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Method for cloning gene by glutathion peroxidase

A glutathione peroxidase gene technology, applied in the field of glutathione peroxidase gene cloning, can solve the problem of no glutathione peroxidase gene cloning

Inactive Publication Date: 2015-04-29
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is still no relevant method and technology for cloning the glutathione peroxidase gene in Spiraea chinensis by RACE method

Method used

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  • Method for cloning gene by glutathion peroxidase
  • Method for cloning gene by glutathion peroxidase
  • Method for cloning gene by glutathion peroxidase

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Experimental program
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specific Embodiment approach 1

[0013] Specific embodiment one: the method for cloning the gene by glutathione peroxidase in this embodiment is carried out according to the following steps: one, extract the total RNA of spirea; two, design degenerate primers: according to glutathione peroxidation The degenerate primers DP1 and DP2 of the middle fragment of the gene were designed according to the amino acid sequence of the biodegradable enzyme; 3. Cloning of the middle fragment of the gene: cDNA was synthesized by reverse transcription reaction: 20 μl reverse transcription reaction system consisted of 10 μl 2×TS Reaction Mix, 3 μl RNase-free H 2 O, 1 μl of Anchored Oligo (dT), 5 μl of Total RNA, 1 μl of TransScript RT / RI Enzyme Mix is ​​composed of RT / RI Enzyme Mix, mixed and put into PCR machine for reverse transcription reaction, and then PCR amplification reaction for the synthesized product: 25 μl amplification reaction system consists of 2 μl cDNA, 1 μl DP1 primer, 1 μl DP2 primer, 2.5 μl of 10×LA PCR...

specific Embodiment approach 2

[0016] Specific embodiment two: the difference between this embodiment and specific embodiment one is: in step one, the pot seedling of Spiraea chinensis with a growth height of 15cm, a branch quantity of 3-5, and a leaf quantity of 20 or less is carried out. 3-6 days of dark cultivation to remove the wax powder layer on the leaf surface. Other steps and parameters are the same as those in Embodiment 1.

specific Embodiment approach 3

[0017] Embodiment 3: The difference between this embodiment and Embodiment 1 is that in step 1, gel electrophoresis is used to detect that the total RNA concentration extracted reaches A260 / A280 of 1.90, or when A260 / A230 is 2.20 or more. Do PCR templates on request. Other steps and parameters are the same as those in Embodiment 1.

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Abstract

The invention discloses a method for cloning gene by glutathion peroxidase and relates to a method for cloning a glutathione peroxidase gene of a landscape plant spiraea fritschiana. Compared with conventional gene cloning methods, the method disclosed by the invention for cloning a cold-resist gene through identified cold-resist glutathion peroxidase has high specificity and accuracy. The method comprises the following steps: I, extracting total RNA of spiraea; II, designing a degenerate primer; III, cloning a middle fragment of a gene; IV, carrying out gene 3'RACE cloning; V, carrying out gene 5'RACE cloning; and VI, splicing sequences to obtain gene full length, namely finishing cloning of the glutathion peroxidase gene. The expression quantity of the glutathion peroxidase gene cloned by the method is remarkably increased due to low temperature induction, so that the gene has a function of enhancing the cold resistance of spiraea, lays a foundation for improving the cold resistance of spiraea and cultivating novel varieties of spiraea and can be applied to the field of cold-resist gene engineering and molecular breeding of spiraea.

Description

technical field [0001] The present invention relates to a method for cloning the gene from glutathione peroxidase. Background technique [0002] Spiraea ( Spiraea L.) Plant resources are abundant, its flowering period is long-lasting, the flower color is pure and bright, and the leaf color changes. It integrates flower viewing and colorful leaf viewing. It has high garden ornamental value and strong ecological adaptability. It has an ideal application prospect in gardens. Due to the limitation of climate factors in northern my country, there are few types of greening plants, monotonous greening tree species, and lack of greening resources. In the construction of plant landscapes, it is impossible to form rich, beautiful and changeable landscaping plant landscapes, which directly affects the effect of urban landscaping. . If the cold resistance of spirea resources is improved so that it can be used in northern gardens, it will provide new greening resources for northern gar...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/10
Inventor 刘慧民刘计璇王大庆聂颖苏青房莉仉茜张娇李小婷刘冰冰
Owner NORTHEAST AGRICULTURAL UNIVERSITY