Application of Humic Acid in Improving the Phytoremediation Effect of Heavy Metals in Water

Abstract

Disclosed is the application of humic acid in improving the phytoremediation effect of heavy metals in water, and relates to the technical field of environmental ecological engineering. The invention discloses the application of humic acid in improving the phytoremediation effect of heavy metals in water, wherein the phytoremediation of heavy metals is to reduce the content of heavy metals in water by planting Vallisneria chinensis. Adding humic acid can slow down the chlorosis of Vallisneria vallissima leaves under heavy metal poisoning and increase the accumulation of heavy metals in Vallisneria vallissima leaves and roots. At the same time, it can also reduce the leaching ability of heavy metals in water, enhance the enzyme activities related to active oxygen metabolism in plants by stimulating the synthesis of protein and enzymes in various organs of plants, reduce the MDA concentration in plants, adjust the active oxygen content in plants, reduce the degree of membrane lipid peroxidation and enhance the resistance of plants to heavy metals.

Description

TECHNICAL FIELD The invention relates to the technical field of environmental ecological engineering, in particular to an . BACKGROUND Phytoremediation is the most commonly used method of bioremediation of heavy metals, which uses plants to remove pollutants from water or soil. Phytoremediation is often used to selectively remove low-concentration heavy metals, and has the advantages of high efficiency, environmental protection, low cost and no secondary pollution. As a typical representative of phytoremediation of water, submerged plants can not only maintain the diversity of aquatic species and functions, but also play a significant role in purifying water, and play a huge ecological role. Vallisneria natans is a typical submerged plant with no erect stem and banded leaves. It has strong environmental adaptability, recovery ability and water purification function, and is considered as important vegetation for aquatic ecosystem reconstruction. Compared with other types of aquatic plants, Vallisneria val / issima is particularly prominent in heavy metal tolerance and enrichment. However, high concentrations of heavy metals have a great acute toxic effect on submerged plants, which can easily cause irreversible damage to plant cells and then affect the effect of phytoremediation. Therefore, it is particularly important to improve the enrichment efficiency of heavy metals and plant tolerance. In recent years, natural organic acids existing in humus have attracted extensive attention of researchers. Humus is considered as a natural chelating agent, and humic acid (HA), as the main component of humus, contains a large number of functional groups with various properties, such as carboxyl, phenolic hydroxyl, alcoholic hydroxyl, carbonyl and so on, so HA can change the morphology and bioavailability of heavy metals. At present, most studies are focused on the effects of HA on soil plants, which indicates that HA can directly interact with soil components to change soil characteristics, thus affecting the forms and bioavailability of heavy metals, and can also affect the retention capacity and mobility of heavy metals in soil through nonspecific and specific adsorption and precipitation, complexation and interaction with heavy metals. For aquatic plants, the application of HA in remediation of heavy metals in water is relatively rare, and there are still many controversies about the role of HA due to the differences between plants and heavy metals. SUMMARY The purpose of the invention is to provide the , so as to solve the problems existing in the prior art. To achieve the above objective, the present invention provides the following scheme: The invention provides an , wherein phytoremediation of heavy metals in water is to reduce the content of heavy metals in water by planting Vallisneria chinensis. Further, the application is to slow down the chlorosis of Vallisneria natans leaves under heavy metal poisoning. Further, the application is to increase the accumulation of heavy metals in leaves and roots of Vallisneria natans. Further, the application is to reduce the leaching ability of heavy metals in water. Further, the application is to enhance the resistance of Vallisneria chinensis to heavy metals. The invention also provides a method for improving the remediation effect of Vallisneria vallissima on heavy metals in water by using humic acid, which comprises the following steps: adding humic acid into the water, wherein the concentration of humic acid in the water is 0.5-2 mg-l'1. The invention discloses the following technical effects: According to the research of the invention, by adding humic acid, the chlorosis of Vallisneria vallissima leaves under heavy metal poisoning can be slowed down, the accumulation of heavy metals in Vallisneria vallissima leaves and roots can be increased, the leaching ability of heavy metals in water can be reduced, the enzyme activity related to active oxygen metabolism in plants can be enhanced, the MDA concentration in plants can be reduced, the active oxygen content in plants can be adjusted, the degree of membrane lipid peroxidation can be reduced, and the resistance of plants to heavy metals can be enhanced. BRIEF DESCRIPTION OF THE FIGURES In order to more clearly explain the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention, and for ordinary technicians in the field, other drawings can be obtained according to these drawings without paying creative efforts. Fig. 1 is a schematic diagram of a device for studying the influence of humic acid on Vallisneria chinensis and biofilm under heavy metal stress; Fig. 2 shows the growth of Vallisneria vallissima under different conditions on the 0th, 10th and 20th day. Fig. 3 shows the change of fresh weight increase of Vallisneria vallissima in each group. Different letters (a-c) indicate significant differences (P < 0.05). Fig. 4 shows the changes of total chlorophyll and Fv / Fm value of Vallisneria vallissima in each group, in which (a) is the change of total chlorophyll and (b) is the change of Fv / Fm value. Fig. 5 shows the change of Pb and Cd concentrations in water, in which (a) the change of Pb concentration and (b) the change ofCd concentration; Fig. 6 shows the changes of Pb and Cd concentrations in the leaves and roots of Vallisneria natans, in which (a) the changes of Pb concentration and (b) the changes ofCd concentration, and the letters a-c indicate significant differences (P < 0.05). Fig. 7 is the three-dimensional fluorescence spectrum ofDOM in water samples ofCT group; Fig. 8 is the three-dimensional fluorescence spectrum ofDOM in HA1 water samples; Fig. 9 is the three-dimensional fluorescence spectrum ofDOM in HA2 water samples; Fig. 10 is the three-dimensional fluorescence spectrum ofDOM in HA3 water samples; Fig. 11 is the three-dimensional fluorescence spectrum ofDOM in M water samples; Fig. 12 is the three-dimensional fluorescence spectrum ofDOM in HA1_M water samples. Fig. 13 is the three-dimensional fluorescence spectrum ofDOM in water samples of group HA2_M; Fig. 14 is the three-dimensional fluorescence spectrum ofDOM in water samples of group HA3_M; Fig. 15 shows the changes of total protein (TPr) and metallothionein (MTs) in the leaves of Vallisneria natans in each group, in which (a) is the change of TPr, (b) is the change of MTs, and the letters a-c indicate significant differences (P < 0.05). Fig. 16 shows the effect of humic acid on SOD(a) and POD(b) activities of Vallisneria chinensis under Pb and Cd stress, and the letters a-b represent significant differences (P < 0.5). Fig. 17 shows the effect of HA on MDA content of Vallisneria chinensis under Pb and Cd stress, and the letters a-e represent significant differences (P < 0.5). Fig. 18 shows the effects of different concentrations of HA on the methylation level of Vallisneria natans leaves. Fig. 19 shows the effects of different concentrations of HA on DNA methylation level of Vallisneria natans leaves under Pb and Cd stress. DESCRIPTION OF THE INVENTION Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description ofsome aspects, characteristics and embodiments of the present invention. It should be understood that the terms used in this invention are only for describing specific embodiments, and are not used to limit the invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Any stated value or intermediate value within the stated range, as well as any other stated value or each smaller range between intermediate values within the stated range, are also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by the ordinary technicians in the field of this invention. Although the present invention only describes the preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated documents, the contents of this specification shall prevail. Vlthout departing from the scope or spirit of the present invention, it is obvious to those skilled in the art that many modifications and changes can be made to the specific embodiments of the present invention. Other embodiments obtained from the description of the present invention will be obvious to the skilled person. The description and example of that present invention are exemplary only. The words "including", "including", "having" and "containing" used in this paper are all open terms, that is, they mean including but not limited to. The submerged plants used in this invention, Vallisneria angustifolia, are all from Shanghai Yuetian Biotechnology Co., Ltd. (Shanghai, China). Before the experiment, the obtained Vallisneria vallissima should be washed with tap water, and put into the same incubator for 2 weeks for later use. The incubation temperature is 25 i 2°C, and the illumination: dark time is 12: 12 h. Example 1 !. Experimental methods As shown in Fig. 1, 7 g of Vallisneria chinensiswas transplanted into a 5 ! plexiglass container (ADAaquAsoil, AquADesign Amano Company, Japan), 3 ! tap waterwas added into the container, and the roots of Vallisneria chinensis were inserted into 50mm quartz sand. During the experiment, the growth condition of Vallisneria vallissima was 25 i 2°C, the ratio of light to darkness was 12:12 h, and the light intensity was 80umol.m'2.s1. Add 1.0 mg-l'1 Pb, Cd mixture and humic acid (HA) with different concentrations into the container. Each experimental group and the control group are set in three groups in parallel. The experimental grouping settings are shown in Table 1, and the experimental period is 20d. Table 1 (mg'l'1) ___- ___- ___- __ ___- ||. Main detection indicators and characterization methods 2.1 Determination of main water quality parameters (1) TN and TOC Use TOC-L analyser to measure the concentration ofTN and TOC in samples. Before each water sample is put on the machine, it needs to be filtered by 0.45 umol ofwater system membrane, and each parallel sample is repeatedly measured for 3 times. (2) P (TP and soluble phosphate) The concentration ofTP in waterwas determined by potassium persulfate digestion- phosphorus molybdenum blue spectrophotometry, and the content of soluble phosphate in water samples was determined by molybdenum blue microplate method. (3) pH value Use a portable pH meter (YSI, USA) to measure the pH value, and repeat the measurement for 3 times for each parallel sample. 2.2 Measurement of biomass and photosynthesis related indexes (1) Fresh weight (FVV) Use analytical balance to measure FW of Vallisneria vallissima. Before weighing, it is necessary to dry the moisture and impurities in the leaves. (2) Total chlorophyll Chl(a+b) content Wash 0.2 g of Vallisneria vallissima leaves, dry them, put them into 10 ml of96% ethanol solution for dark leaching for 24 hours, and measure their absorbance values at 649 nm and 665 nm by spectrophotometer respectively. Total Chl(a+b) content is calculated as follows: c = 12.7.4553 2.59.4545 Cb = 223501545 46814553 C = Ca + ch (3) Pprimary light energy conversion efficiency of 3)PS|| (Fv / Fm) Cut the leaves of Vallisneria vallissima of each group by about 3cm, and measure Fv / Fm value by hand-held chlorophyll fluorescence measuring instrument, and repeat the measurement for 3 times for each sample. 2.3 Detection of heavy metals (1) Plant digestion At the end of the experiment, the harvested leaves and roots of Vallisneria vallissima were dried and ground. A certain amount of powder of leaves and roots was weighed, and a certain proportion ofHNO3 and HCIO4 were added to digest them. The digested sample was filtered by a 0.45um water system membrane, and then tested on the computer after constant volume. (2) On-board detection lnductively coupled plasma atomic emission spectrometry (ICP-MS) was used to determine the concentrations of Pb and Cd. Both water samples and plant-digested samples need to be filtered by 0.45um water system membrane before being put on the machine. 2.4 Three-dimensional fluorescence spectrum detection The water sample was filtered by a 0.45 uM water system membrane, and then the corresponding fluorescence spectrum of the sample was obtained by using a three-dimensional fluorescence spectrometer. ln which the scanning ranges of excitation wavelength and emission wavelength are set to 240-800nm and 250-800nm respectively. The scanning speed was set to 1000 nm / min and the slit width was set to 10 nm. 2.5 Determination of protein and related enzyme activities (1) Sample pre-treatment At the end of the experiment, 1g of Vallisneria vallissima leaves were collected, washed with deionized water and frozen in liquid nitrogen to prevent inactivation. The obtained leaves and 0.1MPBS solution were ground at 4°C according to the weight (g): volume (mL) of 1: 9, centrifuged at 3500rpm for 10min, and the supernatantwas collected and stored in an ultra-low temperature refrigerator at -80°C for later use. (2) Determination of related enzyme activities The activities of total protein (TPr), Metallothioneins (MTs), Superoxide dismutase (SOD), Peroxidase (POD), Catalase (CAT) etc. are all determined by relevant kits, and the specific operation steps are carried out according to the corresponding kit instructions. (3) Determination of Malondialdehyde (MDA) concentration The concentration ofMDA in the leaves of Vallisneria val / issima was determined by using related kits. The principle is that thiobarbituric acid (TBA) is condensed with MDA in lipid peroxide degradation products, and the red product has the maximum absorption peak at 523nM. 2.6 Extraction and determination ofDNA from plant leaves At the end of the experiment, the leaves of each group of Vallisneria val / issima were collected and stored in an ultra-low temperature refrigerator at -80°C for later use. In this invention, the HP Plant DNAKit ofOmega company is used to extract the DNA of each group of Vallisneria chinensis leaves. The specific extraction process was carried out according to the instructions of the kit. 2.7 Detection of methylation of whole genome DNA The extracted sample DNA was used to detect DNA cytosine methylation (5-mC) level. Use methyl flash TM global DNA methylation (5-MC) ELISA easykit rapid kit for detection. The formula of DNA methylation (5-mC) level is as follows: 5 mania =13%:+ 5 >< lüüüfu 2.8 DNA methylation sensitivity analysis (MSAP) The MSAP analysis experiment is carried out, in which the sequence of linker and primer is shown in Table 2. Table 2 Information of linker and primer sequence The amplified PCR products were denatured at 94°C for 10min, then analysed by vertical electrophoresis with 6% denatured polyacrylamide gel, and then used for subsequent band analysis after silver staining. 2.9 Data analysis Origin 8.5 is mainly used for drawing, and SPSS 22.0 is used for Analysis of Variance (ANOVA) to compare the significant differences of each group of data. Among them, the bands ofMSAP spectrum are counted by Quantity One software and converted into 0 / 1 matrix of phenotypic data for subsequent statistical analysis. lll. Experimental results 3.1 Analysis of changes in growth and photosynthesis of Vallisneria vallissima Fig. 2 shows the growth of Vallisneria vallissima on the 0th, 10th and 20th days under different conditions. It can be observed that Vallisneria val / issima has been growing vigorously in the groups without exogenous heavy metals (CT, HA1, HA2, HA3 groups), which shows that humic acid (HA) has no obvious inhibition on the growth of Vallisneria vallissima. However, in the groups with exogenous heavy metals (M, HA1_M, HA2_M, HA3_M groups), the degree of leaf chlorosis became more and more serious from the 0th day to the 20th day. On the 20th day, compared with the M group, with the increase of exogenous HA concentration, the number of leaves of Vallisneria invo / ucrata that lost their green decreased relatively, and the chlorosis was alleviated. Therefore, it can be inferred that adding HA may slow down the phenomenon of leaf chlorosis of Vallisneria invo / ucrata, and alleviate the toxicity of Pb and Cd heavy metals to Vallisneria invo / ucraza. Fig. 3 reflects the effect of adding different concentrations of HA on the fresh weight (FVV) increase of Vallisneria natans in each group. Compared with CT group, in the treatment group without exogenous heavy metals, with the increase of HA concentration, the FW of Vallisneria invo / ucrata increased at first and then decreased, and both groups HA1 and HA2 were significantly higher than CT group (P <0.05). This shows that exogenous HA may promote the growth of Vallisneria val / issima in a certain concentration range. In the exogenous heavy metal treatment group, compared with the M group, with the increase of HA concentration, the increase of Vallisneria chinensisFWshowed an upward trend. This shows that HA can alleviate the toxic effects of heavy metals on Vallisneria invo / ucrata to a certain extent. Fig. 4 (a) and (b) respectively reflect the effects of different concentrations of HA on the photosynthesis of Vallisneria vallisneriana under different conditions, and the total chlorophyll content (Chl(a+b)) and Fv / Fm values of its leaves were measured. Fv / Fm is the index of photoinhibition in leaves, and the decrease of Fv / FM indicates that the photosynthesis system is destroyed. It can be seen from the figure that in the treatment group without exogenous heavy metals, compared with the CT group, the content of Chl(a+b) was not significantly increased by adding different concentrations of HA alone. However, with the increase of HA concentration, Fv / Fm values of groups HA1, HA2 and HA3 showed an upward trend, which indicated that proper amount of HA could promote the photosynthesis of plants. Compared with CT group, Chl(a+b) content and Fv / Fm value of Vallisneria vallissima in group M were significantly lower (P<0.5), indicating that heavy metals would destroy the photosynthesis system of plants. In the treatment group with exogenous heavy metals, compared with the M group, with the increase of exogenous HA concentration, the Chl(a+b) content of Vallisneria chinensis showed a trend of first increasing and then decreasing, but itwas always higher than that of the M group. In addition, Fv / Fm value was significantly increased (P>0.5). To sum up, the results show that a certain concentration of HA can alleviate the chloroplast damage caused by heavy metals. The chlorophyll content first increased and then decreased, which may be due to the decrease of chlorophyll content caused by the influence of higher concentration of HA on the metabolism of related carbohydrate enzymes. 3.2 Changes of heavy metals in water and plant tissues 3.2.1 Changes of Pb and Cd concentrations in water Fig. 5 reflects the changes of the concentrations of Pb (a) and Cd (b) in water at the Oth, 10th and 20th days. It can be seen from the figure that in the treatment group with exogenous heavy metals, the concentrations of heavy metals Pb and Cd decreased significantly on 10th and 20th day (P<0.05). lt shows that the decrease of the concentration of heavy metals in water is due to the absorption and enrichment of heavy metals by Vallisneria natans. On the 20th day, compared with M group, with the increase of HA concentration, the concentrations of Pb and Cd in water decreased, so it can be inferred that adding HA can reduce the leaching ability of heavy metals in water. 3.2.2 Changes of Pb and Cd concentrations in Vallisneria natans leaves and roots In order to further study the effect of HA on the absorption and enrichment of heavy metals by Vallisneria natans, the contents of Pb and Cd in the leaves and roots of Vallisneria natans were determined, as shown in Fig. 6. Obviously, in the treatment group with exogenous heavy metals, compared with the M group, with the increase of exogenous HA concentration, the concentrations of Pb and Cd in the leaves of Vallisneria nata showed a trend of first increasing and then decreasing, especially the Pb content in the leaves decreased significantly (HA3_M group). This shows that the appropriate concentration of HA can promote the absorption of heavy metals by Vallisneria natans leaves, and the effect of HA on plant accumulation is different for different kinds of heavy metals. However, the concentrations of Pb and Cd in the roots of Vallisneria chinensis increased significantly with the increase of exogenous HA concentration (P<0.05), and both ofthem were higher than those in M group, which indicated that exogenous HA could significantly increase the accumulation of heavy metals Pb and Cd in the roots of Vallisneria chinensis. It is considered that, on the one hand, the interaction between HA and organic acids secreted by plant roots and rhizosphere microbial activities will decompose HA into small molecular units easily absorbed by plants, thus promoting the absorption of the complex of HA and heavy metals by plants; On the other hand, HA can increase the ductility of plant cell walls, thus promoting the accumulation and transport of Pb and Cd. 3.3 Three-dimensional fluorescence Fig. 7-14 is the three-dimensional fluorescence spectrum of each group ofwater samples obtained at the end of the experiment, in which the peak of fluorescent group in V is the area where humic acid-like acid is located in DOM. Compared with CT group, with the increase of HA concentration, the fluorescence intensity of humic acid-like area is increasing. This shows that with the increase of the added HA concentration, the residual HA concentration in the water increases, and HA is difficult to degrade in the water. Compared with HA1-M group and HA1 group, HA2-M group and HA2 group and HA3-M group and HA3 group, the fluorescence intensity of ha1-m group and ha2-m group is relatively weak. The results show that HA in water may be complexed with heavy metals. In addition, compared with the CT group, the fluorescence of other groups was enhanced at the position of Ex / Em=270 / 330nm, which was considered to be the dissolved microbial metabolites produced by the degradation process of microorganisms and bacteria. Some studies suggested that tryptophan-Iike components were closely related to the activities of bacterial communities, and they were particularly sensitive to environmental changes. 3.4 Changes in protein As shown in Fig. 15, in the treatment group without exogenous heavy metals, compared with the CT group, the concentrations of total protein (TPr) and metallothionein (MTs) did not change significantly with the increase of the concentration of added HA (P<0.5), which indicated that adding HA alone would not change the synthesis of protein in Vallisneria vallissima. Compared with CT group, TPr concentration and MTs concentration of Vallisneria vallissima leaves in group M increased (P<0.5), which indicated that Vallisneria vallissima would resist heavy metal stress by increasing the synthesis of protein. In the treatment group with exogenous heavy metals, compared with the M group, with the increase of external HA concentration, TPr concentration of Vallisneria natans leaves first decreased and then increased, while MTs concentration gradually decreased with the increase of HA concentration. It can be concluded that HA can enhance the resistance of plants to heavy metals by regulating the synthesis of protein in various organs of plants under stress such as heavy metals. 3.5 Changes of antioxidant enzyme system and MDA concentration As shown in Fig. 16, compared with CT group, SOD and POD of HA group increased in the treatment group without exogenous heavy metals, indicating that HA can improve the activity of antioxidant enzymes of Vallisneria natans. In the treatment group without exogenous heavy metals, compared with the M group, the activities ofSOD and POD both increased, which indicated that under Pb and Cd stress, HA could stimulate the synthesis of Vallisneria angustifolia enzyme, enhance the activities ofSOD and POD to eliminate the free radicals generated by heavy metals stress, thus reducing the toxic effects. As shown in Fig. 17, the MDA concentration in M group was significantly higher than that in CT group (P<0.5). Compared with CT group, the MDA concentration in Vallisneria natans leaves decreased with the increase of HA concentration in the treatment group without exogenous heavy metals. In the treatment group with exogenous heavy metals, compared with the MDA concentration in M group, with the increase of HA concentration, the MDA concentration in Vallisneria natans leaves decreased continuously. It can be inferred that the alleviating effect of HA on Vallisneria vallissima under Pb and Cd stress increases with the increase of HA concentration. To sum up, under the stress of heavy metals, HA can stimulate the synthesis of protein and enzymes in various organs of plants, enhance the activities of SOD, POD and other enzymes related to the metabolism of active oxygen in plants, reduce the concentration in plants, adjust the content of active oxygen in plants, reduce the degree of membrane lipid peroxidation, and keep the plants growing faster, thus maintaining the permeability of cell membranes and enhancing the resistance of plants to heavy metals. The positive effects of HA on the growth of submerged plants are influenced by HA concentration and plant species. Through the above research, we can draw the following conclusions: (1) The addition of HA has a positive effect on the growth of Vallisneria natans, which can slow down the chlorosis of Vallisneria natans leaves under heavy metal poisoning. (2) HA can increase the accumulation of Pb and Cd in leaves and roots of Vallisneria natans, and reduce the leaching ability of heavy metals in water. (3) Under the stress of heavy metals, HA can stimulate the synthesis of protein and enzymes in various organs of plants, enhance the activities of SOD, POD and other enzymes related to active oxygen metabolism in plants, reduce the concentration ofMDA in plants, regulate the content of active oxygen in plants, reduce the degree of membrane lipid peroxidation, and enhance the resistance of plants to heavy metals. IV. Effect of humic acid on the apparent genetic diversity of Vallisneria chinensis DNA under heavy metal stress 4.1 Effects of HA, Pb and Cd stress on DNA methylation of Vallisneria natans leaves Electrophoretic position of PCR amplification products in a certain position of the gel with the same mobility, there are DNA bands recorded as 1, and no DNA bands recorded as 0, which are then transformed into 0,1 data matrix, and the bands ofDNA methylation types in each group of samples are counted and analysed. As shown in Table 3, the number of type I and IV bands obtained by Vallisneria vallissima is large, with an average of 228.9 and 236.6 bands respectively. The methylation status types of groups without exogenous heavy metals (CT, HA1, HA2 and HA3 groups) are similar, and those with exogenous heavy metals (M, HA1_M, HA2_M and HA3_M groups) are similar, while the hemimethylation status types (type II) of Vallisneria chinensis are significantly different between groups without exogenous heavy metals and groups with exogenous heavy metals. Table 3 __-___ As shown in Fig. 18, compared with the CT group, the total methylation level, total methylation level and hemimethylation level of Vallisneria natans leaves under the condition of HA1 increased significantly, while the total methylation level, total methylation level and hemimethylation level under the conditions of HA2 and HA3 did not change significantly. It can be concluded that different concentrations of HA may have different effects on the methylation of Vallisneria natans leaves. In order to further study the effect of HA on DNA methylation of Vallisneria natans leaves under Pb and Cd stress, as shown in Fig. 19, compared with CT group, the total methylation level, total methylation level and semi-methylation level ofM group decreased significantly, especially the total methylation level, from 63.71% to 57.47%. Previous studies have pointed out that environmental factors such as temperature, heavy metals and water stress often lead to the reduction of DNA methylation level in plant genome, and DNA methylation can participate in regulating the expression of heavy metal detoxification transporters and endow plants with the ability to resist heavy metal toxicity. Vlth the addition of different concentrations of HA, the total methylation level, total methylation level and semi-methylation level have an upward trend, especially in the HA3_M group, the total methylation level and semi-methylation level are obviously increased, which are 1.1 and 1.4 times of the original level respectively. Therefore, it can be inferred that adding different concentrations of HA will bring different levels ofDNA methylation to Vallisneria vallissima leaves under Pb and Cd stress. We concluded that the positive effect of HA on Vallisneria vallissima under heavy metal stress may be related to the expression of related genes caused by the change of DNA methylation level. 4.2 Changes ofDNA methylation status in Vallisneria natans leaves under HA, Pb and Cd stress There are four types ofMSAP methylation analysis results, as shown in Table 4. Type !: HAP ll and MSP ! have bands, which are (1,1); Type ll: HAP ll has band and MSP ! has no band, which is (1,0); Type lll: HAP ll has no band, MSP ! has band, which is (0,1); Type IV: HAP II and MSP I have no band, which is (0,0). Table 4 Type of enzymatic Type OfDNA Mode ofDNA methylation __ ____ __ ...- medial and lateral cytosme ___ The MSAP method was used to analyse the possible methylation status changes of genomic DNA of Vallisneria vallissima leaves in each group. There were 15 types, of which A, B and C were related methylation band types, while D was unchanged band type, as shown in Table 5. Among the five methylation patterns of type A (demethylation type, methylation enhancement type), taking CT group as control, the highest proportion under Pb and Cd stress (M group) was the control type l treatment type IV (1,1,0,0), accounting for 12.45%; The lowest is the control type I treatment type lll (1,1,0,1), accounting for 3.16%. This data shows that under Pb and Cd stress, the methylation pattern of genomic DNA of Vallisneria natans is mainly no methylation or medial cytosine hemimethylation, and the pattern of no methylation or medial cytosine hemimethylation to medial cytosine complete methylation is the least. Among the five methylation patterns of type B (demethylation type, methylation weakening), the highest proportion of group M is that of control type IV treatment type l (0,0,1,1), accounting for 20.16%; The lowest is the control type iv treatment type lll (0,0,0,1), accounting for 3.16%. This data shows that under Pb and Cd stress, the pattern of DNA methylation of Vallisneria natans genome is mainly from inside and outside cytosine complete methylation to no methylation or inside cytosine semi-methylation, and the pattern from inside and outside cytosine complete methylation to inside cytosine complete methylation is the least. In addition, in the HA-only group, with the increase of HA concentration, the proportion ofDNA methylation pattern in each group of Vallisneria natans did not show significant correlation with HA concentration. Among the types with re-methylation, the proportion of control type I treatment type IV (1,1,0,0), control type I treatment type lll (1,1,0,1) and control type lll treatment type IV 0,1,0,0) in M group was higher than that in HA group alone. After adding different concentrations of HA, the proportion of type IV (1,1,0,0) of control type l treatment in HA_M group increased, while that of control type I treatment type lll (1,1,0,1) and control type lll treatment type IV 0,1,0,0) decreased. Among the demethylation types, the proportion of control type IV treatment type l (0,0,1,1) in group M was significantly higher than that in group HA alone, and the proportion of control type IV treatment type I (0,0,1,1) in group HA_M showed a downward trend after adding different concentrations of HA. lt indicated that HA contributed to the demethylation of Vallisneria vallissima under heavy metal stress. In the two methylation patterns of type C (methylation amorphous), the proportion of each group is low. Among the three methylation patterns of D-type (methylation invariant), the ratio of the three methylation invariant forms in HA group and HA_M group is higher than that in M group, which indicates that HA can play an important role in maintaining methylation of Vallisneria natans under heavy metal stress. Table 5 Analysis of methylation pattern of Vallisneria vallissima leaves under Pb and Cd stress Type of group group M M M In A 8.73 3.84 4.04 3.16 3.49 3.04 4.61 . 4.48 2.93 3.82 5.93 4.93 3.85 3.81 10.85 6.77 9.21 12.45 12.73 12.96 13.03 _u-uu 1.89 1.81 2.47 4.35 3.29 3.04 3.21 3.54 6.09 5.84 4.94 3.70 3.64 3.01 _... 4.25 4.06 3.37 3.95 3.90 4.05 3.61 " 4.48 6.32 7.19 20.16 19.10 19.84 14.83 5.42 8.35 6.52 5.14 3.49 3.24 6.81 '-.lllllllaau _ 1.42 0.90 1.35 0.40 0.00 0.40 1.60 .-'"'......." 28.07 36.34 32.58 22.13 24.23 24.90 22.85 'uu 6.37 4.97 5.39 1.78 1.85 3.24 2.20 9.20 9.26 8.76 5.34 6.37 6.88 5.81 Under the stress of external environment, plants will produce epigenetic variation. DNA methylation can regulate the growth and development of plants without changing the genome sequence, which is the main way of epigenetic effect. There are two main modes for plants to regulate their gene expression: demethylation and demethylation. This is a way to protect plants from external environmental stress. The demethylation process is thought to be linked with genome imprinting, transcription regulation genes, etc., which can regulate the growth and development of plants and play an important role in genome defence, while the demethylation process is thought to affect life activities such as chromosome activity, embryo growth, cell differentiation and canceration, which is beneficial to gene expression. Studies have shown that under the stress of heavy metals, the methylation pattern of plant DNA is mainly changed by re- methylation, and it is pointed out that genomic DNA may turn off the expression of some related genes and inhibit transcription through methylation, thus reducing the toxicity of heavy metals and enhancing the adaptability of plants to heavy metals stress. However, in this study, the addition of HA changed the proportion ofsome demethylation and demethylation types of Vallisneria val / issima under heavy metal stress, and increased the proportion of maintenance methylation types, indicating that the positive effect of HA on the response of Vallisneria vallissima under heavy metal stress may be to regulate its gene expression by changing the types of demethylation and demethylation and increasing maintenance methylation types, thus enhancing the adaptability and tolerance of Vallisneria vallissima under heavy metal stress. The above-mentioned embodiments only describe the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design 5 spirit of the present invention, all kinds of modifications and improvements made by ordinary technicians in the field to the technical scheme of the present invention should fall within the protection scope determined by the claims of the present invention. SequenceList10> FudanUniversity10> FuhuanQingyunTechnology(Zhejiang)DirectCo.,LTD20> ApplicationofHumicAcidinImprovingthePhytoremediationEffectof avyMetalsinWater30> SHX-HumicAcidNL50> CN202210254262.551> 2022-03-1560> 670> SIPOSequenceListing1.010> 111> 1712> DNA13> ArtificialSequence00> 1cgtagactgcgtacc 1710> 211> 1812> DNA13> ArtificialSequence00> 2ttggtacgcagtctac 1810> 311> 1612> DNA13> ArtificialSequence00> 3cgatgagtcctgag 1610> 411> 1412> DNA13> ArtificialSequence00> 4ctcaggactcat 1410> 511> 1612> DNA13> ArtificialSequence