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Nepenthes wcor413-like protein and its application

A pitcher plant and protein technology, which is applied in the fields of biochemistry and molecular biology, can solve the problems of low temperature-induced protein cloning and application of pitcher plants that have not been reported, and achieve the effect of high expression abundance

Active Publication Date: 2022-06-03
济南瑞之源生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a tropical plant, Nepenthes is sensitive to temperature changes. Low temperature will cause it to secrete a large number of low-temperature-induced proteins. There are no reports on the cloning and application of Nepenthes low-temperature-induced proteins.

Method used

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  • Nepenthes wcor413-like protein and its application
  • Nepenthes wcor413-like protein and its application
  • Nepenthes wcor413-like protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Cloning of Nepenthes wcor413-like protein

[0024] Degenerate primers were designed according to the conserved region sequence of the wcor413-like protein (LOC107779465) gene of tobacco (Nicotiana tabacum):

[0025] Fw: 5'-attgattcagatttgaaggaaatt-3' (SEQ ID NO: 5);

[0026] Rv: 5'-acgatcccaacagtgtttgaaat-3' (SEQ ID NO: 6);

[0027] Using homologous cloning technology, the cDNA obtained by reverse transcription of the extracted Nepenthes RNA was used as a template, and a gene fragment of about 500 bp was obtained by PCR amplification, and then the full-length gene was cloned by 3' and 5' RACE technology. cDNA sequence, sequence analysis showed that: the full length of the gene cDNA is 913bp (SEQ ID NO: 7), the open reading frame is 612bp, and the precursor protein of 204 amino acids is encoded (electrophoretogram detection such as figure 1 shown). The low temperature-inducible gene of Nepenthes was named Nwcor413.

[0028] The cloning process of Nwcor413 g...

Embodiment 2

[0097] Example 2 Mutant acquisition

[0098] Using the p2300-35S-Nwcor413 plant expression vector as a template, the mutants were obtained by overlapping extension PCR, and the mutation sites were the 40th and 41st amino acid residues IM were mutated to TT. The plant expression vector p2300-35S-Nwcor413' was reconstructed and transformed into tobacco by Agrobacterium-mediated method to obtain transgenic tobacco plants with Nwcor413' gene driven by 35S promoter.

Embodiment 3

[0099] Example 3 Cold resistance test

[0100] Membrane structure of plants was damaged at low temperature, and the degree of damage increased with the decrease of temperature. The degree of membrane damage can be judged by the intracellular electrolyte extravasation rate, that is, the relative conductivity. The cold tolerance of plants is inversely proportional to relative conductivity. The semi-lethal temperature obtained when the electrolyte exudation rate reaches 50% can more accurately reflect the cold resistance of plants.

[0101] During the test, select wild-type tobacco, trans-Nwcor413 tobacco and trans-Nwcor413' tobacco leaves, the test material was washed twice with deionized water, and the surface moisture was absorbed with clean filter paper, cut into segments of the same size, and weighed 0.5g (each (repeated 3 times for each sample), put it into a penicillin vial, add 10ml of deionized water, put it in a vacuum drying box and vacuumize it. When the material is...

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Abstract

The invention discloses a Nepenthes wcor413-like protein. The amino acid sequence of the Nepenthes wcor413-like protein is shown in SEQ ID NO: 1 in the sequence table. The present invention has cloned a wcor413-like protein gene in the tropical plant Nepenthes, the wcor413-like protein is expressed in large quantities under cold-induced conditions, the expression abundance is high, and it has the application potential of resisting low temperature stress in plants, wherein wcor413-like A mutant of the protein also has the function of insect resistance.

Description

technical field [0001] The invention belongs to the technical field of biochemistry and molecular biology, in particular to a pitcher plant wcor413-like protein and its application in cold resistance. Background technique [0002] Plants have adaptability and resistance to changes in the environment. Among many adverse environmental factors, temperature is the most important factor limiting plant growth and development. The study found that exposure of plants to gradually lowering temperatures increases their cold resistance, a process known as low temperature acclimation. Cold acclimation of plants involves many physiological and molecular responses. The physiological and biochemical reactions of plant cold resistance are caused by the change of gene expression at low temperature, and many cold resistance-related genes have been cloned. Exploring the genetic mechanism of plant cold resistance is not only important in basic theory, but also has extensive application value ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82C12N15/11A01H5/00A01H6/82
CPCC12N15/8273C12N15/8286C07K14/415
Inventor 徐立利
Owner 济南瑞之源生物科技有限公司
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