Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Coding gene sequence with adenosine triphosphate tunneling metalloenzyme activity

A technology of adenosine triphosphate tunneling and encoding gene, which is applied in the field of adenosine triphosphate tunneling metalloenzyme, and can solve the problem of insufficient understanding of TTM protein and other problems

Pending Publication Date: 2021-10-01
HEBEI AGRICULTURAL UNIV.
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] With the development of sequencing technology, research has discovered more TTMs genes, but the information obtained so far is far from enough to fully understand the characteristics of TTM proteins, so more research and data are needed to identify them

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Coding gene sequence with adenosine triphosphate tunneling metalloenzyme activity
  • Coding gene sequence with adenosine triphosphate tunneling metalloenzyme activity
  • Coding gene sequence with adenosine triphosphate tunneling metalloenzyme activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Amplification of the ZjAC1 gene

[0020] The RNA of winter jujube fruit was extracted, and then reverse-transcribed into cDNA as a PCR amplification template for PCR amplification; wherein, the forward primer: 5'-ATGAGAGGGAAAAATCACAT-3', the reverse primer: 5'-TCAGCTAGGCAACTTACGGG-3'.

[0021] PCR amplification program: 94°C, 4min; (94°C, 40s; 55°C, 45s; 72°C, 50s) 35 cycles; 72°C, 10min.

[0022] The PCR products were subjected to agarose gel electrophoresis to observe the amplification results, and the candidate target bands were recovered by agarose gel, and the recovered products were connected into the pMDTM19-T cloning vector for sequencing to obtain the ZjAC1 sequence as a candidate date adenylate cyclization Enzyme gene for functional verification.

Embodiment 2

[0024] coli-deficient complementation assay

[0025] Cultivate Escherichia coli cyaA-deficient SP850 strain, SP850 strain lacks adenylyl cyclase gene (AC), transform the pET-15b recombinant plasmid with ZjAC1 gene into SP850 after heat shock, coat with ampicillin and kana antibiotics On a petri dish, pick a single clone and culture it until the OD600 is 0.5, then induce expression and culture it for 4 hours, culture it by streaking on MacConkey medium, and observe.

[0026] It was found that, if figure 1 , the color of the colonies of the SP850-deficient strain and the wild-type E. coli strain with the recombinant plasmid was red, while the color of the SP850-deficient strain was white in MacConkey medium, indicating that the ZjAC1 gene made up for the lack of cyaA in the SP850 strain gene, which functions as the adenylyl cyclase gene.

Embodiment 3

[0028] Protein Catalytic Test in Vitro

[0029] The pET-15b recombinant vector carrying the ZjAC1 gene was transformed into Escherichia coli BL21 strain, cultured at 37°C and 200rpm, and then the protein was extracted and purified, and the obtained protein was used for the detection of enzyme catalytic activity. The results were as follows: Figure 2-3 . It was found that when manganese ions were used as coenzyme ions, cAMP was produced in the catalytic product, which indicated that ZjAC1 protein could catalyze ATP to form cAMP in vitro, and played the role of AC.

[0030] AC enzyme activity detection method:

[0031] Catalytic system: 50mM TRIS-HCl (pH 7.5), 0.5mM ATP and 20mM coenzyme metal ion, the catalytic condition is 30°C, 20min. After the catalysis is completed, high-performance liquid chromatography (HPLC) is used to detect the cAMP content in the catalytic system, so as to determine the catalytic activity and efficiency of the enzyme.

[0032] The detection parame...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a coding gene sequence with adenosine triphosphate tunneling metalloenzyme activity, a gene for coding adenosine triphosphate tunneling metalloenzyme is ZjAC1, and the ZjAC1 has a DNA sequence as shown in SEQ ID NO.1 in a sequence table. According to the invention, the coding gene sequence with adenosine triphosphate tunnel metalloenzyme activity is adopted, and the coded protein has the activity of adenylate cyclase, NTP hydrolase activity, triphosphatase activity and relatively weak pyrophosphatase activity.

Description

technical field [0001] The invention relates to the technical field of adenosine triphosphate tunneling metalloenzyme, in particular to a coding gene sequence having the activity of adenosine triphosphate tunneling metalloenzyme. Background technique [0002] The adenosine triphosphate tunnel metalloenzymes (TTMs) superfamily is composed of a group of proteins with the ability to hydrolyze a variety of tripolyphosphates, which are ubiquitous in organisms. The catalytic characteristics of this protein superfamily are: the substrates are all triphosphate substrates, and divalent metal co-ions are required to exert enzyme activity. Moreover, the structure of this protein superfamily is similar, and all TTM proteins have 8 antiparallel β-sheet strands and an EXEXK motif. [0003] Three AtTTMs genes were found in Arabidopsis, among which the proteins encoded by AtTTM1 and 2 showed pyrophosphatase activity and weak ATP hydrolase activity, while the protein encoded by AtTTM3 showe...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/55C12N9/16
CPCC12N9/16
Inventor 刘孟军刘志国袁野
Owner HEBEI AGRICULTURAL UNIV.
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com