阅读说明：本技术 菌株Rhodanobacter sp.LX-100及其在氧化有机硒或单质硒中的应用 (Rhodanobacter sp.LX-100 strain and application thereof in oxidizing organic selenium or elemental selenium ) 是由 郑世学 李明顺 王革娇 安立进 罗雄 汪依婷 于 2021-07-09 设计创作，主要内容包括：本发明属于农业微生物技术领域,具体公开了菌株Rhodanobacter sp.LX-100及其在氧化有机硒或单质硒中的应用,所述菌株的保藏编号为CCTCC NO：M 2021544。该菌对单质硒以及硒代蛋氨酸(SeMet)均具有氧化作用。LX-100菌株能在120 h将初浓度100μM的SeMet转化生成12μM左右的Se(Ⅳ),还能在120 h将过量单质硒(Se(0))氧化生成100μg/L左右的Se(Ⅳ)。因此,LX-100菌株不仅具有将有机硒SeMet氧化为Se(Ⅳ)的能力,而且具有将单质硒氧化成Se(Ⅳ)的能力,在土壤有机硒转化、单质硒氧化及植物富硒方面具有很好的应用前景。(The invention belongs to the technical field of agricultural microorganisms, and particularly discloses a strain Rhodanobacter sp.LX-100 and its application in oxidizing organic selenium or simple substance selenium, the preservation number of the strain is CCTCC NO: m2021544. The strain has oxidation effect on elemental selenium and selenomethionine (SeMet). LX-100 strain can convert SeMet with the initial concentration of 100 mu M into Se (IV) with the concentration of about 12 mu M within 120h, and can oxidize excessive elemental selenium (Se (0)) into Se (IV) with the concentration of about 100 mu g/L within 120 h. Therefore, the LX-100 strain not only has the capacity of oxidizing the organic selenium SeMet into Se (IV), but also has the capacity of oxidizing the simple substance selenium into Se (IV), and has good application prospects in the aspects of soil organic selenium conversion, simple substance selenium oxidation and plant selenium enrichment.)

1. One separated strainRhodanobactersp. saidRhodanobactersp. preservation number is CCTCC NO: m2021544.

2. The method of claim 1RhodanobacterUse in selenium oxidation.

3. The use of claim 2, wherein the selenium is elemental selenium and/or organic selenium.

4. The method of claim 1Rhodanobactersp. in the preparation of selenium-enriched agent for plants.

Technical Field

The invention belongs to the technical field of agricultural microorganisms, and relates to a strain Rhodanobacter sp.LX-100 and application thereof in oxidizing organic selenium or elemental selenium.

Background

Selenium (Se) is located in the VI main group of the periodic table and exists in nature in four valence states of Se (-II), Se (0), Se (IV) and Se (VI). Se (-II) is mainly organic selenium, and is commonly found in organisms, such as selenocysteine (SeCys), selenomethionine (SeMet), and monomethyl selenium (CH)₃-Se), dimethylselenium (CH)₃-Se-CH₃) And methylselenocysteine (SeMeCys), and the like. Se (IV) and Se (VI) are primarily inorganic selenium, mostly present in non-living organisms, and usually present as oxyanions, such as selenite (SeO)₃ ^2-) And selenate (SeO)₄ ^2-) The selenium is a main source for plants to absorb and utilize selenium, and soil organic selenium accounts for about 56-81% of the total selenium, wherein SeMet accounts for about 60% of the organic selenium. The chemical elemental selenium Se (0) is less common in nature and most exists in selenium-rich ores.

At present, various methods for enhancing the selenium enrichment of plants exist, and the methods are most widely applied by applying selenium in soil, spraying selenium on leaf surfaces, dressing seeds and the like. The soil contains elemental selenium, selenite, selenate, organic selenium and the like, and plants mainly absorb the selenite and the selenate. Therefore, the method is a safe, reliable and low-cost way to add microorganisms into the soil to convert a large amount of organic selenium into a bioavailable state or selenium oxide so as to increase the absorption of plants to selenium.

The microbial reduction of selenate and selenite is reported more, the mechanism of selenium reduction is well explained, and the research of microbial selenium oxidation is rarely reported. The applicant previously separates and identifies two strains of the photinia T5M6 and the rhizobium T3F4 with the simple substance selenium oxidation effect, and the two strains can oxidize insoluble chemical simple substance selenium or selenium mineral powder into soluble tetravalent selenium which is further absorbed and utilized by plants to promote the selenium enrichment of the pakchoi; all have obtained the national invention patent. In addition, there are few reports on the promotion of selenium enrichment in plants by microbial oxidation of selenium.

Some strains of Rhodanobacter have denitrification and anisole degradation functions, and no report is provided at present on selenium oxidation.

The Rhodanobacter sp.LX-100 provided by the invention has good biological oxidation function on elemental selenium and organic selenium, and the Rhodanobacter sp.LX-100 can oxidize both elemental selenium and selenomethionine, so that the Rhodanobacter sp.LX-100 can more effectively oxidize the elemental selenium or the organic selenium into soluble tetravalent selenium in selenium-rich or selenium-poor soil, has good application potential for promoting selenium enrichment of plants, and can be used as a good material for researching selenium oxidation and biological selenium enrichment.

Disclosure of Invention

The invention aims to provide a separated bacterial strain Rhodanobacter sp.LX-100, wherein the preservation number of the bacterial strain is CCTCC NO: m2021544.

Another object of the present invention is to provide the use of Rhodanobacter sp.lx-100 for selenium oxidation.

In order to achieve the purpose, the invention adopts the following technical measures:

the applicant separates and screens a bacterial strain Rhodanobacter sp with the capability of oxidizing organic selenium and elemental selenium from selenium-rich soil in Enshi of Hubei province in China, and the bacterial strain is delivered to a China Center for Type Culture Collection (CCTCC) for preservation in 2021, 5 and 17 months, and the preservation number is CCTCC NO: m2021544, classification name: rhodanobater sp.lx-100, address: wuhan university in Wuhan, China.

The culture characteristics of Rhodanobacter sp.LX-100 are as follows:

rhodanobacter sp.LX-100 has a rod-like cell shape with a length of 1.4-1.5 μm and a width of 0.2-0.4 μm, and is cultured on R2A agar plate medium at 28 deg.C for 72h to form yellow, round, raised and smooth colony.

The application of Rhodanobacter sp.LX-100 in selenium oxidation comprises that Rhodanobacter sp.LX-100 is taken as one or the only effective component to prepare a selenium oxidant, in particular to be used for the oxidation of organic selenium and elemental selenium.

The invention also comprises a plant selenium-rich agent prepared from Rhodanobacter sp.LX-100.

Compared with the prior art, the invention has the following advantages:

(1) the Rhodanobacter sp.LX-100 strain obtained by the invention has the capability of oxidizing organic selenium SeMet to generate selenite, and belongs to the discovery for the first time. The soil selenium contains the most organic selenium, and the organic selenium is oxidized into quadrivalent selenium which is easy to be utilized by plants, so that the selenium enrichment of the plants can be promoted.

(2) The LX-100 can oxidize insoluble elemental selenium into the capability of absorbing and utilizing selenite by plants, thereby promoting the selenium enrichment of the plants; is a supplement to the limited selenium oxidation strain resource.

(3) The strain LX-100 can oxidize Se (-II) and Se (0) simultaneously to generate a strain of Se (IV), and has scientific research significance and agricultural application potential of a selenium conversion mode strain.

(4) The invention provides a novel method for promoting selenium enrichment of plants by using beneficial microorganisms, and the method is safe and environment-friendly.

Drawings

FIG. 1 is a microscopic transmission electron microscope image of the strain LX-100.

FIG. 2 is a schematic representation of the growth curve of strain LX-100;

wherein: control represents the Control of treatment of strain LX-100 without selenium;

100 μ M SeMet represents the growth curve of the strain LX-100 after the addition of 100 μ M SeMet to the medium;

the 100 μ M Se (IV) represents a growth curve of the strain LX-100 after the Se (IV) is added to the culture medium to a final concentration of 100 μ M;

0.1g L^-1se (0) indicates that 0.1g L is added to the medium to the final concentration^-1Growth curve of strain LX-100 after Se (0). FIG. 3 is a schematic representation of the transformation of SeMet by strain LX-100.

Wherein: a is the oxidation curve of SeMet to Se (IV) by strain LX-100;

b is an efficiency graph of SeMet utilization in transformation of the strain LX-100;

100 μ M SeMet represents an unlinked control, and 100 μ M SeMet LX-100 represents an inoculated LX-100 treatment. FIG. 4 is a schematic diagram of the oxidation of elemental selenium by strain LX-100.

0.1g L^-1Se (0) represents control treatment without addition of the strain LX-100;

0.1g L^-1se (0) represents additionStrain LX-100 represents the time-dependent profile of the Se (IV) concentration in the latter system.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, but the protection scope of the invention is not limited by the following embodiments. The methods used in the following examples are conventional methods unless otherwise specified.

Example 1:

isolation and identification of Strain LX-100

(1) Sample collection: the applicant collected the selenium-rich soil sample used by the invention from the surface soil of the selenium-rich area of the Rishi soil family of Hubei province, autonomous State of the Ministry of the soil family in 2018.

(2) And (3) separating and purifying thalli: weighing 10g of soil sample in a triangular flask filled with 90mL of sterile physiological saline, placing the soil sample in a shaking table at 28 ℃ for half an hour, and sequentially adding 1mL of soil sample into 9mL of sterile physiological saline to gradually dilute the soil sample to 10^-1，10^-2，10^-3，10^-4Respectively taking 0.1mL of the bacterial suspension, coating the bacterial suspension in a 0.5mM Se (IV) R2A solid culture medium, coating 3 flat plates on each gradient, culturing the bacterial suspension at the constant temperature of 28 ℃ for a week, selecting bacterial colonies which do not produce red nano-selenium (i.e. do not turn red) after selenium reduction, primarily determining the bacterial colonies as non-reduced selenium strains, and facilitating the screening of selenium-oxidized strains in the later period. And picking single colonies, and streaking for purification. After purification, the mixture is preserved in a screwed pipe according to the ratio of 50% of glycerol to bacteria liquid (the volume ratio is 1:1) and preserved at the temperature of minus 80 ℃.

(3) Selenium conversion and screening of oxidizing bacteria: the strains are added according to the inoculation amount of 1 percent and the final concentration is respectively 0.1g L^-1Elemental selenium powder and 100. mu.M R2A liquid medium of SeMet (medium sterilization treatment), shaking culture at 28 deg.C and 150rpm for 120h, and sampling at fixed point in the middle (supernatant). Centrifuging the obtained supernatant at 12000rpm for 10min, and detecting Se (IV) content and SeMet content or other organic selenium content by using high performance liquid chromatography-hydride generation-atomic fluorescence spectrometer (HPLC-HG-AFS). If the Se (IV) content is obviously increased, the bacterium has oxidability to elemental selenium; if the SeMet content is reduced or the content of other organic selenium is increased, the bacterium is indicatedHas the capability of assimilating and transforming SeMet.

(4) And (3) classification and identification of selenium oxidizing bacteria: 16S rRNA gene sequencing is carried out on the obtained bacterial strain, a phylogenetic tree of the bacterial strain is constructed, and the bacterial strain is Rhodanobacter sp. The strain is delivered to China Center for Type Culture Collection (CCTCC) for preservation at 17 months and 5 months in 2021, and is classified and named: rhodanobacter sp.LX-100 with a preservation number of CCTCC NO: m2021544 address: china, Wuhan university.

Rhodanobacter sp.LX-100 has a rod-like cell shape with a length of 1.4-1.5 μm and a width of 0.2-0.4 μm, and when cultured on R2A agar plate medium at 28 ℃ for 72h, the colony is yellow, round, raised and has a complete and smooth edge (FIG. 1).

(5) Growth curve: the strains are added with SeMet of 100 mu M, Se (IV) of 100 mu M or Se (IV) of 0.1gL respectively according to the inoculation amount of 1 percent^-1Placing the elemental selenium powder in 50mL of R2A liquid culture medium, repeating each treatment for 3 times, taking R2A liquid culture medium without additional selenium as control, performing shake culture at 28 deg.C and 150rpm, sampling every 12h, measuring thallus growth concentration OD with 2mL of ultraviolet spectrophotometer₆₀₀Values, growth curves were plotted as shown in fig. 2.

Example 2: oxidation curve of Strain LX-100 to SeMet

The method comprises the following specific steps:

(1) 3 bottles of sterilized R2A liquid medium were prepared, 50mL each. SeMet, which had been filter-sterilized, was added to each of the solutions to a final concentration of 100. mu.M. Log phase of inoculation (OD)₆₀₀Value about 0.8) LX-100 bacterial liquid with 1 percent of inoculation amount. 2mL of sample was taken immediately after mixing and this sample was taken as the first sample and placed in a clean centrifuge tube. The mixture was incubated at 28 ℃ on a constant temperature shaker at 150 rpm. Samples were taken every 12h and the experiment was set up in triplicate, with no addition of strain LX-100, and only the same final concentration of liquid medium R2A for SeMet as a control. After sampling, the samples were processed as follows.

(2) 2mL of the sample is taken each time, centrifuged at 12000rpm for 10min, and the supernatant is passed through a 0.22 μm filter and stored in a refrigerator at-20 ℃.

(3) The content of SeMet and other organic selenium in the sample is detected by HPLC-HG-AFS of Beijing Jitian instruments Ltd.

(4) SeMet conversion plots were constructed with the measured SeMet (μ M) or Se (IV) as ordinate and time as abscissa (FIG. 3).

(5) As shown in FIG. 3, LX-100 has a strong SeMet oxidation ability, and the Se (IV) content in the sample supernatant tends to increase continuously, while the SeMet content tends to decrease continuously. Se (IV) content in the supernatant is increased from 0h to 120h, and the Se (IV) content in the LX-100 supernatant reaches 13.47 +/-0.56 mu M (A in figure 3) until 120h, while most SeMet is absorbed by cells and assimilated (B in figure 3). The following table specifically shows:

example 3:

oxidation curve of strain LX-100 to elemental selenium

The method comprises the following specific steps:

(1) 3 bottles of sterilized R2A liquid medium were prepared, 50mL each. Adding sterilized elemental selenium powder to a final concentration of 0.1g L^-1. Log phase of inoculation (OD)₆₀₀Value about 0.8) LX-100 bacterial liquid with 1 percent of inoculation amount. 2mL of sample was taken immediately after mixing and this sample was taken as the first sample and placed in a clean centrifuge tube. The mixture was incubated at 28 ℃ on a constant temperature shaker at 150 rpm. Samples were taken every 12h and the experiment was set up in triplicate, with control of R2A liquid medium without addition of strain LX-100, but with addition of the same final concentration of elemental selenium. After sampling, the samples were processed as follows.

(2) 2mL of the sample is taken each time, centrifuged at 12000rpm for 10min, and the supernatant is passed through a 0.22 μm filter and stored in a refrigerator at-20 ℃.

(3) The content of Se (IV) in the sample is detected by HPLC-HG-AFS of Beijing Jitian instruments.

(4) To determine the Se (IV) concentration (. mu. g L)^-1) The graph of elemental selenium oxidation was constructed with the ordinate and time the abscissa (fig. 4).

(5) As shown in FIG. 4, the Se (IV) content in the sample supernatant is increased continuously, and the Se (IV) content in the LX-100 supernatant reaches 97.63 +/-4.28 mu g L until 120h^-1。