CN109402273B

CN109402273B - A kit and method for detecting the dynamic changes of gene expression in the process of bacteria-mediated generation of arsenic

Info

Publication number: CN109402273B
Application number: CN201811269891.5A
Authority: CN
Inventors: 李青竹; 柴立元; 王茜; 廖骐; 王庆伟; 杨志辉; 梁彦杰; 唐崇俭
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2018-10-29
Filing date: 2018-10-29
Publication date: 2021-08-06
Anticipated expiration: 2038-10-29
Also published as: CN109402273A

Abstract

The invention relates to a kit and a method for detecting the dynamic changes of gene expression in the process of bacteria-mediated formation of arsenic. The kit mainly uses real-time fluorescence quantitative PCR technology to detect the relative expression of several functional genes in the process of arsenic removal from the acidophilus ferrooxidans. The functional genes include acidophilus ferrooxidans. Ferrous iron oxidation-related genes (including coxB, coxA, rus, Acop, petA1, cyc2, cyc1), arsenic tolerance-related genes (arsH, arsB, arsC, arsR), EPS metabolism genes (galU), and 16s rDNA genes (rrs) as an internal reference gene. The method for detecting the dynamic changes of gene expression during the generation of bacteria-mediated arsenite is simple, fast, low in cost, high in sensitivity and specificity, accurate in detection results, linking microscopic and macroscopic Practical application prospects.

Description

Kit and method for detecting dynamic change of gene expression in process of generating bacterium-mediated map cyrtomium-arsenicum

Technical Field

The invention relates to a kit and a method for detecting dynamic change of gene expression in a process of generating bacterium-mediated arsenopyrite, belonging to the technical field of microbial detection.

Background

In nature, the migratory transformation of arsenic by microorganisms is a very important part of the biogeochemical action of arsenic. Meanwhile, iron is easy to participate in arsenic circulation, and circulation of arsenic between a water body and minerals is promoted through purification, absorption, oxidation and precipitation.

Acidithiobacillus ferrooxidans is a common iron-circulating microorganism and is widely applied in the field of environmental engineering, and comprises the following steps: acid mine wastewater treatment, heavy metal-containing sludge treatment, biological polyferric preparation and the like. The acidophilic ferrous oxide biomineralization can also be used as a method for inhibiting the migration of arsenic in the environment. The Chaihuang et al disclose a method for directly synthesizing arsenopyrite mineral in wastewater containing trivalent arsenic by utilizing acidophilic thiobacillus ferrooxidans so as to remove arsenic and fix arsenic, but no deep molecular level research is carried out.

At present, the molecular level research on acidophilic thiobacillus ferrooxidans mainly focuses on the aspects of growth and metabolism, bioleaching and the like. However, in the process of biosynthesizing arsenopyrite by utilizing acidophilic thiobacillus ferrooxidans, the dynamic change of relative expression amounts of several functional genes in different time periods is not studied, and particularly, the bacterial ferrous oxide related genes rapidly oxidize ferrous in the process of biosynthesizing arsenopyrite so as to fix arsenic and remove arsenic and the expression change of arsenic-resistant genes in the process need to be concerned, and the bacterial ferrous oxide related genes are closely related to a biological mineralization mechanism so as to regulate and control the arsenic removal efficiency. Therefore, there is a need to develop a detection method based on nucleic acid level for applying to the system.

Disclosure of Invention

The invention aims to provide a kit for detecting dynamic change of gene expression in the process of generating bacterium-mediated arsenopyrite through detecting the dynamic change of functional gene expression of acidithiobacillus ferrooxidans by mainly adopting a real-time fluorescent quantitative PCR technology; the invention also aims to provide a method for detecting the dynamic change of gene expression in the process of generating the bacterium-mediated arsenical oxide ferrovanadium, which is a method for detecting the dynamic change of the functional gene expression of the acidithiobacillus ferrooxidans by adopting a real-time fluorescent quantitative PCR technology.

In order to achieve the purpose, the invention adopts the main technical scheme that:

a kit for detecting dynamic change of gene expression in a process of generating bacterium-mediated arsenopyrite through real-time fluorescence quantitative PCR (polymerase chain reaction) technology, comprises a group of nucleic acids for detecting dynamic change of functional gene expression of acidophilic thiobacillus ferrooxidans, wherein the functional gene comprises any one of ferrous oxidation related genes, EPS (expanded polystyrene) metabolic genes and arsenic-resistant related genes, the ferrous oxidation related genes comprise primer pairs for detecting coxB, coxA, rus, Acop, petA1, cyc2 and cyc1, the EPS metabolic genes are primer pairs of galU, and the arsenic-resistant related genes comprise primer pairs of arsH, arsB, arsC and arsR; wherein, the primer pair of the coxB is shown as SEQ ID NO.3 and SEQ ID NO.4, the primer pair of the coxA is shown as SEQ ID NO.5 and SEQ ID NO.6, the primer pair of the rus is shown as EQ ID NO.7 and SEQ ID NO.8, the primer pair of the Acop is shown as SEQ ID NO.9 and SEQ ID NO.10, the primer pair of petA1 is shown as SEQ ID NO.11 and SEQ ID NO.12, the primer pair of cyc2 is shown as SEQ ID NO.13 and SEQ ID NO.14, the primer pair of cyc1 is shown as SEQ ID NO.15 and SEQ ID NO.16, the primer pair of galU is shown as SEQ ID NO.17 and SEQ ID NO.18, the primer pair of the arsH is shown as SEQ ID NO.19 and SEQ ID NO.20, the primer pair of the arsB is shown as SEQ ID NO.21 and SEQ ID NO.22, the primer pair of the arsC is shown as SEQ ID NO.23 and SEQ ID NO.24, the primer pair of the arsR is shown as SEQ ID NO.25 and SEQ ID NO. 26.

The kit also comprises an internal reference control primer pair, wherein the internal reference gene control primer pair is shown as SEQ ID NO.1 and SEQ ID NO. 2.

The kit as described above, preferably, the kit further comprises: 2 XFastStart Essential DNA Green Master.

A method for detecting dynamic change of gene expression in a process of generating bacterium-mediated map Fexojarosite is used for detecting dynamic change of functional gene expression of Acidithiobacillus ferrooxidans by adopting a real-time fluorescent quantitative PCR technology, and specifically comprises the following steps:

(1) extracting RNA from acidophilic thiobacillus ferrooxidans culture solution cultured in different time periods;

(2) reverse transcribing the extracted RNA to synthesize cDNA;

(3) performing fluorescent quantitative PCR amplification on the synthesized cDNA; the fluorescent quantitative PCR amplification method comprises a functional gene reaction system and an internal reference gene reaction system, wherein the functional gene reaction system comprises a primer pair of any one functional gene of ferrous oxidation related genes, EPS (Expandable polystyrene) metabolic genes and arsenic-resistant related genes and cDNA (complementary deoxyribonucleic acid) synthesized by reverse transcription, and the internal reference gene reaction system comprises sequences shown as SEQ ID No.1 and SEQ ID No.2 of internal reference control primer pairs;

(4) putting into a fluorescence quantitative amplification pcr instrument, and detecting by the instrument to obtain Ct_{Functional gene}And Ct_{Internal reference gene}；

(5) Calculating relative expression amount: the real expression level is 2 with 0h as reference group, different time periods as experimental group, and 16srDNA as reference gene^-△(△CT)Where Δ Ct 1 ═ Ct_{Functional genes in other time periods}-Ct_{Reference gene corresponding to time}，△Ct 2＝Ct_{Functional genes of reference group}-Ct_{Reference genes of the reference group}，△(△CT)＝△Ct1-△Ct 2。

Specifically, for example, the relative expression level of 11h, Δ Ct 1 ═ Ct_{11h expression level of functional Gene}-Ct_{11h reference Gene expression level}，△Ct 2＝Ct_{0h expression level of functional Gene}-Ct_{0h reference gene expression level}，△(△CT)＝△Ct 1-△Ct 2。

Ct as described above_{Functional gene}Ct value, Ct value measured by the PCR instrument for the detection of the functional gene reaction system_{Internal reference gene}Ct value detected by a fluorescence quantitative amplification pcr instrument on an internal reference gene reaction system;

Ct_{functional genes in other time periods}The Ct value and the Ct value are detected by a functional gene reaction system of the acidithiobacillus ferrooxidans in other time periods through a fluorescence quantitative amplification pcr instrument_{Reference gene corresponding to time}The Ct value is detected and measured by a fluorescence quantitative amplification pcr instrument in an internal reference gene reaction system of acidithiobacillus ferrooxidans in the same time period.

The method as described above, preferably, in the step (3), the ferrous oxidation related genes include primer pairs for detecting coxB, coxA, rus, Acop, petA1, cyc2 and cyc1, the EPS metabolic genes are primer pairs for galU, and the arsenic-resistant related genes include primer pairs for arsH, arsB, arsC and arsR; wherein, the primer pair of the coxB is shown as SEQ ID NO.3 and SEQ ID NO.4, the primer pair of the coxA is shown as SEQ ID NO.5 and SEQ ID NO.6, the primer pair of the rus is shown as SEQ ID NO.7 and SEQ ID NO.8, the primer pair of the Acop is shown as SEQ ID NO.9 and SEQ ID NO.10, the primer pair of petA1 is shown as SEQ ID NO.11 and SEQ ID NO.12, the primer pair of cyc2 is shown as SEQ ID NO.13 and SEQ ID NO.14, the primer pair of cyc1 is shown as SEQ ID NO.15 and SEQ ID NO.16, the primer pair of galU is shown as SEQ ID NO.17 and SEQ ID NO.18, the primer pair of the arsH is shown as SEQ ID NO.19 and SEQ ID NO.20, the primer pair of the arsB is shown as SEQ ID NO.21 and SEQ ID NO.22, the primer pair of the arsC is shown as SEQ ID NO.23 and SEQ ID NO.24, the primer pair of the arsR is shown as SEQ ID NO.25 and SEQ ID NO. 26.

The method as described above, preferably, in the step (3), the reaction system for the fluorescent quantitative PCR amplification in 25 μ L system includes:

2×FastStart Essential DNA Green Master：10μL，

each functional gene primer pair: the final concentration of the upstream and downstream primers was 10 pmol/. mu.L, each 1. mu.L,

cDNA template: 1 mu L of the mixture is added into the solution,

nuclease-free water to 25 μ L;

meanwhile, the nuclease-free water is set as a negative control.

The method as described above, preferably, the reaction procedure of the fluorescent quantitative PCR amplification in step (2) is: pre-denaturation at 95 ℃ for 300 s; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles; the melting curve program is an initial stage: 95 ℃ for 60s, final stage: the temperature of 55 ℃ was increased to 95 ℃ at a rate of 0.1 ℃/s, and fluorescence signals were collected from 55 ℃.

The method as described above, preferably, in the step 1), the different time periods refer to 0, 11, 30, 35, 48 hours after the cultivation, wherein the initial arsenic concentration in the used culture solution is 500mg/L, the pH is 2-2.5, the iron-arsenic ratio is 4-6, and the inoculation amount is 10%.

The invention has the following advantages:

(1) a method for detecting the expression of some specific genes of acidophilic thiobacillus ferrooxidans in the mineralization process by utilizing a real-time fluorescent quantitative PCR technology and adopting specific primers of the genes is developed and developed. The invention can detect and calculate the relative expression conditions of several genes of the acidithiobacillus ferrooxidans in the process of generating the cerite in the mediation map through PCR reaction, is simpler, more convenient and quicker than the traditional common PCR and single fluorescence PCR method, and can carry out real-time accurate detection on the expression conditions of target genes.

(2) The kit can rapidly detect the relative expression conditions of several genes of acidophilic thiobacillus ferrooxidans and the expression condition of 16s rRNA genes, and determines the influence of different time periods on the ferrous oxide, arsenic resistance, EPS metabolic function and growth condition of acidophilic thiobacillus ferrooxidans in the process of forming ores. The invention provides a method for detecting the dynamic change of functional gene expression by using a real-time fluorescence quantitative PCR technology, which is simple and rapid to operate and low in cost, has high sensitivity and specificity and accurate detection result, and has wide practical application prospect by associating the microcosmic and macroscopical aspects. The method can be applied to the environment of continuous change of bacteria, and can be used for detecting the gene expression change amount at different time.

Drawings

FIG. 1 is a graph showing the ferrous oxidation rate and arsenic removal rate under the optimum conditions in example 1 of the present invention, wherein (a) is the ferrous oxide concentration and (b) is the arsenic removal rate;

FIG. 2 shows a specific melting peak in a real-time fluorescent quantitative PCR system in example 2 of the present invention, wherein the specific melting peak is exemplified by reference gene 16 srDNA;

FIG. 3 is the relative expression level of ferrous oxide gene of Acidithiobacillus ferrooxidans in each time period for generating arsenopyrite dihydrate in example 3 of the present invention;

FIG. 4 is the relative expression level of the arsenic-resistant gene of Acidithiobacillus ferrooxidans in each time period for producing arsenopyrite dihydrate in example 3 of the present invention;

FIG. 5 shows the relative expression levels of the EPS metabolic genes of Acidithiobacillus ferrooxidans in the respective time periods for producing the map of Feitentium hydrarginatum in example 3 of the present invention.

Detailed Description

For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.

EXAMPLE 1 optimization of Ore-forming System and sample preparation

The first step is as follows: optimization of ore-forming arsenic removal system

In the process of removing arsenic from ferrous oxide sulfate (Acidithiobacillus ferroxidans with the preservation number of ATCC 23270, which is commercially available) to produce arsenopyrite, the group with the best arsenic removal rate stability is selected for the Fe/As ratio, the pH and the inoculation amount respectively, namely Fe/As is 6, pH is 2, the inoculation amount is 10%, and the experiment is carried out under the conditions that the temperature is 30 ℃ and the rotating speed is 170 rpm. The adopted culture medium is 9 k; the arsenic concentration was 500ppm, and the samples were taken at various times under the optimum conditions for production of arsenopyrite.

The second step is that: optimal conditions of ferrous oxide rate and arsenic removal rate

And (4) measuring the ferrous oxide by a potassium dichromate method at different time points under the optimal condition of the first step, and measuring the arsenic concentration by utilizing ICP. The results are shown in FIG. 1, in which Fe is measured in FIG. 1(a)²⁺FIG. 1(b) shows the measured arsenic concentration.

Finally, according to the results, respectively taking the bacterial liquids cultured for 0h, 11h, 30h, 35h and 48h, and carrying out the next experiment.

The third step: preparation of samples

And (3) placing the culture solution of the acidithiobacillus ferrooxidans strain in each time period in a 50mL centrifuge tube for centrifugation, and freezing to-80 ℃ for preservation. In preparation for subsequent extraction of RNA and cDNA.

EXAMPLE 2 specificity test for general PCR detection of several specific Gene primers

The first step is as follows: primer design and Synthesis

Corresponding fluorescent quantitative PCR primers are designed according to the DNA sequence of the gene, and primers of a 16srDNA gene (rrs) are designed to be used as an internal reference gene, primers of related ferrous oxide functional genes (including coxB, coxA, rus, Acop, petA1, cyc2 and cyc1), primers of an EPS metabolic gene (galU), primers of (arsH, arsB, arsC and arsR), primers of the ordinary PCR (table 1), 1 pair of internal reference gene primers, 12 pairs of target gene primers and a total of 26 sequences are synthesized by Shanghai bio-Biometrics, and the synthesis amount is 2 OD.

TABLE 1 PCR primer sequences involved in the present invention

The second step is that: bacterial total RNA extraction

And (3) taking 45mL of culture solution of the acidophilic thiobacillus ferrooxidans strain in each time period, placing the culture solution in a 50mL centrifuge tube for centrifugation, and extracting the total bacterial RNA by using an E.Z.N.A.Bacteria RNA Kit (OMEGA).

The third step: reverse transcription to synthesize cDNA

Reverse transcription reaction: sequentially adding 1 mu L of the total RNA template prepared in the previous step into a 0.2mL PCR tube of RNase-Free, carrying out RT reaction according to the operation instruction of a TIANCcript RT Kit (Tiangen (Beijing) reverse transcription Kit), carrying out reverse transcription to synthesize a cDNA chain by utilizing RNA, carrying out operation by adopting the Tiangen reverse transcription Kit according to an amplification program, and obtaining a reaction solution which is the cDNA template after the reverse transcription reaction is finished.

The fourth step: general PCR amplification

And (3) respectively carrying out PCR amplification by using the cDNA synthesized in the third step as a template and using several gene standard primers synthesized in the first step. The general PCR reaction system was 2 XTaq PCR MasterMix 12.5. mu.L, 1. mu.L each of the upstream and downstream primers (10pmol/uL), 1. mu.L of cDNA template, dd H₂O to a total volume of 25. mu.L. The reaction parameters are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 42s for 40 cycles; extension at 72 ℃ for 7 min. The reaction was carried out in a PCR amplificator (Eppendorf, Germany).

The fifth step: gel electrophoresis

And (3) carrying out agarose gel electrophoresis on the amplification product obtained in the fourth step, and detecting that a single bright band is out, thereby indicating that the primer has specificity and the sample is synthesized. Wherein, the sizes of the product fragments obtained by amplification of each primer pair are shown in the following table 2.

TABLE 2 size of amplified fragment of each primer

Example 3 application of real-time quantitative fluorescent PCR detection technique

The first step is as follows: bacterial total RNA extraction

Total RNA extraction: acidithiobacillus ferrooxidans is cultured in a 9K culture medium containing As (III) until the logarithmic growth phase (ferrous iron is completely consumed), and bacterial total RNA is extracted by using a Kit E.Z.N.A.bacteriosis RNA Kit (OMEGA).

The second step is that: reverse transcription to synthesize cDNA

Reverse transcription reaction: mu.L of the total RNA template prepared in the previous step was sequentially added to 0.2mL of a PCR tube of RNase-Free, and RT reaction was performed using a TIANCcript RT kit (Tiangen (Beijing)). And after the reverse transcription reaction is finished, obtaining reaction liquid which is the cDNA template.

The third step: real-time quantitative fluorescent PCR amplification

Real-time quantitative fluorescent PCR reaction system (total reaction volume 25. mu.L): including the second step of cDNA synthesis and the gene primers of example 2, and finally RNase/DNase Free dH₂O to the total volume of 25. mu.L of the reaction system, and a negative control (RNase-free water) was set. The reaction system of the target gene and the reaction system of the 16s rDNA gene (i.e. the reference gene) are both 2 XFastStart Essential DNA Green Master 10. mu.L, the final concentration of the upstream and downstream primers of each gene is (10pmol/uL) 1. mu.L, cDNA template 1. mu.L, RNase/DNase Free dH₂O to a total volume of 25. mu.L.

Reaction procedure: pre-denaturation at 95 ℃ for 300 s; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s for 40 cycles; the melting curve program is an initial stage: 95 ℃ for 60s, final stage: the temperature of 55 ℃ was increased to 95 ℃ at a rate of 0.1 ℃/s, and fluorescence signals were collected from 55 ℃.

The fourth step: determination of detection result

The amplification result was determined from a melting profile automatically generated by a LightCycler Nano 32-well fluorescence detection Polymerase Chain Reaction (PCR) instrument.

In the invention, the same primer automatically generates an amplification curve, a melting curve graph and a Ct value by a LightCycler Nano 32-hole fluorescence detection Polymerase Chain Reaction (PCR) instrument at different times, and a dye combining SYBR Green I and DNA is utilized.

FIG. 2 shows the amplification of the rrs reference gene at different time periods.

The result shows that the invention can well amplify the corresponding gene amplification product through PCR simultaneously, and further determine the expression condition of the specific gene, and the negative control has no non-specific melting peak, thereby proving that the determination result is reliable.

Example 4 calculation of relative expression amount

The first step is as follows: real-time quantitative fluorescent PCR amplification

According to the real-time fluorescent quantitative PCR amplification method of the embodiment 3, the culture solution of different time periods is taken to extract RNA and then amplified, and after the amplification is successful, the Ct value of each gene in different time periods is obtained.

The second step is that: calculation of relative expression amount

Taking bacterial liquids in different time periods under the same conditions, centrifuging to extract RNA, performing RT-qPCR experiments, taking 0h as a reference group, 11h, 30h, 35h and 48h as experimental groups, taking 16srDNA as an internal reference gene, and calculating a delta Ct value.

Wherein, Delta Ct 1 ═ Ct_{Functional genes in other time periods}-Ct_{Reference gene corresponding to time}，△Ct 2＝Ct_{Functional genes of reference group}-Ct_{Reference genes of the reference group}Δ (Δ CT) is Δ CT 1 to Δ CT 2. The actual expression amount is 2^-△(△Ct)。

Taking the relative expression level of 11h as an example, Δ Ct 1 ═ Ct_{11h expression level of functional Gene}-Ct_{11h reference Gene expression level}，△Ct 2＝Ct_{0h expression level of functional Gene}-Ct_{0h reference gene expression level}，△(△CT)＝△Ct 1-△Ct 2。

The results are shown in Table 2.

TABLE 2

The internal reference gene is used as a reference, and the obtained relative expression quantity changes along with time periods are plotted, wherein the related ferrous oxide functional gene is shown in figure 3, the EPS metabolic gene is shown in figure 4, and the arsenic-resistant related gene is shown in figure 5. Compared with ferrous oxidation on a macroscopic scale, the ferrous oxidation is that the expression quantity of related ferrous oxide functional genes is up-regulated, and the expression quantity almost consumed by ferrous in 30-48 hours is slowly reduced; the genes of arsenic resistance and EPS metabolism are the same, and the expression quantity is maximum in 11h and then gradually decreases.

Through calculation, the relative expression amounts in 11h, 30h, 35h and 48h are ferrous related genes respectively: coxA (32.89, 15.80, 2.87, 0.31), coxB (6.36, 0.86, 0.09, 0.01), rus (2.63, 9.87, 1.42, 0.37), Acop (18.65, 12.48, 6.64, 1.28), petA1(13.58, 12.00, 2.87, 0.19), cyc2(3.60, 6.03, 0.72, 0.28), cyc1(0.16, 0.14, 0.81, 3.45); the EPS metabolism gene comprises galU (18.95, 1.28, 0.64, 0.08), the arsenic-resistant related gene comprises arsH (0, 0.94, 0.72, 0.67), arsR (6.57, 1.73, 0.03, 0), arsB (2.37, 0.03, 0.01, 0), and arsC (17.21, 0.13, 0.27, 0.02).

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in other forms, and any person skilled in the art can change or modify the technical content disclosed above into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Sequence listing

<110> university of south-middle school

<120> kit and method for detecting dynamic change of gene expression in process of generating bacterium-mediated arsenopyrite

<160> 26

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

acactgggac tgagacacgg 20

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

accgcctacg caccctttac 20

<210> 3

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atcccaagca gggcgtaa 18

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tgccacaaat caaggaacac tt 22

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gtggtttccc aaggttactg 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atatcgcaat tctcctgttc 20

<210> 7

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aaggccatgt tggagaaa 18

<210> 8

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ggcataaccg cataaggag 19

<210> 9

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gccgtgggat ggaaagata 19

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atgaagtgtt gacccgtctg 20

<210> 11

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ccgcagcagc ctccatact 19

<210> 12

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tgaaatccgc caagccac 18

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gggtgactgg aactgggagg 20

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gtcaagccat tggagctgag ta 22

<210> 15

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gcgttacggt tacatcattc a 21

<210> 16

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

agccgccaca tccttcat 18

<210> 17

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

aagcccaaac ccgaggacg 19

<210> 18

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ttgtcgccgc agtcaaagc 19

<210> 19

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ggttggggat ggtgag 16

<210> 20

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ttggtagaat ggtcgg 16

<210> 21

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gccgataaca ttggcgtag 19

<210> 22

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gaacaatatg ccagcggta 19

<210> 23

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ccattctcgc cgaagtca 18

<210> 24

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

agccctcacg ctctagcag 19

<210> 25

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

atggaaccac tacaagaccc tg 22

<210> 26

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

aaaggaaatg ccgttgtgc 19

Claims

1. a test kit that detects the dynamic change of gene expression in the generation process of bacteria-mediated map water jarosite, is characterized in that, comprises one group for real-time fluorescence quantitative PCR technology to detect Thiobacillus acidophilus ferrooxidans functional gene expression Dynamically changing nucleic acid, the functional genes include ferrous oxidation-related genes, EPS metabolism genes and arsenic tolerance-related genes, and the nucleic acid includes primers for detecting ferrous oxidation-related genes coxB, coxA, rus, Acop, petA1, cyc2 and cyc1 Right, the primer pair for detecting EPS metabolism gene galU and the primer pair for detecting arsenic resistance-related genes arsH, arsB, arsC and arsR; wherein, the primer pair for coxB is shown in SEQ ID NO.3 and SEQ ID NO.4, The primer pair for coxA is shown in SEQ ID NO.5 and SEQ ID NO.6, the primer pair for rus is shown in SEQ ID NO.7 and SEQ ID NO.8, and the primer pair for Acop is shown in SEQ ID NO. .9 and SEQ ID NO.10, the primer pair for petA1 is shown in EQ ID NO.11 and SEQ ID NO.12, and the primer pair for cyc2 is shown in SEQ ID NO.13 and SEQ ID NO.14 As shown, the primer pair of cycl is shown in SEQ ID NO.15 and SEQ ID NO.16, the primer pair of galU is shown in SEQ ID NO.17 and SEQ ID NO.18, the primer pair of arsH is shown in SEQ ID NO.17 and SEQ ID NO.18. The pair is shown in SEQ ID NO.19 and SEQ ID NO.20, the primer pair for arsB is shown in SEQ ID NO.21 and SEQID NO.22, and the primer pair for arsC is shown in SEQ ID NO.23 and SEQ ID NO.22 ID NO.24, the primer pair of arsR is shown as SEQ ID NO.25 and SEQ ID NO.26;

It also includes an internal reference gene control primer pair, and the internal reference gene control primer pair is shown in SEQ ID NO.1 and SEQ ID NO.2.

2 . The kit of claim 1 , wherein the kit further comprises: 2×FastStartEssentialDNA Green Master. 3 .

3. a method for detecting the dynamic change of gene expression in the generation process of bacteria-mediated map water arsenite, it is characterized in that, adopting real-time fluorescence quantitative PCR technology to detect the dynamic change situation of functional gene expression of Thiobacillus acidophilus ferrooxidans, its Include the following steps:

(1) extract RNA from the culture solution of Thiobacillus acidophilus ferrooxidans in different time periods;

(2) synthesizing cDNA by reverse transcription of the extracted RNA;

(3) performing fluorescence quantitative PCR amplification on the synthesized cDNA; wherein, during fluorescence quantitative PCR amplification, it includes a functional gene reaction system and an internal reference gene reaction system, and the functional gene reaction system includes ferrous oxidation-related genes, EPS The primer pair of any functional gene in the metabolic gene and the arsenic resistance-related gene and the cDNA synthesized by reverse transcription, the internal reference gene reaction system comprises the internal reference control primer pair as shown in SEQ ID NO.1 and SEQ ID NO.2 the sequence of;

(4) Put it into the fluorescence quantitative amplification PCR instrument, and generate the Ct _{functional gene} and the Ct _{internal reference gene} by the instrument detection;

(5) Calculation of relative expression level: take 0h as the reference group, different time periods as the experimental group, and 16srDNA as the internal reference gene, the real expression level is 2- ^△(△CT)) , where, △Ct 1=Ct _{other time Section functional gene-} Ct _{corresponds to the reference gene in time} , △Ct 2= _{functional gene in the} Ct _{reference group-the internal reference gene} in the Ct reference group, △(△CT)=△Ct 1-△Ct 2;

In the step (3), the primer pairs of the ferrous oxidation-related genes include the primer pairs that detect coxB, coxA, rus, Acop, petA1, cyc2 and cyc1, and the primer pairs of the EPS metabolism gene are the primer pairs that detect galU. Yes, the primer pair for the arsenic resistance-related gene includes a primer pair for detecting arsH, arsB, arsC and arsR; wherein, the primer pair for coxB is shown in SEQ ID NO.3 and SEQ ID NO.4, the coxA The primer pair is shown as SEQ ID NO.5 and SEQ ID NO.6, the primer pair of rus is shown as SEQ ID NO.7 and SEQ ID NO.8, and the primer pair of Acop is shown as SEQ ID NO.9 and SEQ ID NO.10, the primer pair of petA1 is shown as SEQ ID NO.11 and SEQ ID NO.12, the primer pair of cyc2 is shown as SEQ ID NO.13 and SEQ ID NO.14, The primer pair of cycl is shown in SEQ ID NO.15 and SEQ ID NO.16, the primer pair of galU is shown in SEQ ID NO.17 and SEQ ID NO.18, and the primer pair of arsH is shown in SEQ ID NO.18 NO.19 and SEQ ID NO.20, the primer pair of arsB is shown as SEQ ID NO.21 and SEQ ID NO.22, and the primer pair of arsC is shown as SEQ ID NO.23 and SEQ ID NO.22. 24, the primer pair for arsR is shown in SEQ ID NO.25 and SEQ ID NO.26.

4. The method according to claim 3, wherein in the step (3), the reaction system of fluorescence quantitative PCR amplification is a 25 μL system, comprising:

2 x FastStart Essential DNA Green Master: 10 μL,

Each functional gene primer pair: the final concentration of upstream and downstream primers is 10 pmol/μL, each 1 μL,

cDNA template: 1 μL,

Nuclease-free water to 25 μL;

At the same time, nuclease-free water was set as a negative control.

5 . The method according to claim 3 , wherein the reaction procedure of fluorescence quantitative PCR amplification in the step (3) is: 95° C. pre-denaturation for 300 s; 95° C. denaturation for 30 s, 58° C. annealing for 30 s, and 72° C. Extend for 30 s, 40 cycles; the melting curve program is initial stage: 95 °C for 60 s, final stage: 55 °C rising to 95 °C with a change rate of 0.1 °C/s, and the fluorescence signal is collected from 55 °C.

6. The method according to claim 3, wherein in the step (1), the different time periods refer to after culturing 0, 11, 30, 35, and 48 h, wherein the initial arsenic concentration in the used culture solution is 500 mg/L, pH 2-2.5, iron/arsenic ratio 4-6, inoculum of Thiobacillus acidophilus ferrooxidans was 10%.