Lantana camara L., also known as Wusemei, Wusexiuqiu, Biansecao etc., in Chinese, is native to South America and now distributed in nearly 50 countries, which is often cultivated for ornamental purposes in gardens throughout the world. It is common in the coastal beaches and open areas of Hainan Province, the tropical region of China. Lantana camara L. is classified as one of the invasive species at hazard level II and has toxicity related to the metabolism of triterpene lipids. Its flowers, leaves and roots have the effects of dispersing knots and relieving pain, clearing heat and removing toxicity, dispelling exogenous wind-cold and relieving itching. Foreign studies have shown that its chemical constituents are mainly lantadene, lantanolic acid, tannins, resin, reducing sugar and alkaloid, and α-Caryophyllene, β-Caryophyllene, γ-Terpinene, α-Pinene and P-Cymene and other volatile oils (Zhou et al., 2009, Journal of Guangxi University (Natural Science Edition), 34(4): 494-497; Tao et al., 2017, Journal of Wenshan Teachers College, (3): 116-120; Vardien et al., 2012). Khan et al. (2003) extracted essential oil from the fruits and stems of Lantana camara. Pradeep et al. (2013) studied the bacteriostatic effect of Lantana camara on Escherichia coli and Pseudomonas aeruginosa. Shriniwas and Subhash (2018) found the antioxidant, antibacterial and potential cytotoxicity of the leaves of Lantana camara, and also found that its extract has anti-mycobacterium activity. There are few studies on the separation and extraction of the active components of Lantana camara and their biological activities in China. Tao et al. (2017, Journal of Wenshan Teachers College, (3): 116-120) have studied the activity of aqueous extracts of different parts of Lantana camara against Panagrellus redivivus. Luo et al. (2015, Heilongjiang Animal Science and Veterinary Medicine, (4): 164-167) found that the water extract and ester extract of Lantana camara have antibacterial effects on Escherichia coli and Staphylococcus aureus.

Protein is not only the functional product of gene expression of organisms, but also the executor of all life activities. It can be more direct and effective to analyze the essence of life activities from the protein level. Two-dimensional electrophoresis (2-DE) is one of the main core technologies of proteomics research and has been widely used in the separation and extraction of animal and plant proteins with its high throughput, high resolution and other characteristics (Han et al., 2020). In addition to several biological macromolecules, the important function of metal ions in plants is that they can absorb a variety of nutrients during the growth process, and the content of metal ions will also vary with the origin or species of different plants (Cai et al., 2001, Guihaia, 21(1): 91-94). Inductively coupled plasma-mass spectrometry (ICP-MS) has the characteristics of high resolution and sensitivity, low detection limit, low sample consumption, and simultaneous detection of multiple elements, which has been widely used for the analysis of metal ions in plants (Zou et al., 2016, Soils, 48(5): 844-853; Wang et al., 2004, Journal of Jilin University (Science Edition), 42(3): 455-457).

The flower colors of Hainan Lantana camara is mainly pink and yellow. Gas chromatography/mass spectrometry (GC/MS) has been used to qualitatively identify and analyze the volatile oil from fresh Hainan Lantana camara flowers, branches and leaves (He et al., 2014), but there is no report on the analysis of protein and metal ions in the flower of Hainan Lantana camara. Lantana camara is a kind of plant medicine with both high ornamental value and good biological and pharmacological activities. It is of great practical and theoretical significance to study the name of plant protein and the types of metal elements contained in Lantana camara for the first time for further development and utilization.

1 Results and Analysis

1.1 Determination of protein concentration in the flower of Lantana camara

In this study, Bradford method was used to determine the protein concentration of Lantana camara (Bradford, 1976). The concentration standard curve of BSA (Figure 1) was prepared by measuring the absorbance value of bovine serum albumin (BSA) at 595 nm at different concentrations. The obtained linear regression equation y=42.8000x+0.00958, where R value is 0.9992, which meets the determination requirements. Repeated the determination of each sample for three times through the standard curve, so that the protein concentration in the yellow flower of Lantana camara was 10.05 mg/mL, and 3.786 mg/mL in the pink flower.

1.2 Two-dimensional electrophoresis of differentially expressed proteins in pink and yellow flowers of Lantana camara

Through the determination of protein concentration, the hydration loading buffer of yellow flower was determined as follows: 129 μLprotein solution+321 μL lysate, while pink flower was 343 μL protein solution+107 μL lysate. In this experiment, the immobolized pH gradients isoelectric focusing of 24 cm (pH 3~pH 10, GE company) was used to remove plant high-abundance protein with the help of BPP + phenol extraction. After optimization of conditions, impurities such as volatile oil were removed, and a relatively high purity protein sample was obtained. The protein spots can be well separated, and the protein 2-DE expression profile of Lantana camara with good stability and repeatability (Figure 2) can be obtained. The protein spots identified by the protein 2-DE expression profile of yellow flower and pink flower are 478±49 and 460±37 (average value of three repeats), respectively. Image Master5.0 was used to compare and analyze the protein expression profile of yellow and pink flower with the molecular weight of 14.4~97.4 kD, and the isoelectric point of 3.0~10.0. Through software automatic detection and combined with manual operation, the two protein spots with a matching rate of more than 85%, strong repeatability and high definition were analyzed and determined as differential display proteins. The results showed that 1-23 were yellow flower protein and 24-48 were pink flower protein (Figure 2).

1.3 Mass spectrometric analysis of differentially expressed proteins in yellow and pink flowers of Lantana camara

The enzymatic hydrolysis products of 48 high abundance protein spots were analyzed and identified with the help of MALDI-TOF-MS. First, the parameters of the enzyme auto-cutting peak and the matrix peak were corrected, and the mass spectrum information of each peptide segment in the enzymatic hydrolysis products was collected respectively. Then analyze the reliable mass spectrometry data through the Mascot Distiller software, search and compare the appropriate mass spectrometry data in the Matrix Science database (http://www. matrixscience.com), and compare the results to evaluate the scores, and obtain the detailed information of each protein point (Table 1).

1.4 Determination of metal elements in the yellow and pink flowers of Lantana camara

The yellow and pink samples of Lantana camara were pretreated by microwave digestion in the same way (Yang, 2016, Contemporary Chemical Industry, 45(2): 429-431; Wang et al., 2020, China Condiment, 45(3): 158-162). Then select 3 yellow and pink flowers respectively as parallel samples. The determination conditions of ICP-MS instrument were optimized. With 5% nitric acid solution as control, 25 inorganic metal elements in yellow and pink flowers were determined under the optimal experimental conditions (Table 2).

2 Discussion

Through the extraction and identification experiment of plant proteins from Hainan Lantana camara by two-dimensional electrophoresis and biological mass spectrometry, 48 protein spots were successfully matched (Table 1), including 9 common proteins of yellow flower and pink flower, 6 differentially expressed proteins specific to yellow flower, and 7 differentially expressed proteins specific to pink flower. A simple biological function analysis of these differentially expressed proteins was carried out.

Among the common proteins, 4, 27 are manganese superoxide dismutase (MnSOD), belonging to antioxidant metalloenzymes, which have specific catalytic functions and can be used to eliminate oxygen free radicals, and have been used to fight diseases caused by oxidative stress (Du et al., 2014). 5, 28 are triosephosphate isomerase (TPI), which is a basic metabolic enzyme. It acts on the glycolysis of sugar and helps to generate effective energy (Chen et al., 2006, Journal of Shanghai Normal University (Natural Sciences), 35(4): 75-81). 7, 30 is Lactate dehydrogenase/glycoside hydrolase, family 4, C-terminal, which plays an important role in the synthesis of oligosaccharides, alkyl glycosides and aromatic glycosides, glycosylation of amino acids and peptides, and glycosylation of antibiotics (Hara et al., 2016). 8, 32 is enoyl-[acyl-carrier-protein] reductase [NADH], chloroplast, which is an enzyme related to mitochondrial metabolic function in living plants and participates in the regulation of chloroplast division and embryo development (Lee et al., 2002). 9,33 is cytochrome c oxidase subunit 6b-1, which belongs to the basic metabolic enzyme of plants. It is the terminal enzyme of the electron transfer chain in mitochondria, which transfers the respiratory substrate electrons from cytochrome C to oxygen through catalytic electrons (Curi et al., 2003). 18,44 is chloroplast chaperone protein 21, whose biological function is beneficial to chloroplast and its stability (Guo et al., 2009). 20, 46 is Cu,Zn-SOD, one of the cofactors of superoxide dismutase (SOD), whose function is to clear the first line of defense of ROS and convert ROS into H₂O₂ and molecular oxygen (Du et al., 2014). 23, 48 are histone H4 proteins, belonging to the basic structural proteins of chromosomes. They are alkaline because they are rich in basic amino acids Arg and Lys and can interact with negatively charged phosphate groups in DNA (Law and Suttle, 2005).

Among the unique proteins of yellow flower, 1 is transcription-associated protein 1-like, whose main biological function is to flow genetic information from DNA to RNA and participate in the process of protein synthesis (Shammas, 2017). 3 is phi class glutathione S-transferase protein, which is a supergene family enzyme encoded by multiple genes and has multiple functions. It is mostly used as the main detoxification system in a variety of organisms. This kind of enzyme can be used to catalyze the combination of glutathione and toxic heterologous to metabolize out of biological body and protect cells from invasion (Dobritzsch et al., 2020). 13 is actin, whose main biological function is the organization and morphology of the cell surface and the basis for maintaining normal growth and development, and is the main component of the cytoskeleton (Liu et al., 2010). 14 is a trehalose-6-phosphate synthetase domain protein, which plays an important role in the protection of various bioactive substances and cell structures against abiotic stresses, and its intermediate products are involved in photosynthesis regulation, embryogenesis, cell differentiation and starch synthesis, which are important signals linking plant metabolism and growth and development (González and Lunn, 2020). 15 is aspartate aminotransferase, cytoplasmic-like, mainly found in the cytoplasm and mitochondria, and is an important functional enzyme. It plays a key role in nitrogen and carbon metabolism in plants, and mainly participates in carbon absorption in the process of photosynthesis (Graindorge et al., 2010). 21 is superoxide dismutase, an antioxidant enzyme with physiological defense function, which is used for plants to resist free radicals and eliminate ROS under biological and abiotic stress (Stephenie et al., 2020).

Among the unique proteins of pink flower, 26 is X3 MAP kinase mkh1-like isoform X3, which plays a role in transmitting specific signals during plant growth and participates in regulating gene expression, growth and development, cell division, differentiation and apoptosis, and is of great significance in post-translational modification (Wang et al., 2020, private communication). 31 is a CAT, whose main biological function is to protect cells from the toxicity of hydrogen peroxide, and to remove H₂O₂ produced in the metabolism process of plant cells such as β-oxidation, Photorespiration, or Mitochondrial electron transfer (Liang et al., 2020, private communication). 34 is an APRT (Adenine Phosphoribosyltransferase) protein, which plays an important role in the process of generating adenylate-5-monophosphate (AMP) by catalyzing the reaction of adenine and 5'-phosphoribosylpyrophosphate in the remedy metabolic pathway of plants (Li, 2013). 35 is a kind of heat shock proteins (HSPs), whose biological function is mainly to regulate the redox state of cells, and repair and protect damaged proteins in the pathways related to plant cell stress signal transduction. For example, under high temperature growth environment, it can induce plants to produce new proteins to prevent protein denaturation and to resist environmental changes (Xu et al., 2020, Jiangsu Journal of Agricultural Sciences, 36(1): 243-250). 36 is serine hydroxymethyl transferase, mitochondrial, whose main biological function is to locate in mitochondria, and is the key enzyme involved in photorespiration (Hou et al., 2019). 39 is glutamine synthetase cytosolic isozyme 1, which plays a role in nitrogen assimilation during plant growth, and can make plants grow under nitrogen barren conditions (Liu et al., 2018). 45 is the putative plastid lipid-associated protein 13, chloroplast. Its main biological function is that the pigment body is the organelle of carotenoid biosynthesis and storage in plants, and plays a key role in regulating the activity, stability and pigment diversity of carotenoid synthesis genes (Sun et al., 2018).

It can be seen from the species belonging to the flower protein of Lantana camara (Table 1; Figure 3) that the largest proportion of cereal grains in the family of Poaceae is 14.6%, followed by Genlisea aurea (10.4%) and Cynara cardunculus var. scolymus (8.3%).

Among them, five kinds of protein with serial numbers of 4, 21, 27, 20 and 46 belong to superoxide dismutase, which has specific catalytic function and can be used to eliminate oxygen free radicals and fight diseases caused by oxidative stress (Du et al., 2014; Stephenie et al., 2020). No. 3 protein is phi class glutathione S-transferase protein, belonging to a supergene family of enzymes with multiple functions. Its main function is to catalyze the combination of glutathione and toxic heterologous to metabolize out of the biological body and protect cells from damage. The above two proteins are first discovered from the flower of Hainan Lantana camara, which can supplement the pharmacological activity of Lantana camara as a plant drug from the perspective of protein (Vardien et al., 2012). In particular, the 48 protein belongs to the genus of Thraspi caerulescens, whose main biological function is to repair the important genes of plants contaminated by heavy metals in the soil. Due to the typical and super-high tolerance and heavy metal accumulation ability of the plants of the genus Thraspi caerulescens, they are often referred to as heavy metal hyperaccumulation plants (Ree et al., 2020). The results showed that Lantana camara not only contains rich plant protein, but also may be further developed and utilized in the treatment of environmental pollutants.

In addition, some metal ions such as copper ions play an important physiological role in plant growth and development, but most of the heavy metals have adverse effects on plant seed germination, seedling growth, pollen germination and pollen tube elongation (Zhou and Chen, 2016, Jiangsu Agricultural Sciences, 44(12): 225-227). There are some differences in the content of some metal elements between yellow flowers and pink flowers of Lantana camara, but they contain high content of K, Mg, Fe, Al and other metal ions. Among them, the K with the highest metal content in the yellow flower of Lantana camara is 14139.7224 µg/g, while in the pink flower is 12151.6284µg/g. The lowest content of Be in the yellow flower and pink flower of Lantana camara is 0.0195 µg/g and 0.0075 µg/g, respectively (Table 2). And it can be seen that the content of Zn and Cu in the yellow flower and pink flower of Lantana camara showed the characteristics of high Zn and low Cu, and has a high Zn/Cu ratio, which is consistent with the Zn/Cu in the serum of normal people. On the one hand, it showed that the animals and plants in nature have similar characteristics, and it also suggested that this ratio is important to maintain the normal function of Lantana camara. The high Fe/Mn value is also a characteristic of trace elements in Lantana camara. The high Fe/Mn value also indicated that Lantana camara has the effect of relieve superficies and regulating blood (Yang et al., 2011, Yunnan Chemical Technology, 38(5): 25-28).

In summary, this experiment reported for the first time the content of protein and metal ions in the yellow and pink flowers of Hainan Lantana camara and obtained obvious comparative research results. Hainan belongs to the tropical region with the perennial temperature and humidity, which are suitable to produce Lantana camara. The results of this study not only provide scientific and reasonable theoretical guidance for the further development and utilization of plant protein or metal elements in Lantana camara, but also provide some reference for the in-depth study of the pharmacological or biological activities of Lantana camara.

3 Materials and Methods

3.1 Test materials, instruments and reagents

In this study, Lantana camara L. was taken as a sample and collected on the Campus of Guilinyang of Hainan Normal University 110°30'46.224"E, 19°58'48.792"N), which was identified by Professor Zhong Qiongxin from the School of Life Sciences of Hainan Normal University (Figure 4).

Protein extraction instruments and reagents were as follows: Protein isoelectric focusing instrument (Ettan IPGphor3), 24 cm ReadyStrip IPG (pH 4~pH 7); Image scanner III (GE Healthcare); Gel image analysis software (ImageMaster 5.0); Low temperature overspeed centrifuge (SIGMA 3-18K, SIGMA of Germany); Ultraviolet spectrophotometer (UV-2700, Shimadzu, Japan); Time-of-flight mass spectrometer (Voyager-DE PRO ABI4700, ABI, USA). Acrylamide, Bis-acrylamide, mineral oil, TEMED, Coomassie brilliant blue R-250 and G-250, polyvinylpyrrolidone (PVPP), etc. were purchased from Sigma. CHAPS, DTT were purchased from Bio-Rad company. Other reagents were domestic analytical reagents. All experimental water was secondary distilled water.

Metal ion measuring instruments and reagents were as follows: Microwave digestion chemical system (ETHOSOME, MILESTONE),Agilent 7700X ICP-MS (Agilent Technologies Inc., Japan), AUW120 analytical balance (SSL), Purified water system (Cascada RO WATER), Glassware -- Air dryer (C-30, EXCEED), micro plant grinding machine (Tianjin Taisite Instrument Co., Ltd.), Electric thermostatic blast drying oven (Shanghai Shenxian Thermostatic Equipment Factory), KQ2200E ultrasonic cleaner, GM-0.33A diaphragm vacuum pump (Tianjin Jinteng Experimental Equipment Co., Ltd).

3.2 Extraction and content determination of protein from Lantana camara

The protein of Lantana camara was extracted by BPP + phenol extraction (Wu, 2016). Rinsed the currently collected yellow and pink Lantana camara with tap water respectively, then rinsed with ultrapure water, and absorbed the water with filter paper. Weighed about 3 g of Lantana camara sample, added 1% PVPP, put it in a precooled mortar, added liquid nitrogen, grinded it into a fine powder, and then transferred it to a 30 mL centrifuge tube, then added 5 mL of precooled BPP at 4 °C, shook for 10 min at room temperature, and added equal volume of tris saturated phenol to shake it for 10 min (room temperature). Centrifuged for 15 min under the condition of 16000×g at 4 °C. Took 1 mL of supernatant and put it into a new 30 mL centrifuge tube (dry), and added 2 mL of precooled BPP extraction. Centrifuged for 15 min under the condition of 16000×g at 4 °C after 5 min of high-speed vibration. Combined the supernatant, added quantitative precipitator in the proportion of 1 mL of supernatant/5 mL of AM precipitator (supersaturated ammonium sulfate methanol solution), mixed, and then stood at -20 °C overnight. The next day, the sample was centrifuged at 4 °C for 15 minutes under the condition of 16000×g. Removed the supernatant, added 3 mL of precooled methanol into the precipitation, and then centrifuged at 4 °C for 15 minutes under the condition of 16000×g. This step repeated for twice. Removed the supernatant and added 3 mL of precooled acetone into the sediment. And then centrifuged at 4 °C for 15 minutes under the condition of 16000×g. This step repeated for twice. After pouring the acetone, opened the centrifuge tube cover, put it in room temperature to dry naturally, and then added 15 mg of DTT and 15 mg of PMSF to each tube to dissolve it completely. After the protein is completely dried, weighed it, prepared the lysate according to the ratio of 1 mL protein lysate to 10 mg protein sample, and dissolved it in a 20 °C incubator for 2 hours. After the sample protein lysate was completely dissolved, the protein was centrifuged at 20 °C for 30 min under the condition of 20000×g and then stored at -20 °C.

Using BSA as the protein standard curve, some protein samples were taken and the protein content in the samples was determined by the Bradford method.

3.3 Two-dimensional electrophoresis analysis of protein from Lantana camara

Hydration loading buffer: The protein solution of 1.3 mg protein was made up to 455 μL by adding protein lysate. Selected immobolized pH gradients isoelectric focusing (GE, 24 cm, pH 3~10), placed in a constant temperature bath at 20 °C for at least 18 h of hydration.

IEF: Transferred the hydrated adhesive strips to the focusing disk of Ettan IPGphor 3 electrophoresis instrument and performed isoelectric focusing at 18 °C~20 °C, with a current limit of 50 μA for each adhesive strip. The isoelectric focusing parameters were set to 250 V 3 h, 500 V 2 h, 1000 V 1 h, 1 000-8 000 V 3 h, 8000 V up to 120 000 vhr.

Balance of ReadyStrip IPG: After the completion of IEF, put the ReadyStrip IPG in 15 mL of balanced buffer solution (1% DTT solution), put it on the shaking table for balance for 15 min, and then cleaned the rubber strip with distilled water; Then put in the balanced buffer containing 4% IAA to balance slowly for 15 minutes, and washed the strip with distilled water.

SDS-PAGE: Transfer the strip after balance to the top of 12.5% polyacrylamide gel film, seal and fix the strip with the melted hot agarose solution with the pipettor. Set the circulating water bath temperature of the electrophoresis instrument to 16 °C, and the power parameter is as follows: each film is pre-electrophoresis at 5W for 1 h, and then each film is finished at 7W to two-dimensional electrophoresis, that is, electrophoresis until the Bromophenol blue indicator reaches the bottom edge.

Dyeing and decolorization of gel: After two-dimensional electrophoresis, the gel after electrophoresis was stained with the method of Coomassie blue staining (Bradford, 1976). The gel was stripped from the glass plate and transferred to a dyeing box containing 2 L staining solution. It was dyed overnight under mild vibration at 20 °C with shaker speed r of 25 rpm. The next day, the gel was transferred to the destaining solution (Destaining solution: 60% ethanol solution mixed with 10% acetic acid solution) for 30 minutes. After that, the gel was transferred to the newly prepared 7% acetic acid for 1 min, and then soaked in ultrapure water for 1 h for scanning.

Gel scanning and image analysis: Image Scanner III scanner was used to set 300 dpi transmission scanning parameters for gel spectrum scanning. Then the scanned image was analyzed with Image Master5.0 software to screen out the protein spots with obvious differential expression.

Enzymatic hydrolysis and mass spectrometric determination of protein spots: The differentially expressed protein spots screened above were first dug with Capillary and then put into 1.5 mL PE tubes for decolorization. The specific steps were as follows: added 0.15 mL destaining solution (50% ACN50%, 100 mmol/L NH₄HCO₃) into the differential protein PE tube respectively and put it on the shaking table to shake for 0.5 h (set the rotational speed to 150 r/min), then removed the destaining solution from each PE pipe and repeated the above steps until the color of the rubber particles becomes transparent. Finally, added 0.15 mL of ultra-pure water to each PE that removed the destaining solution, shook the shaking table at a speed of 150 r/min for 15 min, and absorbed the ultra-pure water. Repeated this step for 3 times. Added 0.1 mL of acetonitrile to remove the moisture from the rubber particles, put it on the shaking table and shook for 15 minutes, and replaced it with new acetonitrile until the rubber particles were completely white and air dried at room temperature. After centrifuging the colloidal particles, added trypsin (20 ng/µL) to cover the colloidal particles, and put them in a refrigerator at 4 °C for 1 h. Sucked out the enzyme solution with the pipettor and added 6 μL Trypsin Buffer, enzymatic hydrolysis of protein at 37 °C for 13 h on the PCR instrument, and centrifuged for 5 min under 10000×g at 20 °C. After that, the next step of mass spectrometry was carried out.

Mass spectrometry and database search: The MALDI-TOF-MS instrument was used to set the instrument parameters to correct the enzyme auto-cutting peak and matrix peak, and other parameters were operated according to the instructions of manufacturer. The processed enzymatic hydrolysis products were determined by mass spectrometry. The measured data were then analyzed with Mascot Distiller software. The database search and comparison were completed through the Matrix Science website (http://www.matrixscience.com) to collect the detailed information of different proteins, and then classified and sorted the data.

3.4 ICP-MS determination

The collected Lantana camara of two colors were dried in an oven at 60 °C for 5~6 h. Accurately weighed 0.1000 g of yellow and pink flower into the corresponding numbered liner cup and added appropriate amount of digestion solution (3.0 mL H₂O₂, 5.0 mL HNO₃). Then put it into the microwave digestion instrument and digested it according to the procedure set by the instrument. After the digestion was completed for cooling, the sample solutions were then transferred to 50 mL volumetric flasks separately, fixed with deionized water and mixed well. Blank samples were prepared under the same conditions, and the samples and blanks were subjected to on-board determination. Each sample was taken in parallel in 3 portions, and finally 2 mL of each sample solution was placed in 3 clean and dry 10 mL colorimetric tubes, respectively for determination (Yang, 2016, Contemporary Chemical Industry, 45(2): 429-431).

Operating conditions and parameter settings of the ICP-MS instrument: Performance indicators such as background, sensitivity, oxide, and dual-charge stability of the ICP-MS instrument were tuned to achieve a qualified determination. By optimizing the experimental conditions, the analysis mode was set to full quantitative mode, the instrument power was set to 1 200 W, the average isotope integration time was 0.3 s, and the oxide content was <0.3%. Other operating parameters such as sampling depth, intercept cone aperture, and sampling cone aperture were set to 7.0 mm, 0.4 mm, and 1.0 mm, respectively. Aux Gas, Carrier Gas, MU/Dil Gas, Plasma Gas were set to 1.0 L/min, 1.0 L/min, 1.0 L/min, 15.0 L/min, respectively.

Authors’ contributions

HYN and QJ are the experimental designers and executors of this study. HYN completed the data analysis and the first draft of the paper. ZZZ, WD, and LW participated in the experimental design and analysis of the experimental results. CGY and HWY are the designers and leaders of the project, directing the experimental design, data analysis, and paper writing and revision. All authors read and approved the final manuscript.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (21662012;41866005).

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