CN114903049A

CN114903049A - Application of hydroxamic acid type siderophore as rice cadmium reduction preparation

Info

Publication number: CN114903049A
Application number: CN202210607498.2A
Authority: CN
Inventors: 李峰; 易盛炜; 武晨; 梅延成; 魏兰兰; 葛飞; 田江
Original assignee: Xiangtan University
Current assignee: Xiangtan University
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2022-08-16
Anticipated expiration: 2042-05-31
Also published as: CN114903049B

Abstract

The invention discloses application of a hydroxamic acid type siderophore. The hydroxamic acid type siderophore is applied as a rice cadmium reduction preparation, the cadmium content in rice grains can be greatly reduced by applying the hydroxamic acid type siderophore to cadmium-polluted rice field soil for planting rice, and the hydroxamic acid type siderophore is obtained by microbial fermentation, is easy to obtain and low in cost, has the characteristics of good cadmium reduction effect, no ecological risk, simplicity in operation and the like, and is suitable for being applied to cadmium-polluted farmlands in a large scale.

Description

Application of hydroxamic acid type siderophore as rice cadmium reduction preparation

Technical Field

The invention relates to an application of a hydroxamic acid type siderophore preparation, in particular to a method for reducing cadmium content in rice by using the hydroxamic acid type siderophore preparation obtained by fermentation of pseudomonas fluorescens HMP01 in cadmium-polluted rice field soil, belonging to the technical field of rice planting.

Background

At present, about 70% of soil in China belongs to medium and low yield fields, the quantity of grain crops is directly influenced by soil acidification and degradation caused by natural conditions, the quality of the grain crops is also seriously influenced by heavy metal pollution of the soil caused by human activities, and partial fields cannot be fully and safely utilized. At present, the standard exceeding rate of the soil point position of cultivated land in China is 19.4%, and the main pollutants are heavy metals such as cadmium (Cd). In recent years, the 'Cd rice' event is frequent, and the attention of people to the rice quality is further attracted. Therefore, the development of a simple, economic and efficient rice heavy metal inhibition and control technology is urgently needed to realize the safe production on the heavy metal polluted farmland, so that the national grain safety is guaranteed.

The farmland soil heavy metal resistance control technology in China is mainly divided into an agronomic regulation technology and a bioremediation technology. The agricultural regulation and control only comprises water management, optimized fertilization, lime addition, variety adjustment, leaf surface regulation and the like, namely VIP + n technology. The technology still has the problems of poor environmental adaptability, large difference of cultivation modes in different regions and the like, so that the repair cost is overhigh, the management difficulty is high, and further optimization is needed in the future. The biological repair mainly comprises phytoremediation and microbial repair, wherein the phytoremediation is usually extracted by hyper-enrichment plants, such as ciliate desert-grass, sedum alfredii hance and the like, and is usually used for repairing soil in a place with serious pollution due to long repair period. The microbial remediation mainly utilizes bacteria and the like to inhibit and reduce the biological effectiveness of heavy metals through self activities, such as extracellular adsorption, intracellular mineralization and regulation of the formation of a plant root system iron film. Importantly, small molecular compounds produced by microorganisms such as succinic acid produced by enterobacter, indoleacetic acid produced by bacillus cereus and polyamine produced by sphingomonas can directly influence the change of plant root systems and rhizosphere microorganisms to prevent and control the migration of heavy metals. The complexity of the soil environment and the specificity of the microbial host cause that the added microbes are difficult to effectively play the role of the microbes at the rhizosphere of plants, and the large-scale application of microbial remediation is limited. Therefore, researchers focus on small molecular organic matters secreted by microorganisms and having important environmental effects at present, and the small molecular organic matters are expected to be prepared by means of microbial fermentation extraction or chemical synthesis and applied to farmland soil on a large scale, which is also the key for promoting the deep development of future microbial remediation technologies.

The siderophore is secreted by the microorganism for obtaining Fe in the environment ³⁺ And other organic small molecular substances of trace elements play an important role in mineral substance exchange of a soil ecosystem. Siderophores are generally classified into hydroxamic acid type, catechol type, and carboxylate type, due to structural differences. Chinese patent (CN112725221A) discloses a method for preparing hydroxamic acid type siderophore by using pseudomonas fluorescens, and the hydroxamic acid type siderophore is used as an eluent or an extracting agent to be applied to the remediation of heavy metal contaminated soil, or to the remediation of organic contaminated soil, or to the remediation of heavy metal and organic combined contaminated soil. However, no report on the use of the hydroxamic acid type siderophore for preventing and controlling the migration and transformation of heavy metals such as cadmium and the like in rice fields to grains is found so far.

Disclosure of Invention

Aiming at the defects existing in the process of preventing and controlling cadmium enrichment of rice in heavy metal polluted rice field soil in the prior art, the invention aims to provide the application of the hydroxamic acid type siderophore, the hydroxamic acid type siderophore is used as a cadmium prevention and control agent to be applied to cadmium polluted farmland soil for planting rice, the cadmium enrichment amount of rice grains can be effectively reduced, the hydroxamic acid type siderophore is derived from microorganisms, the hydroxamic acid type siderophore is easy to obtain and low in cost, the ecological structure of the soil cannot be damaged when the hydroxamic acid type siderophore is added into the soil, and the hydroxamic acid type siderophore can be applied in a large scale.

In order to achieve the technical purpose, the invention provides an application of a hydroxamic acid type siderophore, which is applied as a rice cadmium reduction preparation; the hydroxamic acid type siderophore has the following chemical structural formula:

the hydroxamic acid type siderophore is prepared by the following specific method: selecting pseudomonas fluorescens HMP01 from a solid LB culture dish, adding the bacteria into an iron-deficient MSM culture medium (the main components and the concentration of the culture medium are 2-8 g/L of sodium acetate, 0.1-0.5 g/L of potassium dihydrogen phosphate, 0.2-1 g/L of ammonium sulfate, 0.2-1 g/L of sodium nitrate, 0.02-0.05 g/L of calcium chloride and 0.02-0.05 g/L of magnesium sulfate), placing the culture medium in a constant-temperature air bath oscillator at 28-32 ℃ for culturing for 72-96 h at 150r/min (when the activity of HDS is the highest), and then centrifuging the bacterial liquid in a high-speed centrifuge at 12000r/min for 10min to obtain a supernatant, namely the hydroxamic acid type iron carrier preparation. The concentration of the hydroxamic acid type siderophore in the preparation is 0.1-0.5 g/L.

The Pseudomonas fluorescens HMP01(Pseudomonas fluorescens strain HMP01) has the preservation number of CCTCC NO: m20191131.

The hydroxamic acid type siderophore of the invention reduces the absorption of cadmium by the roots of the paddy rice in the filling stage and the mature stage, enhances the transfer and accumulation of cadmium from the roots and stems to leaves and ears and finally reduces the content of cadmium in the paddy rice by mainly regulating the expression of related genes during the growth period of the paddy rice. The hydroxamic acid type iron vector is added to the rice rhizosphere in cadmium-polluted rice field soil in the rice growth period (such as the tillering period, the heading period, the filling period, the mature period and the like) to regulate the expression capacity of certain genes, such as OsHMA2 and OsHMA3 genes, the expression level of the OsHMA 3978 and OsHMA3 genes in the tillering period and heading period of rice is obviously reduced, the expression level of the OsNramp5 gene in the tillering period is obviously reduced, and the expression level of the OsNramp5 gene in the other periods is not obviously changed. The change of the OsHMA2 gene shows that cadmium is mainly transported to the overground parts such as stems and leaves in the filling stage and the mature stage of rice, mediates the transfer of the cadmium from xylem to phloem at the neck node of the panicle and distributes the cadmium to the panicle through the node. The change of the OsHMA3 gene indicates that cadmium is mainly chelated and fixed to root vacuoles in the filling stage and the mature stage of rice, and the absorption capacity to cadmium is reduced. The changes of the OsHMA3 gene and the OsNramp5 gene together indicate that the absorption of cadmium by rice roots at the tillering stage and the heading stage is improved.

As a preferred scheme, the hydroxamic acid type siderophore is added into cadmium-polluted paddy soil for planting rice so as to reduce the enrichment of cadmium in rice grains. The hydroxamic acid type siderophore can regulate the distribution of cadmium in the root, leaf, seed and other parts of rice.

As a preferable scheme, the adding amount of the hydroxamic acid type siderophore in the cadmium-polluted paddy soil is 0.2-1 g/m ² And the average cadmium content in the rice is reduced by 47.33% + -15.63%.

As a preferred embodiment, the hydroxamic acid type siderophore is added during the whole growth period of rice. As a more preferable scheme, the hydroxamic acid type siderophore is added in any period or any several periods of the green turning stage, tillering stage, heading stage, filling stage and mature stage of the rice. The hydroxamic acid type siderophore can affect the expression of different genes in different growth periods of rice, but can effectively reduce the enrichment of cadmium in rice grains on the whole.

As a preferable embodiment, the hydroxamic acid type siderophore is added to the rhizosphere of rice. The hydroxamic acid siderophore is mainly absorbed by the rhizosphere of rice, and the enrichment of cadmium in rice grains is reduced by controlling the expression of certain genes of the rice.

As a preferable scheme, Cd in the cadmium-polluted paddy soil ²⁺ The concentration is 0.3-1 mg/kg, and the pH value is 6.5-7.5.

Compared with the prior art, the invention has the beneficial technical effects that:

the hydroxamic acid type siderophore reduces the absorption of cadmium by the roots of the rice in the filling stage and the mature stage by regulating the expression of related genes of the rice, enhances the transfer and accumulation of the cadmium from the roots and stems to leaves and ears, and finally reduces the content of the cadmium in the rice.

The hydroxamic acid siderophore preparation is obtained by fermenting Pseudomonas fluorescens HMP01(Pseudomonas fluorescens strain HMP01), the preparation process is convenient and fast, the cost is low, and the actual field addition operation is simple.

The hydroxamic acid type siderophore preparation provided by the invention is added into cadmium-polluted soil for planting rice, so that the cadmium content in the rice in the cadmium-polluted soil during rice planting can be greatly reduced, ecological risks cannot be caused, and the hydroxamic acid type siderophore preparation is beneficial to popularization and application.

Drawings

FIG. 1 shows the result of the present invention on the cadmium content of various parts of early rice under soil culture conditions; as can be seen from FIG. 1, the cadmium content of the rice in the group added with the hydroxamic acid type siderophore preparation is reduced by 71.09% compared with the blank control group; the cadmium content in the rice roots, stems and ears is reduced by 39.41-45.66%, and the cadmium content in the leaves is increased by 82.73%.

FIG. 2 shows the result of the present invention on cadmium content of various parts of middle rice under soil culture conditions; as can be seen from FIG. 2, the cadmium content of the rice in the group added with the hydroxamic acid type siderophore preparation is reduced by 39.97 percent compared with the blank control group; the cadmium content in the rice roots, stems and leaves is reduced by 13.81-53.04%, and the cadmium content in the ears is increased by 68.53%.

FIG. 3 shows the result of the present invention on cadmium content of various parts of late rice under soil culture conditions; as can be seen from FIG. 3, the cadmium content of the rice in the group added with the hydroxamic acid type siderophore preparation is reduced by 28.54 percent compared with the blank control group; the cadmium content in the rice roots, stems and ears is reduced by 28.93-54.21%, and the cadmium content in the leaves is increased by 24.89%.

FIG. 4 shows the result of the ratio of cadmium concentration in various parts of early rice, middle rice and late rice under the soil culture condition; as can be seen from FIG. 4, after the hydroxamic acid type siderophore is added, the ratio of cadmium in the leaves of early rice and late rice is obviously increased, and the ratio of cadmium in the leaves and ears of medium rice is increased; indicating that HDS promotes cadmium migration and accumulation in rice leaves and ears. FIG. 5 shows the result of the present invention on the cadmium content of each part of early season rice under field conditions; as can be seen from FIG. 5, compared with the blank control group, the cadmium content of the rice added with the hydroxamic acid type siderophore preparation group is reduced by 49.72 percent, which is 0.14mg/kg and is lower than the limit of the national food safety standard-food pollutant limit (GB2762-2017) standard of 0.2 mg/kg; the cadmium content in the rice roots, stems, leaves and ears is reduced by 29.32-74.77%.

FIG. 6 shows the result of the ratio of cadmium concentration in each part of early season rice under field conditions according to the present invention; as can be seen from the figure, the proportion of cadmium in the leaves is obviously increased and the proportion of the cadmium in the leaves is obviously reduced by adding the hydroxamic acid type siderophore preparation group; the result shows that the HDS promotes the cadmium to migrate and accumulate in rice leaves, and the result is consistent with the change result in soil culture.

FIG. 7 shows the results of the expression abundance of related genes in the tillering stage, heading stage, filling stage and maturation stage of rice in the field; the expression levels of the OsHMA2 and OsHMA3 genes at the tillering stage and the heading stage of rice are obviously reduced, while the expression levels at the grouting stage and the maturation stage are obviously increased, and the expression level of the OsNramp5 gene at the tillering stage is obviously reduced, while the expression levels are not obviously changed at other stages; the change of the OsHMA2 gene indicates that the addition of the hydroxamic acid siderophore improves the transport of cadmium to overground parts such as stems and leaves in the filling stage and the mature stage of rice, mediates the transfer of cadmium from xylem to phloem at the neck part of the ear and distributes cadmium to the ear through the joint, and the change is also an important reason for the accumulation of Cd in the leaves and the ear; the change of the OsHMA3 gene indicates that the addition of the hydroxamic acid type siderophore improves the capability of chelating and fixing cadmium to root vacuoles in the filling stage and the mature stage of rice, namely, the absorption capability of cadmium is reduced; the changes of the OsHMA3 gene and the OsNramp5 gene together indicate that the addition of the hydroxamic acid type siderophore only improves the absorption of cadmium by the rice roots in the tillering stage and the heading stage; in general, the hydroxamic acid type siderophore reduces the absorption of cadmium by the roots of the rice in the filling stage and the mature stage by regulating the expression of related genes of the rice, enhances the transfer and accumulation of the cadmium from the roots and stems to leaves and ears, and finally reduces the content of the cadmium in the rice.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

Preparation of hydroxamic acid type siderophore formulations:

the hydroxamic acid type siderophores in the following specific examples were prepared by the following specific methods: pseudomonas fluorescens HMP01 is selected from a solid LB culture dish and added into an iron-deficient MSM culture medium (the main components and the concentration of the culture medium: 7g/L of sodium acetate, 0.1g/L of potassium dihydrogen phosphate, 0.5g/L of ammonium sulfate, 0.5g/L of sodium nitrate, 0.02g/L of calcium chloride and 0.02g/L of magnesium sulfate), the mixture is placed in a constant-temperature gas bath oscillator at 30 ℃ for culture for 96h at the speed of 150r/min (the culture is stopped when the activity of HDS is the highest), then the bacteria liquid is centrifuged in a high-speed centrifuge at the speed of 12000r/min for 10min, and the supernatant is the hydroxamic acid type iron carrier preparation. The concentration of the hydroxamic acid type siderophore in the preparation is 0.1 g/L.

Example 2

Soil culture test of early rice (Zhongjiazao 17):

soil of Hunan Tan Yangkun soil used for experiments is collected in field, and Cd in the soil ²⁺ The concentration is 0.79 plus or minus 0.05mg/kg, and the pH value is 6.71 plus or minus 0.05. The soil is naturally dried and ground through a 20-mesh screen for later use. Loading 10kg of Hunan Tan Yangchun soil into plastic pots, transplanting the Jiazao 17 rice seedlings of the three plants, and adding 0.5L (0.2 g/m) of the soil in tillering stage, heading stage, grouting stage and ripening stage ² Dose addition) were continued until rice was harvested for 100 days. The results are shown in FIG. 1 and FIG. 4 (a).

Example 3

Medium rice (Huanghuazhan) soil culture test:

soil of Hunan pond Yangkun used for experiments is collected on the spot, and Cd in the soil ²⁺ The concentration is 0.79 plus or minus 0.05mg/kg, and the pH value is 6.71 plus or minus 0.05. The soil is naturally dried and ground through a 20-mesh screen for later use. Loading 10kg soil into plastic pot, transplanting three Huanghua seedlings in rice, adding 0.5L (0.2 g/m) at tillering stage, heading stage, grouting stage and ripening stage ² Dose addition) were continued until rice was harvested for 114 days. The results are shown in FIGS. 2 and 4 (b).

Example 3

Soil culture test of late rice (Yuzhenxiang):

soil of Hunan Tan Yangkun used for experiment is collected on the spot, and soilMiddle Cd ²⁺ The concentration is 0.79 plus or minus 0.05mg/kg, and the pH value is 6.71 plus or minus 0.05. The soil is naturally dried and ground through a 20-mesh screen for later use. Loading 10kg soil into plastic pot, transplanting three Huanghua seedlings in rice, adding 0.5L (0.2 g/m) at tillering stage, heading stage, grouting stage and ripening stage ² Dose addition) were continued until rice was harvested for 140 days. The results are shown in FIGS. 3 and 4 (c).

Example 4

Actual field experiments for early rice (Zhongzao 35):

selecting three actual cadmium-polluted paddy fields of Yankeecun, namely Cd ²⁺ Concentration of 0.76 +/-0.03 mg/kg, pH value of 6.21 +/-0.15, sowing the seeds 35% in the middle ten days of 4 months, selecting two experimental points in each field, and adding 5L (1 g/m) of the fertilizer in tillering stage, heading stage, grouting stage and ripening stage ² Dosed) with HDS formulations, managed with actual agronomic measures of the field, continuously cultured until the rice is harvested, for 112 days. The results are shown in FIGS. 5 and 6.

Example 5

Expression of rice Cd transporter:

leaf tips of field early rice in tillering stage, heading stage, filling stage and mature stage are respectively taken and stored at-80 ℃ and transported to a laboratory. Then, the mixture was pulverized with liquid nitrogen, a high-purity rice RNA solution was obtained using a plant total RNA extraction kit (Shanghai Biotech Co., Ltd.), a rice cDNA solution was obtained using a reverse transcription kit (Shanghai Saint Biotech Co., Ltd.), and changes in the expression levels of cadmium transporter genes such as rice OsHMA2, OsHMA3 and OsNramp5 were measured using fluorescent quantitative PCR (Lightcycler 96, Roche).

The technical scope of the invention claimed by the embodiments of the present application is not exhaustive, and new technical solutions formed by equivalent replacement of single or multiple technical features in the technical solutions of the embodiments are also within the scope of the invention claimed by the present application; in all the embodiments of the present invention, which are listed or not listed, each parameter in the same embodiment only represents an example (i.e., a feasible embodiment) of the technical solution, and there is no strict matching and limiting relationship between the parameters, wherein the parameters may be replaced with each other without departing from the axiom and the requirements of the present invention, unless otherwise specified.

The technical means disclosed by the scheme of the invention are not limited to the technical means disclosed by the technical means, and the technical scheme also comprises the technical scheme formed by any combination of the technical characteristics. While the foregoing is directed to embodiments of the present invention, it will be appreciated by those skilled in the art that various changes may be made in the embodiments without departing from the principles of the invention, and that such changes and modifications are intended to be included within the scope of the invention.

The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.

Claims

1. The application of hydroxamic acid type siderophore is characterized in that: the hydroxamic acid type siderophore is applied as a rice cadmium reduction preparation;

the hydroxamic acid type siderophore has the following chemical structural formula:

2. the use of a hydroxamic acid type siderophore according to claim 1, wherein: the hydroxamic acid type siderophore is added into cadmium-polluted paddy soil for planting paddy to reduce the enrichment of cadmium in paddy grains.

3. Use of a hydroxamic acid type siderophore according to claim 1 or 2, wherein: the adding amount of the hydroxamic acid type iron carrier in the cadmium-polluted paddy soil is 0.2-1 g/m ² 。

4. Use of a hydroxamic acid type siderophore according to claim 1 or 2, wherein: the hydroxamic acid type siderophore is added in the whole growth period of the rice.

5. The use of a hydroxamic acid form siderophore according to claim 4, wherein: the hydroxamic acid type siderophore is added in any period or any several periods of the rice at the turning green stage, the tillering stage, the heading stage, the filling stage and the mature stage.

6. The use of a hydroxamic acid form siderophore according to claim 4, wherein: the hydroxamic acid type siderophore is added at the rhizosphere of rice.

7. The use of a hydroxamic acid type siderophore according to claim 1, wherein: cd in cadmium-polluted paddy soil ²⁺ The concentration is 0.3-1 mg/kg, and the pH value is 6.5-7.5.