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Method for improving adenylate cyclase expression activity and increasing cAMP content in plants

A technology for adenylate cyclase and catalytic activity, which is applied in the field of improving adenylate cyclase expression activity and cAMP content in plants, and can solve problems such as blanks

Pending Publication Date: 2021-03-05
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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  • Method for improving adenylate cyclase expression activity and increasing cAMP content in plants
  • Method for improving adenylate cyclase expression activity and increasing cAMP content in plants
  • Method for improving adenylate cyclase expression activity and increasing cAMP content in plants

Examples

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Embodiment 1

[0019] Example 1 Cloning and assembly of adenylate cyclase activity gene AC and construction of its plant expression vector

[0020] 1. Cloning of the DNA sequence fragment encoding the catalytic active center of adenylyl cyclase:

[0021] According to literature records (Al-Younis et al. 2015), the AT5G09400 gene locus in the Arabidopsis genome encodes AtKUP7, and the amino-terminal fragment consisting of 100 amino acids in its protein sequence contains the catalytic active center of adenylyl cyclase. Therefore, according to the DNA sequence of the AtKUP7 gene, design PCR primers that can specifically amplify the DNA fragment encoding the catalytic active center of adenylyl cyclase:

[0022] KUP7-F1, 5'-CACC ATG GCGGAGGAAAGCAGTAT-3', the underlined sequence is the start codon of AtKUP7 gene;

[0023] KUP7-R1, 5'- TTA TTTCCTCCCAACGGTCAAG-3', where the underlined sequence is the added stop codon sequence.

[0024] Prepare wild-type Col-0 Arabidopsis seedlings, grind them ...

Embodiment 2

[0041] Example 2 Genetic transformation of AC gene plant expression vector to Arabidopsis

[0042] 1. Prepare the Agrobacterium transformed with the AC gene plant expression vector:

[0043] The pMDC83-AC and pTA7001-AC plant expression vectors constructed in Example 1 were respectively transformed into Agrobacterium GV3101, and the operation steps of the freeze-thaw transformation method were as follows:

[0044] Mix 1 µg of pMDC83-AC or pTA7001-AC plasmid DNA with 100 µl of GV3101 chemically competent cells, place on ice for 30 minutes, then freeze in liquid nitrogen for 2-3 minutes, then transfer to a 37°C water bath for 3-5 minutes, and then Place on ice for 5 minutes. Add 200 μL of LB liquid medium, culture on a shaker at 28°C for 3-5 hours, and spread the bacterial solution on LB solid medium containing 50 mg / L kanamycin, 25 mg / L rifampicin and 50 mg / L gentamicin , and then placed in a 28°C incubator for 2-3 days.

[0045] Colony PCR identification was carried out acc...

Embodiment 3

[0053] Example 3 Genetic Transformation of Brassica napus with AC Gene Plant Expression Vector

[0054] Explanation: MSB5 in this example refers to the medium composed of the basic salt components of MS (Murashige & Skoog) medium and the vitamin components of Gamborg B5 medium. The pH value of all the configured media is adjusted by potassium hydroxide, and the pH value after sterilization is about 5.6-5.8. The aseptic operation process is carried out on the ultra-clean workbench.

[0055] 1. Cultivate aseptic seedlings: use Brassica napus breeding parent line P300 (provided by the Rapeseed Breeding Laboratory of Henan Academy of Agricultural Sciences) as the experimental material, select healthy seeds and soak them in 70% alcohol for 30 seconds, rinse them with sterile water for 2 -3 times, then use 0.1% HgC1 2 Treat for 5 minutes, rinse with sterile water 3-5 times. Plant the seeds on the germination medium (1 / 2 MSB5 + 10g / L sucrose + 8g / L agar powder), and place them in ...

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Abstract

The invention discloses a method for improving adenylate cyclase expression activity and increasing cAMP content in plants. The method comprises the following steps: selecting a template DNA sequencecapable of encoding a catalytic activity center of adenylate cyclase by using biological information resources, adopting PCR amplification or chemical synthesis or other selectable cloning methods toobtain a DNA product with the sequence fragment, and after transcriptional expression of the DNA product, generating a peptide chain or protein with adenylate cyclase catalytic activity by translation; introducing the obtained DNA product into a selected plant gene expression vector system by using a DNA cloning technology, and inserting the plant gene expression vector system into multiple cloning sites between promoter and terminator element sequences for controlling exogenous gene expression; and carrying out genetic transformation on the constructed plant gene expression vector to obtain atransgenic plant with significantly improved adenylate cyclase expression activity and significantly increased cAMP content. The method provided by the invention provides a new idea for people to develop and utilize cAMP in plants, and fills the blank of the research.

Description

technical field [0001] The invention relates to biotechnology, in particular to a method for increasing the expression activity of adenylyl cyclase and the content of cAMP in plants. Background technique [0002] The discovery and research of cyclic adenosine monophosphate (3',5'-cyclic adenosine monophosphate; cyclic AMP; cAMP) has a history of more than 60 years. It is a second messenger molecule ubiquitous in the biological world, which can play an important regulatory role in life activities, such as gene expression, cell cycle, immune response, metabolism, cognition and memory, environmental adaptability, growth and development, etc. Many biological processes are significantly affected. At present, people have a considerable understanding of the signal regulation and mechanism of cAMP in bacteria, fungi, animals and other species, and are paying more and more attention to exploring its application value in human health and hygiene, disease treatment and drug developmen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/60A01H5/00A01H5/10A01H6/20
CPCC12N9/88C12N15/8243C12Y406/01001
Inventor 徐如强彭颂郭艳辉刘金蕊李盼宇贾文静赵俊恒
Owner ZHENGZHOU UNIV
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