CN111690564B - Millettia speciosa champ cadmium-resistant endophyte RH5 and application thereof - Google Patents
Millettia speciosa champ cadmium-resistant endophyte RH5 and application thereof Download PDFInfo
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Abstract
The invention discloses a beautiful millettia root cadmium-resistant endophyte RH5 which is classified and named as Burkholderia sp RH5 with the preservation number: GDMCC NO: 61030. according to the invention, the millettia speciosa champ cadmium-resistant endophyte RH5 is added in the growth process of the millettia speciosa champ, and the endophyte has the function of repairing cadmium toxicity of the millettia speciosa champ medicinal material, so that the growth of the millettia speciosa champ can be effectively promoted, and the safety of the medicinal material is improved.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a beautiful millettia root cadmium-resistant endophyte RH5 and application thereof.
Background
Endophytes refer to fungi or bacteria that live within the tissues and organs of healthy plants at some or all stages. Endophytic bacteria are ubiquitous in higher plants, woody, herbaceous, monocotyledonous and dicotyledonous plants. Has become a potential microbial pesticide in biological control, yield-increasing bacteria or is used as a potential carrier bacteria for biological control.
Beautiful millettia speciosa (Callerya speciosa) is a Callerya plant of Leguminosae (Ledomiosae) Hemsleya, also called beautiful Millettia speciosa, beautiful millettia speciosa, beautiful Millettia dielsiana and the like, is mainly distributed in Fujian, Guangdong, Guangxi, Guizhou, Hainan and the like in China, and is widely cultivated in the south China. The beautiful millettia root is used as the medicine, has sweet taste and mild nature, enters lung and kidney channels, has the effects of tonifying deficiency, moistening lung, strengthening tendons and activating collaterals, and has better curative effect on chronic diseases such as lumbar muscle strain, rheumatoid arthritis, cough due to lung deficiency, pulmonary tuberculosis, chronic bronchitis, chronic hepatitis and the like. Beautiful millettia root is used as a main raw material and is processed into Chinese patent medicines such as waist strengthening and kidney strengthening pills, strengthening and body building capsules, Guilong ointment and the like by pharmaceutical enterprises, and is widely applied nationwide. Besides being used as the main raw material of various Chinese patent medicines, the bovine megaforce is also widely used as the raw material for cooking soup, making medicated diet, medicated wine and the like.
In recent years, with the continuous expansion of the planting scale of beautiful millettia root, the yield and the quality of the beautiful millettia root medicinal material are seriously influenced by the soil environment, and according to the detection result, the medicinal material at the root of the beautiful millettia root is excessively enriched with heavy metal cadmium. How to reduce the content of heavy metal cadmium in the beautiful millettia root medicinal material and improve the yield and the quality becomes a great problem to be solved urgently.
Disclosure of Invention
It is an object of the present invention to address at least the above problems and to provide at least the advantages described hereinafter.
Still another object of the present invention is to provide a cadmium-tolerant endophyte RH5 of beautiful millettia root, which is classified and named Burkhoideria sp RH5, and which was filed 5/25.2020 to Guangdong province collection of microorganisms under the accession number: GDMCC NO: 61030, the radix Millettiae Speciosae is resistant to cadmium endophyte RH5, and can repair cadmium poisoning caused by medicinal materials.
The invention also aims to provide application of the millettia speciosa champ cadmium-resistant endophyte RH5, which can be used for repairing cadmium toxicity generated by the millettia speciosa champ.
To achieve these objects and other advantages in accordance with the present invention, there is provided a beautiful millettia root cadmium-resistant endophyte RH5, classified under the designation Burkhoideria sp RH5, deposited under the accession number: GDMCC NO: 61030.
preferably, the 16S rDNA gene sequence of the beautiful millettia root cadmium-resistant endophyte RH5 is shown as SEQ ID NO. 1.
Preferably, the beautiful millettia root is resistant to cadmium endophyte RH5 stored in glycerol.
Preferably, the glycerol concentration is 20%.
Preferably, the beautiful millettia root is resistant to cadmium endophyte RH5 and is used for repairing cadmium toxicity generated by the beautiful millettia root.
Provides the application of the beautiful millettia root cadmium-resistant endophyte RH5, and comprises the following specific steps: the preparation method comprises the steps of uniformly mixing the beautiful millettia root cadmium-resistant endophyte RH5 with a liquid LB culture medium to obtain bacterial suspension, and then pouring the bacterial suspension with a proper volume to the root of a beautiful millettia root plant.
Preferably, the volume ratio of the beautiful millettia root cadmium-resistant endophyte RH5 to the liquid LB culture medium is as follows: 1:500.
Preferably, the liquid LB medium comprises: tryptone, yeast extract, NaCl.
Preferably, each liter of the liquid LB culture medium specifically comprises: 10g tryptone, 5g yeast extract, 5g NaCl, and its pH was 7.4.
The invention at least comprises the following beneficial effects:
the radix millettiae speciosae cadmium-resistant endophyte RH5 is added in the growth process of the radix millettiae speciosae, and the cadmium-resistant endophyte has the function of repairing cadmium poison of the radix millettiae speciosae, so that the growth of the radix millettiae speciosae can be effectively promoted, and the safety of the medicinal materials is improved.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a strain morphological feature diagram of cadmium-resistant endophyte RH5 of beautiful millettia root;
FIG. 2 is a phylogenetic tree diagram of the cadmium-tolerant endophyte RH5 for beautiful millettia root constructed based on 16S rDNA gene sequence;
FIG. 3 shows the effect of inoculation of cadmium-tolerant endophyte RH5 on the chlorophyll a content of Millettia speciosa;
FIG. 4 shows the effect of inoculation of cadmium-tolerant endophyte RH5 on the chlorophyll b content of Millettia speciosa champ;
FIG. 5 shows the effect of inoculation of cadmium-tolerant endophyte RH5 on the total chlorophyll content of Millettia speciosa champ;
FIG. 6 shows the effect of inoculation of cadmium-tolerant endophyte RH5 on SOD activity of Millettia speciosa under cadmium stress according to the present invention;
FIG. 7 shows the effect of inoculation of cadmium-tolerant endophyte RH5 on POD activity of Millettia speciosa under cadmium stress according to the present invention;
fig. 8 shows the influence of inoculation of cadmium-resistant endophyte RH5 on the proline content of beautiful millettia root under cadmium stress.
Note: the beautiful millettia root cadmium-resistant endophyte RH5 is classified and named as Burkholderia (Burkholderia arboris) RH5, and is submitted to Guangdong province microorganism strain collection center at 25/5 of 2020 with the collection number: GDMCC NO: 61030;
CK in FIGS. 3 to 8 indicates a group of cadmium-resistant non-inoculated Millettia speciosa RH5, and RH5 indicates a group of cadmium-resistant inoculated Millettia speciosa RH 5.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It is to be noted that the experimental methods in the following embodiments, unless otherwise specified, are all conventional methods, and that reagents and materials, unless otherwise specified, are commercially available. The methanol was a commercially available analytically pure methanol. 2 Xtag Master Mix was purchased from Takara Bio Inc., and Primer-F, Primer-R was synthesized by Okagaku Bio Inc., Beijing.
Examples
Firstly, separating and purifying strains
Test materials: caulis Spatholobi (Callerya speciosa (Champ. ex Benth) Schot) from Guangxi Guangzhou health industry GmbH.
NA medium: each liter of NA culture medium contains 3g of beef extract, 1g of yeast extract, 5g of peptone, 10g of sucrose and 15g of agar, and the pH value of the culture medium is 7.0.
Washing fresh healthy beautiful millettia root with the length of 5-6cm with running water, transferring to a super clean workbench, sterilizing the cleaned beautiful millettia root with 75% ethanol for 15s under aseptic condition, sterilizing with 0.1% mercuric chloride solution for 8-10 min, washing with aseptic water for 4-5 times after sterilization, and sucking out surface water with aseptic filter paper for later use.
Scraping the epidermis of the sterilized bovine high-potency roots, cutting into root sections with the length of 1cm, putting into a sterile mortar, adding 5mL of sterile water for grinding, transferring the grinding liquid into a sterile centrifuge tube after full grinding, standing for 10min, taking 0.5mL of supernatant, performing gradient dilution by 10-106 times, absorbing 100 mu L of diluent per concentration gradient, coating on an NA flat plate, and repeating each concentration gradient for 3 times. Sterile water from the last rinse was pipetted onto NA plates as a negative control. And (3) placing the NA plate in a biochemical incubator for constant-temperature culture at 28 ℃ for 24-96 h, selecting colonies with different forms, repeatedly streaking and purifying, and preserving the purified millettia speciosa champ endophyte strain with 20% of glycerol.
II, identification of the strains
1. Morphological characteristics
Inoculating the purified strain on an NA culture medium, culturing at 28 ℃ for 24h, and observing the colony morphological characteristics of the strain. Meanwhile, the above-mentioned strain cultured for 24h on the NA medium was gram-stained and microscopic. The observed morphological results were photographed, and as shown in FIG. 1, the colonies of the strain were pale yellow on the NA plate, smooth in surface, raised, well-defined, round in shape, and 3-4mm in diameter after being cultured on the NA plate for 5 d.
2. 16S rDNA sequence and phylogenetic analysis thereof
2.1 preparation of DNA template
A. Reagent: (1) lysis buffer: Tris-Ac (pH 7.8)40mM, NaAc 20mM, EDTA 1mM, SDS 1% (w/v); (2)5M NaCl solution.
B. The extraction step comprises:
a. placing 1.5mL of the overnight bacterial culture in a microcentrifuge tube (EP tube), centrifuging at 12000rpm for 0.5 min, discarding the supernatant, and retaining the precipitate;
b. adding 400 μ L lysis buffer solution into the precipitate, and repeatedly blowing with a pipette to resuspend the solution;
c. adding 200ul of 5M NaCl, mixing well, centrifuging at 12000rpm for 10min, and collecting 600 μ L supernatant;
d. c, adding equal volume of phenol/chloroform (1:1) into the supernatant obtained in the step c, uniformly mixing, centrifuging at 12000rpm for 10min, and transferring the supernatant into a clean EP tube after centrifuging;
e. d, adding equal volume of chloroform into the supernatant obtained in the step d, uniformly mixing, centrifuging at 12000rpm for 10min, and transferring the supernatant into another clean EP tube after centrifugation;
f. adding isopropanol with the same volume into the supernatant obtained in the step e, uniformly mixing, placing at room temperature for 10min, then centrifuging at 12000rpm for 10min, removing the supernatant after centrifugation, and keeping the precipitate;
g. washing the precipitate obtained in the step f by using ethanol with the volume concentration of 70%, and airing to obtain DNA;
h. dissolve the dried DNA from step g in 30. mu.L double distilled water (ddH) 2 O) at-20 ℃.
2.2 PCR amplification of 16S rDNA sequences
(1) A PCR instrument: ABI 3730-XL DNA sequencer (Applied Biosystems, USA);
(2) an amplification primer: the sequence of Primer-F is 5'-AGAGTTTGATCCTGGCTCAG-3' shown as SEQ ID NO. 2; Primer-R: 5'-GGTTACCTTGTTACGACT-3' is shown as SEQ ID NO. 3;
SEQ ID NO.2 and SEQ ID NO.3 of the sequences shown in the sequence Listing are sequences corresponding to Primer-F and Primer-R, respectively, and are synthesized by Oakbiology, Inc. of Beijing.
(3) An amplification system: the 16S rDNA sequence PCR amplification system is shown in Table 1:
TABLE 1
2.3 electrophoretic detection of PCR amplification products
1% agarose Gel (containing Gel red 5. mu.L/100 mL), 1 XTAE electrophoresis buffer, 150V electrophoresis for 20min, the Loading amount of PCR product is 3. mu.L, and the sample is applied after mixing with 1. mu.L Loading dye. The result was observed by a gel imaging system, and the amplified fragment length was determined to be 1396bp, using DL1000 DNA Marker from TaKaRa as a nucleic acid standard molecular weight reference. And (3) transmitting and sequencing the bacterial liquid with the correct target band, wherein the sequencing is performed by Shenzhen Huadai gene science and technology limited, and the sequence obtained by the sequencing result is shown as SEQ ID No. 1.
SEQ ID No.1 shown in the sequence table is a 16S rDNA gene sequence of the beautiful millettia root cadmium-resistant endophyte RH5, the classification of the beautiful millettia root cadmium-resistant endophyte RH5 is named as Burkholderia (Burkhoidia sp.) RH5, and the genetic material is submitted to Guangdong provincial microorganism collection center in 29/5 of 2020, and the specific address of the microorganism collection center is as follows: the Guangzhou city first furious Zhonglu No. 100 large yard No. 59 building No. 5 building, the preservation number is: GDMCC NO: 61030.
2.4 construction of phylogenetic Tree
Comparing the 16S rDNA sequence with the 16S rDNA sequence of bacteria determined in GenBank gene library, and downloading related sequences according to the comparison result. Systematic analysis was performed by the Neighbor-joining algorithm (NJ) of MEGA 6.0 and a phylogenetic tree was constructed, as shown in fig. 2, and the measured 16S rDNA sequence was up to 99% similar to Burkhoideria sp, which was identified as Burkhoideria sp.rh5 in combination with morphological features.
Application of beautiful millettia root cadmium-resistant endophyte RH5
1. Callicarpa speciosa cadmium-resistant endophyte RH5 cadmium reduction repair experiment
Beautiful millettia root with basically consistent growth vigor is selected and transplanted into nutrient cups filled with 3kg of soil, and 1 plant is planted in each cup. And (3) after 3 days of seedling rejuvenation, sequentially irrigating the prepared bacterial suspension and LB blank culture solution into corresponding nutrition cups by adopting a root irrigation method, and irrigating 50mL of each plant to the roots of the plants as much as possible. Irrigating the root once a week for 3 times, and repeating the treatment 10 times. The inoculated beautiful millettia roots are managed according to a conventional cultivation management method, and all plant management methods are consistent. And (5) harvesting plants after inoculating the bacteria for 5 months, and measuring each index.
Wherein, the test soil: the quartz sand with the diameter of about 1mm required by the experiment is purchased from water treatment science and technology company of Nanning war department; the soil is taken from the surface soil of suburbs of Nanning city of Guangxi province. The basic physicochemical properties of the soil are as follows: the pH value is 5.2, and the organic matter content is 4.6g kg -1 Total nitrogen content of 0.2mg kg -1 The total phosphorus content was 1.4mg kg -1 The total potassium content is 28mg kg -1 The Cd content was 0.224mg kg -1 ；
Heavy metal resistance medium: taking an LB culture medium as a basic culture medium, adding each heavy metal mother solution subjected to filtration sterilization to prepare screening culture mediums with different concentrations;
heavy metal mother liquor: with CdCl 2 ·2.5H 2 O、ZnSO 4 ·7H 2 O、CuSO 4 ·5H 2 O、MnCl 2 ·4H 2 O、K 2 Cr 2 O 7 (analytically pure) preparation of Cd 2+ 、Zn 2+ 、Cu 2+ 、Mn 2+ 、Cr 6+ Filtering the mother liquor with the concentration of 0.1M through a 0.22 mu M bacterial filter for sterilization;
potting container: the nutrition cup has the size of an upper caliber of 12cm, a lower caliber of 10cm and a height of 30cm, and is wiped with 75% alcohol for disinfection before use;
liquid LB medium: each liter of the liquid LB medium contained 10g of tryptone, 5g of yeast extract, 5g of NaCl, and its pH was 7.4.
2. Influence of inoculation of cadmium-resistant endophyte RH5 on chlorophyll content of beautiful millettia root under cadmium stress
Washing leaves of the beautiful millettia root which are planted through a beautiful millettia root cadmium-resistant endophyte RH5 cadmium reduction repair experiment with distilled water, wiping off surface water, shearing the leaves into long strips with basically consistent width by using scissors and uniformly mixing, randomly weighing 0.2g of the leaves, putting the leaves into a 25mL brown volumetric flask, adding 20mL of a mixed solution of 95% ethanol and acetone (V: V ═ 1:1), ensuring that all the leaves are immersed in the mixed solution, tightly covering a volumetric flask cover, standing the leaves in a dark and dark place, fixing the volume to 25mL by using the mixed solution after the leaves are completely whitened, respectively measuring OD values of an extracting solution at 663nm and 646nm, and calculating the chlorophyll content of each processed sample. As shown in FIGS. 3-5, the heavy metal cadmium has a great influence on the chlorophyll of the beautiful millettia root leaves, and the contents of chlorophyll a, chlorophyll b and total chlorophyll in the beautiful millettia root leaves are gradually reduced along with the increase of the cadmium stress concentration no matter the beautiful millettia root leaves are treated by cadmium-resistant endophyte RH5 or not treated by cadmium-resistant endophyte. The higher the cadmium stress concentration, the faster the chlorophyll a and total chlorophyll decay, the content of which is equal to that of the cadmium-free control group (0 mg. kg) -1 ) The greater the difference. The inoculation of beautiful millettia root cadmium-resistant endophyte RH5 can be significantThe level of chlorophyll in radix Millettiae Speciosae is increased, and the contents of chlorophyll a, chlorophyll b and total chlorophyll are all higher than those in a control group without inoculation of bacteria. Under all cadmium concentrations, the millettia speciosa champ cadmium-resistant endophyte RH5 is inoculated, so that the chlorophyll a content of the plant is increased by 15-40%, the chlorophyll b content is increased by 19-35%, and the total chlorophyll content is increased by 17-37%. The method has the advantages that the cadmium-tolerant endophyte RH5 inoculated on the beautiful millettia root relieves the decomposition of chlorophyll of the beautiful millettia root leaves under the cadmium stress, and the accumulation amount of the chlorophyll is increased, so that the normal growth of beautiful millettia root plants is ensured to a certain extent.
3. Influence of inoculation of cadmium-resistant endophyte RH5 on antioxidant enzyme of radix millettiae speciosae under cadmium stress
3.1 extraction of the enzyme solution
Weighing 0.3g of leaf of the sheared and uniformly mixed beautiful millettia root which is planted through the beautiful millettia root cadmium reduction repair experiment, adding 3mL of phosphoric acid buffer solution with the pH of 7.8 and the concentration of 0.05M, grinding the mixture in an ice bath environment until the mixture is homogenized, then centrifuging the mixture at the low temperature of 4 ℃ and 12000rpm for 20min, taking supernatant after centrifuging the centrifugation, namely the enzyme crude extract, wherein the supernatant is stored in an ice bath.
SOD Activity measurement
SOD activity was measured by a nitroblue tetrazolium (NBT) photoreduction method. 0.1mL of the crude enzyme extract was put in a glass test tube, and a mixed reaction solution consisting of 1.5mL of a phosphate buffer solution having a pH of 7.8 and a concentration of 0.05M, 0.3mL of a methionine solution having a concentration of 30mM, 0.3mL of a nitroblue tetrazolium solution having a concentration of 750uM, 0.3mL of a riboflavin solution having a concentration of 2uM, and 0.2 mL of distilled water was added thereto, and after being sufficiently and uniformly mixed, the glass test tube was uniformly irradiated with 4000Lux light intensity for 10 min. Taking another 2 test tubes as a reference tube, replacing enzyme liquid with equivalent distilled water, taking a non-enzyme liquid and non-lighting test tube as a blank for zero adjustment, and taking a non-enzyme liquid and non-lighting test tube as a maximum light reduction tube. The absorbance at 560nm was measured. The SOD enzyme activity was calculated according to the formula, and one enzyme activity unit was determined when the inhibition of NBT photoreduction by the reaction system per minute was 50%.
POD Activity assay
The guaiacol method is used. 50 mu L of the enzyme crude extract is put into a test tube, and 3mL of guaiacol solution with the concentration of 25mM and 25mM are added in sequenceH of 0Mm 2 O 2 The solution was immediately mixed, and the change in absorbance at 470nm was measured within 10min, and the change in absorbance per minute was used as an indicator of the activity of the enzyme.
3.4 results
As shown in FIGS. 6-7, the activities of antioxidant enzymes SOD and POD of radix Millettiae Speciosae were significantly increased (p < 0.05) with the increase of cadmium concentration. The influence of the inoculation treatment on the activities of the SOD and the POD of the beautiful millettia roots under the cadmium stress has basically consistent trend. When cadmium stress does not exist, the enzymatic activities of POD and SOD of the plants of the treated group inoculated with the millettia speciosa champ cadmium-resistant endophyte RH5 strain are not obviously different from those of the plants of the uninoculated control group; under the condition of cadmium stress of different concentrations, the cadmium-resistant endophyte RH5 strain of beautiful millettia root is inoculated to ensure that the activity of POD and SOD enzyme of the beautiful millettia root is obviously higher than that of an uninoculated control group (0mg kg) -1 ). At 15 mg/kg -1 Under the cadmium stress concentration, the activities of the beautiful millettia root SOD and the POD enzyme are respectively increased by 6.3 percent and 10.3 percent; at 30 mg/kg -1 The activities of the beautiful millettia root SOD and the POD enzyme are respectively increased by 8.0 percent and 7.7 percent under the cadmium stress concentration. The cadmium-resistant beautiful millettia root bacteria RH5 can reduce the oxidative damage of plants, relieve the toxic action of cadmium stress and ensure the normal growth of beautiful millettia root by improving the activity of POD and SOD in beautiful millettia root bodies.
4. Influence of inoculation of cadmium-resistant endophyte RH5 of beautiful millettia root on proline content of beautiful millettia root under cadmium stress
Weighing 0.2g of leaf of beautiful millettia root which is planted by a beautiful millettia root cadmium reduction repair experiment, adding 2mL of 3% sulfosalicylic acid solution, grinding into homogenate, putting into a constant temperature water bath kettle, leaching in boiling water bath for 10min, and frequently shaking. Cooling to room temperature, centrifuging, sucking 2mL of proline extract, 2mL of glacial acetic acid and 2.5% acidic ninhydrin, mixing, and reacting in boiling water bath for 30min to obtain pink solution. After cooling, 4mL of toluene was added and shaken by hand for 30 s. The pink material was extracted into the toluene layer. Standing for a moment, centrifuging the upper layer pink liquid in a centrifuge tube at 4000rpm for 4min, absorbing the solution of the proline in toluene, measuring the light absorption value at 520nm by using a spectrophotometer, and taking toluene as a blank control for zeroing. And calculating the proline content in the beautiful millettia root sample according to a proline standard curve. As shown in fig. 8, the proline content in beautiful millettia root significantly increased with the increase of the cadmium concentration. In the treatment of the non-inoculated endophytic bacteria, the proline content of the cadmium-added treated plants is respectively increased by 28 percent and 59 percent compared with the cadmium-free control plants, and the difference reaches a significant level (p is less than 0.05). When no cadmium stress exists, the MDA content of the inoculated group and the missed group is not different; under the stress of cadmium with the same concentration, the content of proline in the beautiful millettia root leaves is obviously reduced by inoculation treatment, and is respectively reduced by 22 percent and 12 percent, so that the obvious difference is achieved (p is less than 0.05). The cadmium stress induces the beautiful millettia root to make stress response and secrete more proline so as to protect a cell membrane system, thereby keeping the intracellular environment stable; the cadmium-resistant endophyte RH5 strain of beautiful millettia root can reduce the proline content in plant leaves, help plant cells maintain internal and external osmotic pressure balance, and relieve the damage of heavy metal cadmium stress on beautiful millettia root.
5. Influence of inoculation of cadmium-resistant endophyte RH5 on cadmium accumulation of each part of beautiful millettia root under cadmium stress
And crushing the dried and weighed beautiful millettia root sample, sieving the smashed beautiful millettia root sample by a 100-mesh sieve with the aperture of 0.149mm, and uniformly mixing the smashed beautiful millettia root sample and the sieved beautiful millettia root sample. Accurately weighing 0.3g of sample in digestion tube by analytical balance, and adding HNO 3 /HCLO 4 (5:1, v/v) digesting the mixed acid solution at 160 ℃ until the mixed acid solution is clear. Diluting the digested sample solution with deionized water, filtering with 0.22 μm filter membrane, and measuring cadmium concentration in the digested plant sample by ICP-MS. As shown in table 2, the cadmium accumulation amount of each part of beautiful millettia root growing in the cadmium polluted environment is different, the root system of beautiful millettia root has the largest enrichment effect on cadmium in soil, the cadmium accumulation amount is the largest, the stem part is the next, and the leaf part is the smallest. With the increase of the cadmium concentration, the cadmium content of each part of the beautiful millettia root is increased in different degrees, when the cadmium concentration is 30mg kg -1 And then reaches a maximum value. The inoculation of the beautiful millettia root cadmium-resistant endophyte RH5 bacterial strain can reduce the absorption of cadmium by each part of the beautiful millettia root. At 15 mg/kg -1 Under the cadmium stress, the cadmium accumulation amount of the root, the stem and the leaf of the cattle is lower than that of a non-inoculated control plant by inoculation treatment, and is respectively reduced by 25%, 12% and 13%; and 30 mg/kg -1 Under the stress of cadmium, the cadmium accumulation of roots, stems and leaves of beautiful millettia roots is respectively reduced by 24 percent, 29 percent and 17 percent by the inoculation treatment compared with the control group without inoculation, and the difference reaches a significant level (p is less than 0.05). To illustrate high concentrationUnder the stress of cadmium, the cattle high-capacity cadmium-resistant endophyte RH5 is infected, so that cadmium enrichment in plants is effectively reduced, and the toxicity of heavy metals to the plants is reduced.
TABLE 2 cadmium content of various parts of Millettia speciosa champ
Note: "ND" means not detected.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is thus not limited to the details given herein and to the illustrations shown and described without departing from the generic concept as defined by the claims and their equivalents.
SEQUENCE LISTING
<110> university of traditional Chinese medicine in Guangxi
<120> beautiful millettia root cadmium-resistant endophyte RH5 and application thereof
<130> 3
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1396
<212> DNA
<213> Burkhoideria sp.RH5
<400> 1
gtcctccttg cggttagact agccacttct ggtaaaaccc actcccatgg tgtgacgggc 60
ggtgtgtaca agacccggga acgtattcac cgcggcatgc tgatccgcga ttactagcga 120
ttccagcttc atgcactcga gttgcagagt gcaatccgga ctacgatcgg ttttctggga 180
ttagctcccc ctcgcgggtt ggcaaccctc tgttccgacc attgtatgac gtgtgaagcc 240
ctacccataa gggccatgag gacttgacgt catccccacc ttcctccggt ttgtcaccgg 300
cagtctcctt agagtgctct tgcgtagcaa ctaaggacaa gggttgcgct cgttgcggga 360
cttaacccaa catctcacga cacgagctga cgacagccat gcagcacctg tgcgccggtt 420
ctctttcgag cactcccacc tctcagcagg attccgacca tgtcaagggt aggtaaggtt 480
tttcgcgttg catcgaatta atccacatca tccaccgctt gtgcgggtcc ccgtcaattc 540
ctttgagttt taatcttgcg accgtactcc ccaggcggtc aacttcacgc gttagctacg 600
ttactaagga aatgaatccc caacaactag ttgacatcgt ttagggcgtg gactaccagg 660
gtatctaatc ctgtttgctc cccacgcttt cgtgcatgag cgtcagtatt ggcccagggg 720
gctgccttcg ccatcggtat tcctccacat ctctacgcat ttcactgcta cacgtggaat 780
tctacccccc tctgccatac tctagcctgc cagtcaccaa tgcagttccc aggttgagcc 840
cggggatttc acatcggtct tagcaaaccg cctgcgcacg ctttacgccc agtaattccg 900
attaacgctc gcaccctacg tattaccgcg gctgctggca cgtagttagc cggtgcttat 960
tcttccggta ccgtcatccc ccgatcgtat taggaccaag gatttctttc cggacaaaag 1020
tgctttacaa cccgaaggcc ttcttcacac acgcggcatt gctggatcag gctttcgccc 1080
attgtccaaa attccccact gctgcctccc gtaggagtct gggccgtgtc tcagtcccag 1140
tgtggctggt cgtcctctca gaccagctac tgatcgtcgc cttggtaggc ctttacccca 1200
ccaactagct aatcagccat cggccaaccc tatagcgcga ggcccgaagg tcccccgctt 1260
tcatccgtag atcgtatgcg gtattaatcc ggctttcgcc gggctatccc ccactacagg 1320
acatgttccg atgtattact cacccgttcg ccactcgcca ccaggtgcaa gcacccgtgc 1380
tgccgttcga cttgca 1396
<210> 2
<211> 20
<212> DNA
<213> Primer-F
<400> 2
agagtttgat cctggctcag 20
<210> 3
<211> 18
<212> DNA
<213> Primer-R
<400> 3
ggttaccttg ttacgact 18
Claims (9)
1. The beautiful millettia root cadmium-resistant endophyte RH5 is characterized in that the beautiful millettia root cadmium-resistant endophyte RH5 is classified and named as Burkholderia (Burkhoideria sp.) RH5 with the preservation number: GDMCC NO: 61030.
2. the beautiful millettia root cadmium-tolerant endophyte RH5 of claim 1, wherein the 16S rDNA gene sequence of the beautiful millettia root cadmium-tolerant endophyte RH5 is shown in SEQ ID No. 1.
3. The Millettia speciosa champ-resistant endophyte RH5 of claim 1, wherein the Millettia speciosa champ-resistant endophyte RH5 is stored in glycerol.
4. The beautiful millettia speciosa champ-resistant endophyte RH5 of claim 3, wherein the glycerol concentration is 20%.
5. The use of the beautiful millettia root cadmium-tolerant endophyte RH5 as claimed in claim 1, wherein the beautiful millettia root cadmium-tolerant endophyte RH5 is used for repairing cadmium poisoning caused by beautiful millettia root.
6. The use of the beautiful millettia root cadmium-resistant endophyte RH5 as claimed in claim 5, characterized by comprising the following specific steps: uniformly mixing the millettia speciosa champ cadmium-resistant endophyte RH5 with a liquid LB culture medium to obtain a bacterial suspension, and then pouring a proper volume of the bacterial suspension to the root of the millettia speciosa champ.
7. The use of the beautiful millettia root cadmium-tolerant endophyte RH5 as claimed in claim 6, wherein the volume ratio of the beautiful millettia root cadmium-tolerant endophyte RH5 to the liquid LB medium is: 1:500.
8. The use of the beautiful millettia root cadmium-tolerant endophyte RH5 as claimed in claim 6, wherein the liquid LB medium components comprise: tryptone, yeast extract, NaCl.
9. The use of the beautiful millettia root cadmium-tolerant endophyte RH5 as claimed in claim 8, wherein each liter of liquid LB medium specifically comprises: 10g tryptone, 5g yeast extract, 5g NaCl, and its pH was 7.4.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101684452A (en) * | 2009-06-24 | 2010-03-31 | 广东省微生物研究所 | Burkholderia dabaoshanensis and application thereof |
| CN105670980A (en) * | 2016-04-20 | 2016-06-15 | 国家烟草质量监督检验中心 | Microbial strain capable of remedying heavy metal-contaminated soil and application of microbial strain |
| CN111676160A (en) * | 2020-06-16 | 2020-09-18 | 广西中医药大学 | Application of beautiful millettia root endophyte RH5 in promoting strong growth of cows |
-
2020
- 2020-06-16 CN CN202010550296.XA patent/CN111690564B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101684452A (en) * | 2009-06-24 | 2010-03-31 | 广东省微生物研究所 | Burkholderia dabaoshanensis and application thereof |
| CN105670980A (en) * | 2016-04-20 | 2016-06-15 | 国家烟草质量监督检验中心 | Microbial strain capable of remedying heavy metal-contaminated soil and application of microbial strain |
| CN111676160A (en) * | 2020-06-16 | 2020-09-18 | 广西中医药大学 | Application of beautiful millettia root endophyte RH5 in promoting strong growth of cows |
Non-Patent Citations (1)
| Title |
|---|
| 牛大力内生细菌的多样性及其促生特性研究;苏坤等;《广西科学院学报》;20200604;第36卷(第2期);第171-176页 * |
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