ACC deaminase production by strain ES5-4

[0081] strains <![CDATA[OD 540 Value]]> ACC deaminase (mmol / L) ES5-4 1.7058 1.10

[0082] Example 4

[0083] This example is about the effect of Paenibacillus sp. ES5-4 (ie, Paenibacillus sp.) on the growth of 'Shanghai Qing'.

[0084] (1) Field trials

[0085] Sowing: Select large and full 'Shanghai Green' seeds and grow them in hole trays filled with nutrient soil with appropriate humidity, one seed per hole, and then lightly cover them with a layer of nutrient soil.

[0086] Watering: After sowing, water as needed to keep the nutrient soil moist but not too wet.

[0087] Transplanting: About 45 days after sowing, try to select seedlings of the same size and transplant them to the field. Transplant 3 seedlings per row, and try each row as a repeat. After transplanting, water appropriately. Before transplanting, plow the soil to keep it soft, which is convenient for plant transplanting, weed removal, and plant absorption of soil nutrients.

[0088] Inoculation: Culture strain ES5-4 to logarithmic phase, centrifuge, and resuspend in sterile water. The first inoculation treatment was carried out two weeks after transplanting. Each seedling in the experimental group was inoculated with 10 mL of resuspended bacterial solution on the soil surface around the seedling. Two weeks after the first inoculation, the second inoculation was carried out in the same manner and dosage. The control group was sprayed with sterile water in the same manner and dosage, with 3 replicates and a protection row.

[0089] Harvest: About 2 weeks after the second inoculation, carefully remove the whole plant from the soil (take one seedling from each row), separate the aboveground and underground parts, bring to the laboratory, rinse and absorb water. In addition, take the leaves of the other two plants in each row, weigh the corresponding weight (0.1 or 0.2 g) with a 1 / 10,000 balance in a 2mL centrifuge tube, place it in liquid nitrogen, bring it back to the laboratory and place it in a -80℃ refrigerator for the determination of the nutritional quality (Vc and total protein) of pakchoi.

[0090] Drying samples: Place the aboveground and underground plant samples in an oven, sterilize at 105℃ for 15min, bake at 70℃ for 72h until constant weight, and weigh to analyze the dry weight of aboveground biomass. Grind the samples, pass through a 100-mesh sieve, dry and store at room temperature for later determination and analysis of the total selenium content in the aboveground part of 'Shanghai Qing'.

[0091] The field experiment was conducted in the agricultural experimental field of Yichun University. The physical and chemical properties of the tested soil are shown in Table 6.

[0092] Table 6 Physical and chemical properties of soil tested in field experiment

[0093]

[0094] (2) Potted plant test

[0095] The same field test sowing and seedling raising, after sowing for about one month, select seedlings of the same size, transplant them into flower pots, and transplant 3 seedlings per pot. Before transplanting, the flower pot soil is turned over, pesticides are applied, and exposed to the sun to kill eggs, pests, etc. The strain ES5-4 is cultured to the logarithmic phase, centrifuged, and resuspended in sterile water. The first inoculation treatment is carried out two weeks after transplanting, and 30mL (i.e., 10mL per pot) of the experimental group is sprayed with resuspended bacterial solution on the soil surface around the seedling. Two weeks after the first inoculation, the resuspended bacterial solution is sprayed in the same way and dosage for the second inoculation. With sterile water treatment as a control, 3 repetitions are set, and the flower pots are randomly placed in the greenhouse. Two weeks after the second inoculation, the same field test method is sampled, that is, one plant is used to determine the aboveground dry weight, and the leaves of the other two plants are accurately weighed to determine Vc and total protein content.

[0096] The pot experiment was carried out in the greenhouse of the agricultural base of Yichun University. The physical and chemical properties of the test soil are shown in Table 7.

[0097] Table 7 Physical and chemical properties of soil tested in potted plant experiment

[0098]

[0099]

[0100] (3) Hydroponic test

[0101] The same method as (1) and (2) was used for sowing and seedling raising. About 20 days after sowing, the seedlings of the same size were gently removed from the plug tray, the roots were rinsed with tap water, and transplanted into the Hoagland nutrient solution. Five seedlings were transplanted per pot, and the nutrient solution was changed every 3 days. A resuspended bacterial solution was prepared for standby use. One week after transplanting, an inoculation experiment was conducted by dipping the roots for half an hour; one week later, a second inoculation was conducted, in which the prepared resuspended bacterial solution was added to the nutrient solution, 10 mL of bacterial solution per pot, and the nutrient solution was not changed during the inoculation. One week later, sampling was performed. Two seedlings were taken for determination of the aboveground biomass dry weight and total selenium content, and the leaves of the remaining plants were accurately weighed for determination of Vc and total protein content.

[0102] The effects of strain ES5-4 on the dry weight of aboveground biomass of 'Shanghaiqing' were studied by field, potted and hydroponic experiments. Fig.14 Compared with the control, ES5-4 could significantly promote the growth of 'Shanghaiqing' (P<0.05), with the increase rates being 16.57%, 31.89% and 21.33% respectively (Table 8).

[0103] Table 8 Effect of strain ES5-4 on aboveground biomass of 'Shanghaiqing'

[0104]

[0105] Example 5

[0106] This example is about the effect of Paenibacillus sp. ES5-4 (ie, Paenibacillus sp.) on the nutritional quality of 'Shanghai Qing'.

[0107] The test process is the same as described in Example 4. The specific determination methods of the nutritional quality indicators Vc and total protein content are as follows.

[0108] The determination of Vc in 'Shanghai Qing' was performed according to the instructions provided by Nanjing Jiancheng Bioengineering Research Institute. Specifically, 0.2 g of fresh leaves of non-heading Chinese cabbage (completed simultaneously in Example 4 and stored in a -80°C refrigerator) were taken, 0.8 mL of 0.1 mol / L phosphate buffer (pH 7.0) was added, and the mixture was fully homogenized in an ice-water bath. 0.15 mL of the homogenate was added with 0.45 mL of reagent 1 application solution, and the mixture was centrifuged at 3500 rpm for 10 min after standing for 15 min, and the supernatant was taken. After adding 0.25 mL of reagent 4 application solution, the mixture was mixed, and the mixture was bathed in 37°C water for 30 min, and then 0.1 mL of reagent 5 was added and mixed, and the centrifuge tube was allowed to stand for 10 min, and the absorbance value was determined at a wavelength of 536 nm. The Vc content operation table is shown in Table 9.

[0109] Table 9 Vc content operation table

[0110]

[0111] Note: “ / ” means not added.

[0112] The Vc content of non-heading cabbage was calculated according to the following formula:

[0113] Vc content (μg / mL) = (A 测定 -A 空白 ) / (A 标准 -A 空白 )×C 标准 ×N

[0114] In the formula, C 标准 : concentration of standard, 6μg / mL; N: dilution factor of sample pretreatment, 4; A 测定 , A 空白 , A 标准 They are the absorbance values ​​of the sample, blank sample and standard solution measured at a wavelength of 536 nm.

[0115] The total protein content of 'Shanghai Qing' was determined according to the instructions provided by Nanjing Jiancheng Bioengineering Research Institute. Take 0.1g of fresh leaves of non-heading cabbage (completed simultaneously in Example 4 and stored in a -80°C refrigerator), add 0.9mL of 0.1mol / L phosphate buffer (pH 7.0), and prepare a non-heading cabbage tissue homogenate in an ice-water bath. Centrifuge at 2500 rpm for 10 minutes and take the supernatant. After adding the color developing solution, mix well, let the centrifuge tube stand for 10 minutes, and measure the absorbance at a wavelength of 595nm. The total protein content operation table is shown in Table 10.

[0116] Table 10 Total protein content operation table

[0117]

[0118] Note: “ / ” means not added.

[0119] The total protein content of Shanghai Qing was calculated according to the following formula:

[0120] Total protein concentration (g / L) = (A assay - A blank) / (A standard - A blank) × C standard × N

[0121] Wherein, Cstandard: concentration of standard, 0.524 g / L; N: dilution factor of sample pretreatment, 10; Adetermination, Ablank, and Astandard are the absorbance values ​​of sample, blank sample, and standard solution measured at a wavelength of 595 nm, respectively.

[0122] The effects of strain ES5-4 on the nutritional quality of 'Shanghai Qing', including Vc and total protein content, were studied by field, potted and hydroponic experiments. Fig.15 Compared with the control, strain ES5-4 could significantly (P < 0.05) promote the content of Vc and total protein in 'Shanghaiqing' in field, potted and hydroponic experiments, with the increase rates of 56.25%, 31.58%, 91.67% and 37.89%, 45.16%, 43.33% respectively (Table 11).

[0123] Table 11 Effect of strain ES5-4 on Vc and total protein content of 'Shanghai Qing'

[0124]

[0125] Example 6

[0126] This example is the effect of Paenibacillus sp. ES5-4 (ie, Paenibacillus sp.) on selenium enrichment in 'Shanghai Qing'.

[0127] The field test process is as described in Example 4, and the samples for the determination of total selenium content have been prepared in Example 4. In the hydroponic test, the early seedling raising process is the same as that described in Example 4. After the seedlings are transferred to the Hoagland nutrient solution for acclimatization for one week, the roots are dipped for half an hour for inoculation, and then the seedlings are transferred to the new Hoagland nutrient solution with a selenium concentration of 2.0 mg / L, and the bacterial solution is added at the same time, 10 mL per pot, and the treatment is performed for 3 days, with 3 repetitions. During this period, the nutrient solution is not replaced. After the cultivation is completed, the samples are taken, dried, ground, sieved through a 100-mesh sieve, and stored dry at room temperature for the determination of total selenium content. The determination of selenium content in the aboveground part of 'Shanghai Qing' was carried out with reference to GB5009.258-2016 National Food Safety Standard Method for Determination of Multi-Elements in Food.

[0128] Determination principle: After digestion, the sample is determined by inductively coupled plasma mass spectrometer (ICP-MS) and qualitatively analyzed by the specific mass number (mass-to-charge ratio, m / z) of the element; the external standard method is used to perform quantitative analysis in which the intensity ratio of the mass spectrum signal of the element to be measured and the mass spectrum signal of the internal standard element is proportional to the concentration of the element to be measured.

[0129] Operation steps: Weigh 0.1-0.4g (accurate to 0.0001g) of dry sample into a polytetrafluoroethylene digestion inner tank, add 5mL of nitric acid and soak overnight. Cover the inner cover, tighten the stainless steel outer cover, put it into a constant temperature drying oven, keep it at 80℃ for 2h, keep it at 120℃ for 2h, and then increase it to 160℃ for 4h. Cool it naturally to room temperature in the oven, open it and heat it to drive out the acid until it is almost dry, wash the digestion solution into a 25mL volumetric flask, wash the inner tank and inner cover 3 times with a small amount of nitric acid solution (1%), combine the washing solution into the volumetric flask and dilute it to the scale with 1% nitric acid, mix well and set aside. At the same time, perform a reagent blank test. The test solution is measured on the machine for the selenium content, and it is repeated 3 times.

[0130] Result calculation: Selenium content (mg / kg) = (c×V) / m×D

[0131] Wherein, c: concentration of selenium in solution, mg / L; V: extraction volume, mL; D: dilution factor; m: sample mass, g.

[0132] The effect of strain ES5-4 on selenium enrichment in 'Shanghaiqing' was analyzed by field and hydroponic experiments. Fig.16 In the field test and hydroponic test, compared with the control, strain ES5-4 could significantly (P < 0.05) promote the enrichment of selenium in 'Shanghaiqing', with the increase rates of 12.41% and 25.44%, respectively (Table 12)

[0133] Table 12 Effect of strain ES5-4 on the enrichment of total selenium content in 'Shanghaiqing'

[0134]

[0135] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A Paenibacillus strain, characterized in that The Bacillus sp. strain is named Paenibacillus sp., and is deposited in Guangdong Microbial Culture Collection Center with a deposit number of GDMCC No: 65116 and a deposit date of September 12, 2024; the nucleotide sequence of the 16S rRNA of the Paenibacillus sp. is shown in SEQ ID NO.

1.

2. A use of the Paenibacillus strain according to claim 1, characterized in that: The bacillus strain is used for promoting the growth of Shanghai greens, improving the nutritional quality and strengthening the enrichment of selenium.

3. The use according to claim 2, characterized in that: The bacillus strain is used to increase the dry weight of the aerial part of the Shanghai Grandis, improve the Vc content, and improve the total protein content.

4. The use according to claim 3, characterized in that: The growth rate of the aboveground dry weight of the Shanghai green is 16.57% to 31.89%, the growth rate of the Vc content is 31.58% to 91.67%, and the growth rate of the total protein content is 37.89% to 45.16%.

5. The use according to claim 2, characterized in that: When the Bacillus strain is used to enhance the selenium enrichment of Shanghai Green, when the Shanghai Green is cultivated in the field, the growth rate of the selenium content is 12.41%, and when the Shanghai Green is cultivated in hydroponics, the growth rate of the selenium content is 25.44%.

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