Tea tree caffeine synthase gene promoter tcsp and its application

A technology of promoter and caffeine, applied in application, genetic engineering, enzyme and other directions, can solve the problems of insufficient expression of exogenous genes and inability to obtain transgenic plants ideally

Active Publication Date: 2019-05-24
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the main content of plant genetic engineering research is the expression and regulation of genes, but the insufficient expression of exogenous genes is an important reason why transgenic plants cannot be ideally obtained

Method used

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  • Tea tree caffeine synthase gene promoter tcsp and its application
  • Tea tree caffeine synthase gene promoter tcsp and its application
  • Tea tree caffeine synthase gene promoter tcsp and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Cloning of caffeine synthase gene promoter TCSP in embodiment 1 tea tree

[0034] 1.1 Extraction of total genomic DNA (Biomiga kit)

[0035] (1) Weigh about 0.2g of the young leaves of Shucha tea tree into a pre-cooled mortar, add an appropriate amount of liquid nitrogen, and grind to powder.

[0036] (2) Transfer the powder to a 2ml centrifuge tube, immediately add 900μl Buffer P1 and 10μl β-mercaptoethanol, vortex briefly and place in a water bath at 65°C for 10 minutes, mixing by inversion 3-4 times. (Add 5 μl RNaseA before water bath)

[0037] (3) Add 140 μl Buffer P2, vortex and mix well. Centrifuge at 13000 rpm for 10 min.

[0038] (4) Carefully pipette the supernatant and transfer it to a new 2ml centrifuge tube to avoid suctioning up the precipitate. Add 0.5 times the volume of P3 and mix well, then add 0.5 times the volume of absolute ethanol to the mixture, and mix thoroughly by inversion.

[0039] (5) Insert a DNA adsorption column into a 2ml collection t...

Embodiment 2

[0105] Example 2 Construction of Tobacco Transgenic Plants and Verification of Promoter Activity

[0106]Use Gateway technology to introduce the promoter sequence into the plant expression vector (pKGWFS7), construct a recombinant plant expression vector, named pKGWFS7-TCSP, and use the Agrobacterium-mediated tobacco genetic transformation method to perform promoter activity by expressing the GUS gene functional verification.

[0107] 2.1 Vector construction and identification

[0108] (1) BP reaction

[0109]

[0110] The system was incubated overnight at 25°C. After the reaction, 2 μl of 2 μg / μl Proteinase K was added and treated at 37°C for 10 minutes, and then the BP reaction product (entry vector) was transferred into 50 μl of E. coli Trans1-T1. After screening on the LB plate, the plasmid was extracted after the positive single colony was detected and stored at -20°C.

[0111] (2) LR reaction

[0112]

[0113]

[0114] The system was incubated overnight at 2...

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Abstract

The invention provides tea tree caffeine synthetase gene promoter TCSP and application thereof in the preparation of transgenic plants. Tea tree DNA is used as a template to acquire a tee tree TCS gene promoter sequence by cloning through Genome Walking method, recombinant plant expression vector pKGWFS7-TCSP is constructed by using Gateway technology, Agrobacterium tumefaciens-mediated tobacco genetic transformation is utilized to carry out promoter activity function verification by means of expressing GUS (glucuronidase) gene. The specific caffeine synthetase gene promoter TCSP is acquired by cloning from tea trees for the first time, its activity is verified, and the promoter is significant to disclosing the synthetic mechanism of tea tree caffeine and biologically controlling the content of tea tree caffeine.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a promoter TCSP of a tea tree caffeine synthase gene and an application thereof. Background technique [0002] Caffeine (1,3,7-trimethylxanthine) is a nitrogen-containing compound. The alkaloids in tea tree are mainly purine bases, and caffeine is the main body of purine bases. Therefore, caffeine plays an important role in the secondary metabolism of tea trees. play a pivotal role. Caffeine can not only protect plants from the damage of the natural environment and promote the growth of plants in plants, but also is a common substance with certain medicinal value. Appropriate intake of caffeine can strengthen the stomach and eliminate food, diuresis, reduce high blood pressure, improve memory, fight cancer, reduce the risk of type II diabetes, etc.; but excessive intake can cause insomnia, anxiety, osteoporosis and fetal malformation. Therefore, how to regulate the synt...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/11A01H5/00
CPCC12N9/1007C12N15/8205C12N15/8243C12Y201/0116
Inventor 邓威威王荣秀张正竹江丽娜袁艳
Owner ANHUI AGRICULTURAL UNIVERSITY
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