CN113897324B

CN113897324B - jcVIPP1 recombinant escherichia coli used as anti-manganese agent and construction method thereof

Info

Publication number: CN113897324B
Application number: CN202111194678.4A
Authority: CN
Inventors: 郝大海; 徐紫薇; 唐湘华; 龚明; 王莎莎; 杨双龙; 刘啟巧; 孔垂凡
Original assignee: Yunnan Normal University
Current assignee: Yunnan Normal University
Priority date: 2021-10-13
Filing date: 2021-10-13
Publication date: 2023-07-28
Anticipated expiration: 2041-10-13
Also published as: CN113897324A

Abstract

The invention provides a jcVIPP1 recombinant escherichia coli serving as an anti-manganese agent and a construction method thereof, wherein the jcVIPP1 recombinant escherichia coli is constructed by recombining a plastid 1 vesicle induction protein gene jcVIPP1 in jatropha curcas into a vector and introducing the vector into the escherichia coli. The engineering bacterium of the escherichia coli constructed by the invention has wide application range, can grow rapidly after a delay period at normal temperature, has an optimal growth temperature of 37 ℃, ensures that the thallus can treat Mn at high temperature ²⁺ The pollution is removed at the same time, and the cost is very low; the growth speed of thalli is high, so that the period for removing pollution is short; after the generated thalli are further concentrated, the absorbed Mn can be easily extracted, and the biomass smelting method has the advantage of biological smelting.

Description

jcVIPP1 recombinant escherichia coli used as anti-manganese agent and construction method thereof

Technical Field

The invention belongs to the technical field of biochemistry, and particularly relates to a jcVIPP1 recombinant escherichia coli serving as an anti-manganese agent and a construction method thereof.

Background

VIPP1 was first found in pea in 1994 and was named membrane associated protein (membrane association protein) because it binds to chloroplast envelope (envelope) and thylakoid (thyakoid) membranes and has a molecular weight of 30kDa, and is also known as IM30 (30 kDa inner-membrane association protein). In 2001 was renamed as Vesicle-inducing protein in plastid 1 (Vesicle-inducing Protein in Plastids, vipp 1). Phylogenetic analysis indicated that VIPP1 originated from prokaryotes and may evolve from bacteriophage shock protein a (Phage Shock Protein A, pspA). This is the first plant homologous protein of E.coli (Escherichia coli). VIPP1 is involved in the formation or remodeling of green plants and blue algae cyst membranes in a manner that alters local structure. In chloroplast-containing plants, VIPP functions by relying on magnesium ions. In an experiment for expressing VIPP-his in an in vitro experiment of escherichia coli, the VIPP1 is depended on being combined with GTP to play a role of GTPase. Thereby promoting the movement of the VIPP1 and maintaining the transmembrane proton gradient of the cell membrane of the escherichia coli. This may have some relevance to fusion repair of the plasma membrane. Meanwhile, experimental study proves that the VIPP1 can save defective proton leakage in the escherichia coli PspA mutant and play a role in membrane repair. In adverse stress, under the condition that the membrane is damaged, the VIPP1 can participate in bacterial stress reaction, specifically interact with the surface of the negatively charged membrane to trigger membrane fusion, so that the membrane stabilizing effect is achieved.

Manganese is a trace element necessary for organisms. It is an important component of superoxide dismutase, phosphopyruvate decarboxylase, arginase, glutamate enzyme, etc., and manganese plays an important role in electron transfer in water splitting of photosynthesis. Because of the important role of manganese, researchers have been focusing on the effects of Mn deficiency in the past. Biological manganese deficiency can lead to the occurrence of various adverse symptoms, but with the development of industry and agriculture, the use of Mn is increasingly common. In industry, mn is one of important alloying elements, and is incorporated into various steels to increase hardness of the materials, so that both welder and smelter are exposed to a high-definition environment. Because of exploitation, storage and transportation, manganese in manganese mines permeates soil and water to pollute Mn in the environment. In agriculture, mn-containing pesticides, such as mancozeb and mancozeb, are used for crop antibiosis, enter the environment and the crop body through various application modes, and are taken into the body by people. In addition, with the rapid development of economy and transportation, organic lead has been used as an antiknock agent, and is now replaced by MMT (methylcyclopentadienyl manganese tricarbonyl ), and the octane number of oil can be improved. Thus Mn enters the engine body through the contact of the automobile exhaust and oil.

The harm of excessive manganese to living beings is most reported to be human. Mn can enter human body through 3 routes: (1) food enters the human body through the digestive tract: (2) through the respiratory tract, across the blood brain barrier and into the brain; (3) the manganese-containing material is contacted through the skin into the human body. Mn is mainly toxic to damage pallidum and substantia nigra of basal nuclei of brain, affect secretion and degradation of dopamine, and produce symptoms such as ataxia like parkinsonism. Because of the similarity to iron, excessive manganese also competes for Fe ²⁺ Is responsible for iron deficiency anemia. In addition, excessive Mn can also cause harm to hearing loss, stillbirth, cardiopulmonary insufficiency, and finger loss.

At present, the existing treatment methods for heavy metal pollution of water body include adsorption, ion exchange, precipitation/coprecipitation, various separation technologies (filtration, membrane and even magnetic methods), biological methods and natural restoration. Wherein the ion exchange technique is carried out by H carried on the resin ⁺ The metal ions are exchanged with metal ions to remove the polluted metal ions in the water body, and the ion exchange capacity is required to be recovered periodically after a certain time, so that the cost is high; chemical precipitation forms more sludge, which is costly and difficult to treat. The membrane treatment technology and the electrochemical method have high cost. Phytoremediation, limited by environmental conditions, takes too long.

Disclosure of Invention

The invention aims to provide a recombinant Escherichia coli of JCVIPP1 used as an anti-manganese agent and a construction method thereof, and the constructed Escherichia coli engineering bacteria have wide application range, can rapidly grow after a delay period at normal temperature, have an optimal growth temperature of 37 ℃, ensure that thalli are treated with Mn at high temperature ²⁺ The pollution is removed at the same time, and the cost is very low; the growth speed of thalli is high, so that the period for removing pollution is short; the generated thallus is introduced intoAfter one-step concentration, the absorbed Mn can be easily extracted, and has the advantage of biological smelting.

The invention provides a recombinant Escherichia coli of JCVIPP1 used as an anti-manganese agent, which is constructed by recombining a plasmid 1 vesicle induction protein gene JCVIPP1 in Jatropha curcas into a vector and introducing the vector into the Escherichia coli.

The invention also provides a construction method of the JCVIPP1 recombinant escherichia coli, which comprises the following steps:

primer Vipp-F5'-CCG ATGGCCGCAAAATCACAGTTAATT AC-3'; RT-PCR is carried out on the drought treated Jatropha curcas cDNA library to obtain a JCVIPP1 full-length coding gene, wherein the full length of the coding gene is 1005bp, and the method comprises the following steps:

ATGGCCGCAAAATCACAGTTAATTACAGGATTGACCTTGCCATTGCCACCGCCGCATTCTTCCACTTCCTCAACCTCCAATAACAGCAGCAACACTCTCTGTATGGTCAAGCGGCCGCAACTTACGACTTCGTTTTTCAATGGCGGAGTTGAAGCTCTAAAATTTTCTAGGATAAGGACTTGTTCTACTAGGTCCCATTGCTACAGACAAGGTGGAGGTGCTCTTGGCACTCGTATGAATCTTTTTGATCGGTTTGCTAGAGTTGTCAAGTCATATGCAAATGCAATCTTGAGTGGTTTTGAGGACCCGGAAAAAATTCTAGATCAGACGGTTCTTGAAATGAATGATGACTTGACAAAGATGCGTCAGGCCACAGCACAAGTATTGGCATCTCAAAAACGTTTGGAAAATAAATACAAAGCTGCGGAACAAGCTTCTGAGGATTGGTACCGTAAAGCACAACTTGCTCTTCAGAAAGGAGAGGAAGATCTCGCTCGGGAAGCTCTTAAGAGGCGTAAATCTTATGCTGACAATGCAAATTCCTTGAGAGCTCAACTTGATCAACAGAAAAGTGTTGTTGAGAATCTTGTCTCTAATACTCGGCTTTTGGAGAGCAAGATACAGGAGGCAAAGTCTAAAAAAGATACTCTGAAAGCGCGTGCCCAATCTGCAAAAACTCAAACCAAAGTGAATGAGATGCTGGGGAATGTAAATACAAGCAACGCTCTTTCAGCTTTCGAGAAAATGGAAGAGAAAGTATTGCAAATGGAATCAGAAGCTGAAGCACTTGGCCAGTTAGCTACAAGTGAATTGGATGGAAAGTTTGCTTTACTTGAGAGCTCATCTGTTGATGATGATCTTGAGAACCTGAAGAAGGAAATTTCTGGTAGCAAAAAGAGAGGAGAACTGCCGCCCGGTAGAACAGTTGTCAGCAGCTCTGCATTGAGAGATCCTGAGATTGAGATGCAGCTCAATGAATTAAGACAAAAGAGAAAGGAATTCTAA；

the amino acid composition of the protein consisting of 334 amino acid residues is shown in table 1:

TABLE 1jcVIPP1 amino acid composition

Amino acids	Quantity of	Percentage of	Amino acids	Quantity of	Percentage of
						Ala(A)	31	9.30％	Leu(L)	39	11.70％
Arg(R)	22	6.60％	Lys(K)	30	9.00％
						Asn(N)	18	5.40％	Met(M)	9	2.70％
Asp(D)	15	4.50％	Phe(F)	9	2.70％
						Cys(C)	3	0.90％	Pro(P)	9	2.70％
Gln(Q)	20	6.00％	Ser(S)	35	10.50％
						Glu(E)	29	8.70％	Thr(T)	20	6.00％
Gly(G)	15	4.50％	Trp(W)	1	0.30％
						His(H)	2	0.60％	Tyr(Y)	5	1.50％
Ile(I)	7	2.10％	Val(V)	15	4.50％

The amplified product is digested with restriction enzymes HindIII and BamH I, and then ligated to vector pYES2, and introduced into E.coli DH 5. Alpha. To construct the recombinant E.coli of JCVIPP 1.

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

mn in clean environment ²⁺ In the aspect of pollution, the invention has the advantages of low use cost, wide application and high speed. Compared with the ion exchange technology in the prior art, the latter needs to periodically recover the ion exchange capability, and requires time, manpower, material resources and financial investment, and the use cost of engineering bacteria constructed by using the JCVIPP1 is low. The chemical precipitation method can form more sludge, the cost is high, and the sludge treatment is difficult. The membrane treatment technology and the electrochemical method have high cost. Phytoremediation, limited by environmental conditions, takes too long.

The engineering bacterium of the escherichia coli constructed by the invention has wide application range, can grow rapidly after a delay period at normal temperature, has an optimal growth temperature of 37 ℃, ensures that the thallus can treat Mn at high temperature ²⁺ The pollution is removed at the same time, and the cost is very low; the growth speed of thalli is high, so that the period for removing pollution is short; after the generated thalli are further concentrated, the absorbed Mn can be easily extracted, and the biomass smelting method has the advantage of biological smelting.

Drawings

FIG. 1 shows strains obtained by screening in accordance with an embodiment of the present inventionRow Mn ²⁺ A comparison graph of the minimum inhibitory concentration experiment of (2);

FIG. 2 is a graph showing experimental comparison under the condition of normal LB medium culture in an embodiment of the invention;

FIG. 3 shows the Mn at 25mM in one embodiment of the invention ²⁺ Experimental comparison diagrams under the condition of culture medium culture;

FIG. 4 shows the effect of CAT activity under manganese stress in Escherichia coli JcVIPP1 according to one embodiment of the present invention;

FIG. 5 shows the effect of JCVIPP1 on E.coli POD activity according to one embodiment of the present invention;

FIG. 6 shows the effect of JCVIPP1 on SOD activity of E.coli according to one embodiment of the present invention.

Detailed Description

The present invention will be described in detail below with reference to the embodiments shown in the drawings, but it should be understood that the embodiments are not limited to the present invention, and functional, method, or structural equivalents and alternatives according to the embodiments are within the scope of protection of the present invention by those skilled in the art.

This example provides a recombinant Escherichia coli of JCVIPP1 as an anti-manganese agent, in which a plastid 1 vesicle-inducing protein (VIPP 1) gene JCVIPP1 of Jatropha curcas (Jatropha curcas) was recombined into a vector and introduced into Escherichia coli (strain name abbreviated as JcV) and having resistance to high concentration of manganese ions (Mn ²⁺ ) Through a growth curve, the change condition of Peroxidase (POD), catalase (CAT) and superoxide dismutase (Superoxide Dismutase, SOD) in an in-cell antioxidant system; in contrast to the control, jcvpp 1 recombinant escherichia coli had an effect on environmental repair under manganese stress.

The engineering bacterium of the escherichia coli constructed by the invention has wide application range, can grow rapidly after a delay period at normal temperature, has an optimal growth temperature of 37 ℃, ensures that the thallus can treat Mn at high temperature ²⁺ The pollution is removed at the same time, and the cost is very low; the growth speed of the thalli is high, so that the period of pollution removalShort; after the generated thalli are further concentrated, the absorbed Mn can be easily extracted, and the biomass smelting method has the advantage of biological smelting.

The construction method of the recombinant Escherichia coli of the JCVIPP1 comprises the following steps: primer Vipp-F5'-CCG tgatacatatgcccg ATGGCCGCAAAATCACAGTTAATTAC-3'; vipp-R5'-CCG agcaatcagaattcg TTAGAATTCCTTTCTCTTTTGTCTTAATTCATTG-3', RT-PCR was performed on the drought-treated Jatropha curcas cDNA library to obtain the full-length coding gene of JCVIPP1 (JCCAP 30). The whole length of the coding gene is 1005bp (as follows), the coding gene codes for protein consisting of 334 amino acid residues, and the amino acid composition is shown in table 1. The amplified product was digested with the restriction enzymes HindIII and BamHI, ligated to vector pYES2 (ThermoFisher, USA), and introduced into E.coli DH 5. Alpha.

Strains containing the recombinant plasmid were selected with selection medium. First, positive clones were screened with LB solid medium containing 50mg/l ampicillin (Amp), colony PCR was performed with Vipp-F and Vipp-R primers, and the PCR products were subjected to agarose gel electrophoresis to verify whether the positive clones were false positive. After the positive colony is subjected to secondary single colony streak culture and purification, the positive colony is subjected to Mn-containing culture and purification ²⁺ Screening again, verifying the positive clone by PCR again, sequencing the PCR product, and confirming that the gene sequence is correct.

ATGGCCGCAAAATCACAGTTAATTACAGGATTGACCTTGCCATTGCCACCGCCGCATTCTTCCACTTCCTCAACCTCCAATAACAGCAGCAACACTCTCTGTATGGTCAAGCGGCCGCAACTTACGACTTCGTTTTTCAATGGCGGAGTTGAAGCTCTAAAATTTTCTAGGATAAGGACTTGTTCTACTAGGTCCCATTGCTACAGACAAGGTGGAGGTGCTCTTGGCACTCGTATGAATCTTTTTGATCGGTTTGCTAGAGTTGTCAAGTCATATGCAAATGCAATCTTGAGTGGTTTTGAGGACCCGGAAAAAATTCTAGATCAGACGGTTCTTGAAATGAATGATGACTTGACAAAGATGCGTCAGGCCACAGCACAAGTATTGGCATCTCAAAAACGTTTGGAAAATAAATACAAAGCTGCGGAACAAGCTTCTGAGGATTGGTACCGTAAAGCACAACTTGCTCTTCAGAAAGGAGAGGAAGATCTCGCTCGGGAAGCTCTTAAGAGGCGTAAATCTTATGCTGACAATGCAAATTCCTTGAGAGCTCAACTTGATCAACAGAAAAGTGTTGTTGAGAATCTTGTCTCTAATACTCGGCTTTTGGAGAGCAAGATACAGGAGGCAAAGTCTAAAAAAGATACTCTGAAAGCGCGTGCCCAATCTGCAAAAACTCAAACCAAAGTGAATGAGATGCTGGGGAATGTAAATACAAGCAACGCTCTTTCAGCTTTCGAGAAAATGGAAGAGAAAGTATTGCAAATGGAATCAGAAGCTGAAGCACTTGGCCAGTTAGCTACAAGTGAATTGGATGGAAAGTTTGCTTTACTTGAGAGCTCATCTGTTGATGATGATCTTGAGAACCTGAAGAAGGAAATTTCTGGTAGCAAAAAGAGAGGAGAACTGCCGCCCGGTAGAACAGTTGTCAGCAGCTCTGCATTGAGAGATCCTGAGATTGAGATGCAGCTCAATGAATTAAGACAAAAGAGAAAGGAATTCTAA。

TABLE 1jcVIPP1 amino acid composition

Subsequently, mn was applied to the strain obtained by screening ²⁺ Minimum inhibitory concentration experiments of (c). The wild E.coli DH 5. Alpha. And the strain containing empty vector pYES2 (designated pYES 2) were used as controls, the strain carrying the gene of JCVIPP1 (designated JCVIPP 1) was confirmed as the experimental strain, and after activation of these 3 strains, DH 5. Alpha. And pYES2 and JCVIPP1 were cultured in LB liquid medium and 50mg/l amp+LB liquid medium, respectively. After overnight culture at 37℃and 200rpm, the bacterial solutions of 3 strains were diluted to OD ₆₀₀ =1.00±0.01, 10 to 10 ⁵ After gradient dilution, the mixture contains 5 mM-30 mM Mn ²⁺ A minimum inhibitory concentration experiment is carried out on an LB solid medium, and the result shows that the JCVIPP1 gene can greatly improve the Mn resistance of the strain ²⁺ Stress ability to E.coli at 25mM Mn ²⁺ Is grown in the environment (fig. 1).

In FIG. 1, the JCVIPP1 gene enhances the Mn resistance of E.coli ²⁺ Stress ability. The 3 strains are escherichia coli (jcvpp 1) containing vesicle induction protein (jcvpp 1) genes in jatropha curcas plastid 1; a strain containing empty vector pYES2 (pYES 2); wild type E.coli DH 5. Alpha. InGrowth conditions on LB plates of different mn2+ concentrations, a:5mM Mn ²⁺ +LB solid medium; b:10mM Mn ²⁺ +LB solid medium; c:15mM Mn ²⁺ +LB solid medium; d:20mM Mn ²⁺ +LB solid medium; e:25mM Mn ²⁺ +LB solid medium; f:30mM Mn ²⁺ +LB solid medium.

According to FIG. 2, it is shown that under the condition of normal LB medium culture, wild type E.coli DH5 alpha, E.coli with empty vector pYES2 recombined, E.coli with JCVIPP1 recombined are basically consistent in growth condition, so that the transfer of pYES2 and JCVIPP1 has no obvious influence on E.coli under LB medium culture, and basically enters the growth phase from 2 hours to 18 hours.

As can be seen from FIG. 3, the concentration of Mn was 25mM ²⁺ Under the condition of culture medium culture, the wild type escherichia coli DH5 alpha, the escherichia coli recombined with the empty vector pYES2, the escherichia coli recombined with the JCVIPP1 are obviously inhibited, the time for entering the growing period and the platform period is obviously delayed, the DH5 alpha is most obviously inhibited, the faint growth is started from 32h, and after the empty vector pYES2 is recombined, the recombinant escherichia coli is subjected to 25mM Mn ²⁺ The resistance under stress is improved to a certain extent, and the growth starts from 28h, which shows that the transfer of the pYES2 vector is carried out on the escherichia coli at 25mM Mn ²⁺ Growth under stress has an effect on 25mM Mn after recombination of JCVIPP1 ²⁺ The best performance under stress, the growth period starts from 24h to 4 and the platform period starts from 40 h. Coli at 25mM Mn for elimination of vector pYES2 ²⁺ The effect of growth and physiological response under stress, so that subsequent experiments selected pYES2 as a control. The enzyme activity change at 26h and 32h was selectively determined to observe the E.coli strain with JCVIPP1 at 25mM Mn ²⁺ Physiological response in culture.

Effect of CAT Activity under manganese stress in Escherichia coli JcVIPP1

In the case of LB culture and 25mM Mn2+ culture for 26 hours, DH 5. Alpha. Was not significantly different from pYES2 in CAT enzyme activity, but 25mM Mn ²⁺ When cultured for 32h, pYES2 CAT enzyme activity was significantly improved compared with DH5 alpha (FIG. 4). Compared with pYES2, CAT activity expression of Escherichia coli transformed with JCVIPP1 and cultured in LB medium for 26h is extremely remarkableInfluence (p)<0.01 At 25mM Mn) ²⁺ Both cultures 26h and 32h had significant effects (p<0.05)。

Compared with CAT activity under LB culture, the catalyst has the following characteristics of 25mM Mn ²⁺ In culture, the CAT activities of DH 5. Alpha. And pYES2 were reduced as a whole (FIG. 4), indicating that CAT expression in E.coli antioxidant system was inhibited under manganese stress. The transfer of JcVIPP1 counteracts this inhibition to some extent at 25mM Mn ²⁺ No significant decrease in POD activity occurred in culture. The jcVIPP1 gene obviously improves the activity of CAT in escherichia coli. Compared with CAT activity under LB culture, the catalyst has the following characteristics of 25mM Mn ²⁺ Under culture, CAT activity was reduced, indicating that CAT expression in E.coli antioxidant system was inhibited under manganese stress.

Effect of JCVIPP1 on E.coli POD Activity

Either in LB or 25mM Mn ²⁺ In the culture, DH5 alpha has no obvious difference compared with pYES2 POD enzyme activity. Compared with pYES2, the POD activity expression of E.coli transformed with JCVIPP1 cultured in LB medium for 26h showed a significant difference (0.01)<p<0.05 No significant difference by 32h (p)>0.05 (fig. 5).

At 25mM Mn ²⁺ Under culture, POD activity expression of E.coli for 26h and 32h had extremely significant effects (p<0.01 (fig. 5). Compared with POD activity under LB culture, the Mn at 25mM ²⁺ In culture, the POD activities of DH 5. Alpha. And pYES2 were reduced as a whole (FIG. 5), indicating that the expression of POD in E.coli antioxidant system was inhibited under manganese stress. The transfer of JcVIPP1 counteracts this inhibition to some extent at 25mM Mn ²⁺ No significant decrease in POD activity occurred in culture.

Effect of JCVIPP1 on SOD Activity of E.coli

Either in LB or 25mM Mn ²⁺ There was no significant difference between the SOD enzyme activity of pYES2 and that of the medium culture `DH 5. Alpha.' (FIG. 6). Compared with pYES2, the expression of SOD activity of E.coli transformed with jcVIPP1 cultured in LB medium at 26h and 32h showed extremely significant differences (p<0.01 (p) and significant differences (p)<0.05 (fig. 6).

At 25mM Mn ²⁺ Culturing for 26hPOD activity expression was not significantly affected (p>0.05 32h POD activity showed significant differences (p)<0.05 (fig. 6). But compared with SOD activity under LB culture, the catalyst has the Mn of 25mM ²⁺ Under culture, the activity of SOD is obviously doubled (figure 6), which shows that under the stress of manganese, the expression of SOD in an escherichia coli antioxidant system is obviously enhanced.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

1. Application of plastid 1 vesicle induced protein gene JCVIPP1 in Jatropha curcas in improving manganese stress resistance of escherichia coli, wherein the nucleotide sequence of the JCVIPP1 is as follows: