在阿特拉津应激和影响土壤微生物量和DNA因素影响下土壤硫循环的基因进化和多样性分析

发布时间：2016-09-28 06:34

1 Introduction

The impact of pesticides on soil is mainly reflected in its impact on soil fertility, plantgrowth and plant diseases and insect pests associated with the microbial species, quantity andactivity of the impact (Chowdhury A, et al. 2008). These effects are either direct or indirect,inhibited or promoted, temporary or permanent, reversible or irreversible, etc.. Largeamounts of pesticides and herbicides are used to kill or inhibit the activity of soil m icrobes.Soil fungicide and fumigation agent affect the balance of soil microbial system(Serna‐Chavez S, et al. 2013). The effect of pesticides on soil microorganisms, and thenaffect the activity of enzymes in the soil and the transformation of nutrients, change theefficiency and speed of the nutrient cycle of agricultural ecosystem, so that the sustainableproductivity of land (Malicki M A and Bieganowski A. 1999). At the same time, the pesticideresidues in the soil will cause heavy metal pollution. Once the soil is polluted by heavymetals, it will be difficult to recover.pesticides on pests and their day dare to effect in nature, insect pests and their naturalenemies is complementary to each other, a large number of application makes the pests itsdrug content high, predation of pests in bioaccumulation in the pesticide accumulate,eventually poisoning death (Dequiedt , et al. 2011). At the same time, accumulate in thenatural enemies of the pesticide can poison their offspring, the offspring of natural enemiesoccur in various diseases and even death, cause, pest, natural enemy, there is a significantreduction or extinction, coupled with the pest resistance to pest population sharp growth, t heharm to the crops greatly increased (Drenovsky R E, et al. 2010). Pesticides in the majorityof the currently used with broad-spectrum insecticidal activity, while killing pests to nontarget insects also brought disaster.

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2 Materials and methods

2.1 Materials and Methods in Experiment 1

The content of H2O2was determined using the method described by Jena andChoudhuri[117]with minor modifications. Plant material (1g) was homogenized in 5 mL ofprecooled acetone at 4oC. The homogenate was centrifuged for 10 min at 12 000 g and thesupernatant was collected for sample extraction. 1.0 mL of supernatant was mixed thoroughlywith 0.1mL of 5% titanium sulphate and 0.2mL ammonia solution and the mixture wascentrifuged at 3000×g for 10 min at room temperature. The absorbance of H2O2wasmeasured at 415 nm; the content of H2O2was calculated from a standard curve.For the analytical quantification of superoxide anion radicals, plant leaves (1 g) werehomogenized in 10 mL of precooled phosphate buffer saline (PBS) (pH 7.8). The homogenatewas centrifuged for 10 min at 12 000 g and the supernatant was used for sample extraction. Areaction mixture containing PBS (pH 7.8) and 1 mM hydroxylamine hydrochloride wasformed in addition to 0.5 mL of the supernatant. This mixture was incubated at 25oC for 20min. Then appropriate volumes of 17 mM sulanilic acid and 7 mM α –naphthylamine wereadded to the incubated mixture and allowed to stay for another 20 min at 25oC. Thedeveloping solution was shaken with an equal volume of n-butanol and subsequently allowedto separate into two phases. The upper n-butanol phase was measured at 530 nm.

2.2 Materials and Methods in Experiment 2

 All enzyme extraction procedures were conducted at 4oC. Fresh leaves (0.5 g) wereground to fine powder in a chilled pestle and mortar. Five mL 50 mM potassium phosphatebuffer (pH 7.8) containing 1mM EDTA and 1.5% w/w polyvinylpyrrolidone was added to thepowder. The homogenate was centrifuged at 12,000×g for 20 min at 4°C. The supernatantobtained was used for enzyme assays.POD (EC 1.11.1.5) activity was determined by the method of[121]. The enzyme activitywas assayed by the determination of guaiacol oxidation by hydrogen peroxide. Five mL ofthe assay mixture comprised: 100 mM potassium phosphate buffer (pH 7.0), 20 mM guaiacol,10 mM hydrogen peroxide, and 1 ml of the enzyme extract. Changes in the absorbance (470nm) of the reaction solution were measured after 1 min with a Spectrophotometer (UV-1800,Shimadzu, Japan). One enzyme activity unit was defined as the amount of enzyme that couldmake the value of the absorbance decrease by 0.01 during the 1 min. The results were givenas the unit of enzyme activity per gram of fresh leaf weight.APX (EC 1.11.1.11) activity was measured by the decrease in the absorbance at 290 nmas described by[122], with minor modification. The reaction mixture consisted of 1.8 mL potassium phosphate buffer (pH 7.0, 50 mM), 0.1 mL ascorbate (15 mM), 1.0 mL hydrogenperoxide (0.3 mM) in a final volume of 3 mL. The reaction was started by adding hydrogenperoxide and the decrease in absorbance at 290 nm was recorded for 1 min to determine theoxidation rate of ascorbate. One enzyme activity unit was defined as the amount of enzymethat could make the value of the absorbance decrease by 0.01 during the 1 min. The resultswere given as the unit of enzyme activity per gram of fresh leaf weight .The catalase (CAT, EC 1.11.1.6) activity was measured according to the methoddescribed by[123]. The reaction mixture contained 1 mL extraction buffer (pH 7.0 potassiumphosphate buffer containing 1 % polyvinylpyrrolidone), 0.3 mL 30 % H2O2, and 0.2 Mlsupernatant. One unit of CAT was defined as the amount of enzyme that decomposes 1 μmolof H2O2in 1 min. Activity was expressed as units per gram of fresh weight.

3 Result analysis..............19
3.1 Soil total DNA extraction: ....... 19
3.2 PCR products of aprA gene with soil total DNA as template ..................................... 19
3.3 PCR product purification.......... 20
3.4 Cloning and sequencing ............... 21
3.5 Gene Bank Accession number and Identification of Closet Sequence ............... 22
3.6 Similarity Analysis .................. 23
3.7 Phylogenetic Analysis Based on aprA gene Sequence ................. 25
3.8 Variability of Soil Microbial Biomass and DNA in Summer Season .......................... 26
3.9 Variability of Soil Microbial Biomass and DNA in Spring Season ............................ 28
3.10 Impact of Seasonal Variations and Fertilizers on Soil MB, DNA and ODR .............. 29
3.11 Correlation between Spring and Summer Variations on Selected Parameters ........... 31 

4 Discussion........................33

4.1 influence of atrazine on soil microbial communities ............... 33

4.2 Selection of Soil sulfur 16S rRNA genes.................... 33

4.3 Evaluation of microbial diversity by using aprA gene............................................... 35
4.4 Sulfate Reducing and oxidizing prokaryotic diversity by cloning of aprA gene.......... 36
4. 5 Soil biological Indicators .......................... 37
4.6 Soil Microbial Biomass....... 37 
5 Conclusion.............................39

4 Discussion

4.1 Influence of Atrazine on Soil Microbial Communities

Herbicide is a class of drugs used to kill plants, there are about 233 kinds of herbicides.These agents are able to selectively target specific targets and to make other crops useful tohumans not to be harmed, or less damaged. Some herbicides can interfere with the growth ofweeds, which are usually based on plant hormones. Herbicide used to clean up waste landscan kill all plants that are exposed to it. Some plants themselves also have the ability toproduce herbicides, such as walnuts. Herbicides are widely used in agriculture as well as inturf management, or to control the growth of vegetation on roads and railways (Malicki M Aand Bieganowski A. 1999: Serna‐Chavez S, et al. 2013). Atrazine is one of three triazineherbicides are widely used in many countries. Persistent organic pollutants, because of theproblem of pollution has been banned in the European union. China mainland use atrazine asthe name of the product. Over the years, maize production in Northeast parts of the extensiveuse of long residue herbicides atrazine to Tianjin or its mixture, resulting in atrazine in thesoil residue accumulation, affecting crop growth, become crop rotation, planting structureadjustment of key constraints. Atrazine is very harmful for human health ( Cai Q Y, et al.2008: Devers M, et al. 2007). Keeping this in view, researchers focused on safe and efficientmethod of atrazine degradation. Many studies investigated that soil microbes can play animportant role in atrazine degradation. There are many mutually beneficial and mutuallybeneficial interactions among the various microorganisms in the soil. For example, soil existsome antibiosis microorganisms, they are able to produce antibiotics, inhibit the propagationof pathogenic microorganisms, so that you can control and reduce the harm of pathogenicmicroorganisms in the soil to crops, so soil microbial actuall y have adverse side, such aspathogenic microorganisms (Struthers J K , et al. 1998).Microbes also residues in soil degradation of organic pesticides, urban sewage and plantwaste, they decomposed into less harmful or harmless substances reduce the harm o f residue.Of course, all of these functions are done by different populations of microbes, and therealization of each function requires a large number of microbes to work together ( Ley R E,et al. 2004: Kanissery R G. and Sims G K. 2011).

4.2 Selection of Soil Sulfur 16S rRNA Genes

Microbes in the soil so capable, we might think, if we can let them lis ten to humanwords, what they want to do, they will play what function is good. Well, that's right,scientists think so. Now, in the pharmaceutical and health industry, industrial and agriculturalproduction, there have been a lot of examples of the applic ation of microorganisms (Dunbar J, et al. 2000: Klindworth A, et al .2012). Take the microorganisms in the soil, throughdevelopment and screening of effective strain, cultivate, strain, we can for the remediation ofcontaminated soil, producing microbial fertilizer, biological pesticide, and so on.In spite of this, most of the soil microbial population is still unknown to the populationof resources, scientists are currently on the understanding of soil microorganisms, andunderstanding and utilization are still very limited. Isolation and screening of soil microbialresources and development of functional microorganisms will be an important work in thefuture (Gregersen L H, et al. 2001).With the progress of science and technology, molecularbiotechnology for scientists to explore the role of microorganisms, and the relationshipbetween the community and the environment to find a key to the mystery of the great worldof microorganisms in human eyes (Friedrich C G, et al. 2001).In order to fully exploit theresources of microorganisms (especially bacteria), the United States launched a program ofmicrobial genome research (MGP) in 1994. Through study the complete genomic informationfor the development and utilization of microbial functional gene can not onl y deepen ofmicrobial pathogenesis, important metabolic and regulatory mechanism of understanding,based on the development of a series of our life is closely related to the gene engineering.

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5 Conclusion

phylogenetic and diversity analysis of genes (AprA, dsr and rdsr) performing differentfunctions in soil sulfur cycle showed that mostly prokaryotes and proto-bacterium wereinvolved in sulfate reduction and sulfur oxidation. Some previous studies also reported suchspecies from environment performing their role in sulfurcycle. Many species identified inthis study were not specified with their role in sulfur cycle. Study indicated that impact onsoil microbes performing role in sulfur cycle was directly proportional to atrazineconcentration. Higher the concentration, higher the impact, decreased matching withsequences of closet relatives already registered in database. aprA gene from control soilsample ( with no atraine addition) demonstrated the highest similarity rate with closetrelatives in database. For making analysis easy of nucleotide matching in all sequences, thesum of r values was taken equal to 100. Rates of various transitional substitutions showedthat nucleotide matching frequencies were 25.30% for base A, 23.31% for T/U, 27.66% for C,and 23.72% for G. Furthermore, this was concluded that type of fertilizer had a great impacton soil microbial biomass and DNA contents. Soil microbial biomass and DNAconcentrations showed significant correlation. Soil treated with organic fertilizers, microbialbiomass concentration ranged between 0.0051-0.0098 g/g of soil in summer season, whilesoil treated with inorganic fertilizers demonstrated microbial biomass concentration rangebetween 0.0010-0.0090 g/g of soil in summer. Similarly, in the spring soil treated withorganic fertilizers revealed microbial biomass concentration between 0.0053 -0.0085 g/g ofsoil and DNA contents ranged between 0.5367-1.49 ug/g of soil, whereas application ofinorganic fertilizers resulted in soil microbial biomass concentration from 0.0022 to 0.0045g/g of soil and DNA concentrations ranged between 0.2133 -1.1667 ug/g of soil. Thestatistical correlation between soil microbial biomass, DNA and ODR in spring and summerseasons along with organic and inorganic fertilizers were found highly was significant(p>0.01).

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参考文献（略）

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