阅读说明：本技术 一种具有三磷酸腺苷隧道金属酶活性的ZjAC2编码基因序列 (ZjAC2 encoding gene sequence with adenosine triphosphate tunnel metalloenzyme activity ) 是由 刘孟军 刘志国 袁野 于 2021-08-05 设计创作，主要内容包括：本发明公开了一种具有三磷酸腺苷隧道金属酶活性的ZjAC2编码基因序列,编码三磷酸腺苷隧道金属酶的基因为ZjAC2,ZjAC2具有如序列表中SEQ ID NO.1的DNA序列。本发明采用上述的一种具有三磷酸腺苷隧道金属酶活性的ZjAC2编码基因序列,编码的蛋白除腺苷酸环化酶活性外,还具备NTP水解酶活性、三聚磷酸酶活性以及较弱的焦磷酸酶活性。(The invention discloses a ZjAC2 coding gene sequence with adenosine triphosphate tunnel metalloenzyme activity, wherein the gene coding the adenosine triphosphate tunnel metalloenzyme is ZjAC2, and ZjAC2 has a DNA sequence shown as SEQ ID NO.1 in a sequence table. The ZjAC2 coding gene sequence with the adenosine triphosphate tunnel metalloenzyme activity is adopted, and the coded protein has NTP hydrolase activity, tripolyphosphate phosphatase activity and weaker pyrophosphatase activity besides adenylate cyclase activity.)

1. A ZjAC 2-encoding gene sequence having adenosine triphosphate tunneling metalloenzyme activity, characterized in that: the gene for coding the adenosine triphosphate tunnel metalloenzyme is ZjAC2, and ZjAC2 has a DNA sequence shown as SEQ ID NO.1 in a sequence table.

2. The gene sequence encoding ZjAC2 having atpc-tunnellin metalloenzyme activity according to claim 1, wherein the core region sequence of ZjAC2 is obtained by amplification using the following nucleotide sequences as primers:

forward direction: 5'-ATGGAGGTCGAAGTCAAGCT-3', respectively;

and (3) reversing: 5'-CTAAGGCAGCTTTCCGGATC-3' are provided.

3. A derivative sequence of a ZjAC 2-encoding gene sequence having atp tunneller activity, characterized by: the derivative sequence is an allosteric variant of the gene sequence obtained by substitution, insertion or deletion of one or several bases of the gene sequence according to claim 1.

Technical Field

The invention relates to the technical field of adenosine triphosphate tunnel metalloenzyme, in particular to a ZjAC2 encoding gene sequence with adenosine triphosphate tunnel metalloenzyme activity.

Background

The adenosine Triphosphate Tunnel Metalloenzymes (TTMs) superfamily is composed of a group of proteins with the ability to hydrolyze multiple tripolyphosphates, and is ubiquitous in organisms. The catalytic characteristics of this protein superfamily are: substrates are all triphosphate substrates, and divalent metal auxiliary ions are needed to exert enzyme activity. Furthermore, the superfamily of proteins is structurally similar, with all TTM proteins having 8 antiparallel beta sheet strands and an EXEXK motif.

In Arabidopsis thaliana, 3 AtTTMs were found, of which AtTTM1, 2 encoded proteins showed pyrophosphatase activity and weaker ATP hydrolase activity, while AtTTM3 encoded proteins showed tripolyphosphate activity, and none of the three proteins had Adenylate Cyclase (AC) activity.

With the development of sequencing technology, more TTMs genes are discovered in research, but the information obtained at present is far from sufficient to fully understand the characteristics of TTM protein, so more research and data are needed for identifying the TTMs.

Disclosure of Invention

The invention aims to provide a ZjAC2 encoding gene sequence with adenosine triphosphate tunneling metalloenzyme activity, and the encoded protein has NTP hydrolase activity, tripolyphosphate phosphatase activity and weaker pyrophosphatase activity besides adenylate cyclase activity.

In order to achieve the aim, the invention provides a ZjAC2 encoding gene sequence with adenosine triphosphate tunneling metalloenzyme activity, the gene encoding the adenosine triphosphate tunneling metalloenzyme is ZjAC2, and ZjAC2 has a DNA sequence shown as SEQ ID NO.1 in a sequence table.

Preferably, the core region sequence of ZjAC2 is obtained by amplification with the following nucleotide sequences as primers:

forward direction: 5'-ATGGAGGTCGAAGTCAAGCT-3', respectively;

and (3) reversing: 5'-CTAAGGCAGCTTTCCGGATC-3' are provided.

A derivative sequence of ZjAC2 coding gene sequence with ATP tunneller metalloenzyme activity, which is an allosteric variant of said gene sequence obtained by substituting, inserting or deleting one or several bases of said ZjAC2 gene sequence.

Therefore, the invention adopts the ZjAC2 encoding gene sequence with adenosine triphosphate tunneling metalloenzyme activity, and the encoded protein has NTP hydrolase activity, tripolyphosphate phosphatase activity and weaker pyrophosphatase activity besides adenylate cyclase activity.

The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.

Drawings

FIG. 1 is a schematic diagram of a complementation test culture dish for E.coli cyaA deficiency;

FIG. 2 is a bar graph of adenylate cyclase activity in an in vitro protein catalysis assay;

FIG. 3 is a bar graph of a hydrolase activity assay;

FIG. 4 is Mn²⁺、Mg²⁺、Ca²⁺Under the conditions, a histogram of the change of the activity of the ATP hydrolase before and after the addition of pyrophosphatase;

FIG. 5 is a line graph showing the time-dependent changes in the expression level of ZjAC2 gene and cAMP content in the in vivo gene transient expression test of Zizyphi fructus.

Detailed Description

The technical solution of the present invention is further illustrated by the accompanying drawings and examples.

Example one

Amplification of ZjAC2 Gene

Extracting RNA of winter jujube fruits, then carrying out reverse transcription to obtain cDNA serving as a PCR amplification template, and carrying out PCR amplification; wherein, the forward primer: 5'-ATGGAGGTCGAAGTCAAGCT-3', reverse primer: 5'-CTAAGGCAGCTTTCCGGATC-3' are provided.

PCR amplification procedure: 94 ℃ for 4 min; (94 ℃, 40 s; 56 ℃, 45 s; 72 ℃, 50s)35 cycles; 72 ℃ for 10 min.

And observing an amplification result of the PCR product through agarose gel electrophoresis, carrying out agarose gel recovery on the candidate target band, and connecting the recovered product into a pMDTM19-T cloning vector for sequencing to obtain a ZjAC2 sequence as a candidate jujube adenylate cyclase gene for functional verification.

Example two

Escherichia coli defect complementation test

Culturing Escherichia coli cyaA deficient strain SP850, wherein the SP850 strain lacks Adenylate Cyclase (AC) gene, respectively thermally shocking pET-15b recombinant plasmid with ZjAC2 gene into SP850, coating ampicillin and Carna antibiotic culture dishes, selecting and performing monoclonal culture until OD600 is 0.5, performing induced expression culture for 4h, performing streak culture in Macconka culture medium, and observing.

As a result, as shown in FIG. 1, it was found that the colonies of the SP850 deficient strain harboring the recombinant plasmid and the wild type E.coli strain were both red in color, whereas the SP850 deficient strain was white in color in the MacConkey medium, indicating that the ZjAC2 gene complements the cyaA gene lacking in the SP850 strain and functions as an adenylate cyclase gene.

EXAMPLE III

Protein in vitro catalysis assay

The recombinant vector pET-15b with the ZjAC2 gene is transferred into Escherichia coli BL21 strain, cultured at 37 ℃ and 200rpm, protein extraction and purification are carried out after IPTG induces protein expression, and the obtained protein is used for enzyme catalytic activity detection, and the result is shown in figure 2-4. As a result, it was found that Mn²⁺And Ca²⁺When the protein is used as coenzyme ions, cAMP is generated in a catalytic system, which shows that ZjAC2 protein can catalyze ATP to form cAMP and exert the function of AC; furthermore, in Mg²⁺When a large amount of Pi was present in the reaction product as a coenzyme ion, the amount of Pi was not increased by the addition of pyrophosphohydrolase, indicating that ZjAC2 protein hydrolyzed ATP to ADP and PPi, while Mn was present²⁺And Ca²⁺The Pi content in the reaction product was small as the coenzyme ion, but increased dramatically upon the addition of pyrophosphatase, indicating that the ZjAC2 protein hydrolyzes ATP to AMP and PPi under both ionic environments.

AC enzyme activity detection method:

the catalytic system is as follows: 50mM TRIS-HCl (pH 7.5), 0.5mM ATP and 20mM coenzyme metal ion, under catalytic conditions at 30 ℃ for 20 min. After the catalysis is finished, High Performance Liquid Chromatography (HPLC) is used for detecting the cAMP content in the catalytic system, so that the catalytic activity and efficiency of the enzyme are determined.

The detection parameters were as follows: the column was an Agilent Eclipse XDB-C18 (4.6X 250mm,5 μm), the column temperature was 30 ℃ and the mobile phase was methanol: 20mmoL potassium dihydrogen phosphate in a volume ratio of 10: 90, the detection wavelength is 254nm, the injection volume is 20 μm, and the flow rate is 0.8 mL/min.

The detection method of the phosphohydrolase activity comprises the following steps:

using a monophosphorus detection Kit (Malachite Green phosphor Assay Kit (POMG-25H)) of Sigma company to detect the monophosphorus content for the first time, adding pyrophosphatase into the reaction system to continue the reaction for 20min, and detecting the monophosphorus content in the reaction system again to determine that the product of the hydrolase activity is ADP or AMP.

Example four

Jujube tree in vivo transient expression test

The ZjAC2 gene was cloned into pCG3301 plant overexpression vector, transferred into Agrobacterium GV3101 by heat shock transformation and plated on LB solid medium containing rifampicin (50. mu.g/mL) and kanamycin (50. mu.g/mL), after which a single colony was picked up and cultured in LB liquid medium containing the same antibiotic at 28 ℃ 200rpm until the bacterial liquid OD600 became 0.6.

The cells were collected and resuspended in 10mM MES and 10mM MgCl₂And 200mM acetosyringone, pH6.0, and 2mL of the injection staining solution is injected into the winter jujube fruits in the white ripe stage, the winter jujube fruits are taken 24h and 72h after injection, and the expression level of the ZjAC2 gene and the cAMP content are detected, and the result is shown in figure 5, compared with the no-load group (pCG3301) and the blank control group, the expression level of ZjAC2 in the test group (namely pCG3301 after injection recombination) is remarkably improved, and the cAMP content is also remarkably improved.

Therefore, the present invention employs the above-mentioned ZjAC 2-encoding gene sequence having adenosine triphosphate tunneling metalloenzyme activity, and has NTP hydrolase activity, tripolyphosphate activity and weak pyrophosphatase activity in addition to adenylate cyclase activity.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the invention without departing from the spirit and scope of the invention.

Sequence listing

<110> university of agriculture in Hebei

<120> ZjAC2 encoding gene sequence having adenosine triphosphate tunneling metalloenzyme activity

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<213> adenosine triphosphate Tunnel metalloenzyme genes (TTMs) (SIPHOSEQUENCEListing 1.0)

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atggaggtcg aagtcaagct tcggctccca gacgccgccg cccatcgccg cgtcaacgac 60

ctcctgtctc ccttccgcgt cgccactttc cggcaagaga atctcttctt tgacggggcc 120

gccgctgagc tctcgtcgcg ccgagccgtc ctccgcctga ggttctacga cgacgacacc 180

cgttgcgtcg tgtcgctcaa ggccagggcg gttctcgtcg acggcatcag ccgggtcgag 240

gaggacgagg aggagctgga gccggcggtg gggcgtgcct gcgttgccga gccggagaag 300

cttctggcgg cggagtgtag gatcttgaag agggtgaagg atgaatttgg ggtttcgggg 360

tttgttgggc ttggagggtt tgggaatgtg agggatgtgt atgagtggaa aggattgaaa 420

ttggaggtcg atgagactcg gtatgggttc gggacttgct atgagattga atgcgagagt 480

gctgaccctg aaggagtcaa gaaggtgctc gaggagtttt tgaaggagaa tgggattgaa 540

tattcttact cagaggcctc caagtttgcg gtttttcgat ccggaaagct gccttag 597