Chimonanthus praecox GDSL lipase gene CpGLIP1 and application thereof

A technology of lipase gene and wintersweet, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of late plant GDSL lipase and no wintersweet GDSL lipase, etc., to improve drought tolerance and cold resistance effect of ability

Active Publication Date: 2021-06-11
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The research on plant GDSL lipase started relatively late, and there are few studies on GDSL participating in abiotic stress. At present, there is no research report on the GDSL lipase of Wintersweet, which has certain research and application potential.

Method used

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  • Chimonanthus praecox GDSL lipase gene CpGLIP1 and application thereof
  • Chimonanthus praecox GDSL lipase gene CpGLIP1 and application thereof
  • Chimonanthus praecox GDSL lipase gene CpGLIP1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Isolation of Wintersweet CpGLIP1 Gene

[0042] Total RNA was extracted from the young leaves of Wintersweet by Trizol method, and cDNA was synthesized by reverse transcription. According to the known sequence fragments in the Wintersweet transcriptome database (the database is provided by the Chongqing Flower Engineering Technology Research Center), use the software Primerprimer 5.0 to design primers across the two ends of the largest ORF frame for PCR amplification. The primer sequences are as follows:

[0043] GLIP1-ORF-F: 5'-gatcaagtgaccttagcccatc-3' (SEQ ID No. 3)

[0044] GLIP1-ORF-R: 3'-aggagatgagatgatactgaggc-5' (SEQ ID No. 4)

[0045] Using Wintersweet cDNA as a template to amplify the largest ORF fragment of Wintersweet CpGLIP1, the PCR reaction system and reaction conditions are as follows: ddH 2 O 17.8 μL, 10×HiFi Taq PCR Buffer Ⅱ 2.5 μL, dNTP (10 mM) 1.5 μL, GLIP1-ORF-F (10 μM) 1 μL, GLIP1-ORF-R (10 μM) 1 μL, Wintersweet cDNA 1 μL, TaKaRa Ex Taq ...

Embodiment 2

[0050] Example 2 Analysis of the expression characteristics of Wintersweet CpGLIP1 gene

[0051] 1 Collection of different stress treatment materials for different stages of wintersweet flower development, each tissue and its seedlings

[0052] The roots, stems, cotyledons, young leaves, mature leaves, inner perianth segments, outer perianth segments, stamens and pistils of Wintersweet were taken and stored in a -80°C refrigerator for later use. The wintersweet flowers in the germination stage, flower bud stage, dew petal stage, early bloom stage, full bloom stage and decay stage were collected respectively, and samples from three parallel experiments were taken in each stage, which were quick-frozen in liquid nitrogen and stored in a -80°C refrigerator. The same size and robust wintersweet seedlings cultured in the greenhouse were selected to be treated with low temperature (4°C), high temperature (42°C), drought (30% PEG6000), high salt (150mM NaCl) and JA (100μM), GA 3 (10...

Embodiment 3

[0079] Example 3 Analysis of Prokaryotic Expression of Wintersweet CpGLIP1 Gene

[0080] In order to verify whether the gene can perform its function through correct translation into lipase-like protein after transcription, a prokaryotic expression experiment was carried out first.

[0081] 1 Prokaryotic expression vector construction

[0082] A pair of specific primers were set according to the largest ORF frame of the CpGLIP1 gene, and the restriction sites BamH I and Hind III of the downstream primers and corresponding protective bases were added respectively.

[0083] The primer sequences are as follows:

[0084] PG-CpGLIP1-F:5'- cgggatcc agacccatggccggttctt-3' (SEQ ID No.11), the underline shows the enzyme cutting site,

[0085] PG-CpGLIP1-R:5'- cccaagctt aggagatgagatgatactgaggc-3' (SEQ ID No. 12) is underlined as the enzyme cutting site.

[0086] PCR amplification was carried out using the positive colony plasmid DNA of the positive colony cloned by the correct Cp...

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Abstract

The invention belongs to the technical field of plant gene functions, and particularly relates to a chimonanthus praecox GDSL lipase gene CpGLIP1 and application thereof. The invention provides a chimonanthus praecox CpGLIP1 gene. An amino acid sequence of a coding protein of the chimonanthus praecox CpGLIP1 gene is shown as SEQ ID No. 1. The chimonanthus praecox CpGLIP1 gene is obtained through cloning for the first time, expression characteristic analysis and prokaryotic expression are carried out, and the function of the gene is verified in a plant (arabidopsis thaliana) through a transgenic technology. The overexpression of the chimonanthus praecox CpGLIP1 gene in the arabidopsis thaliana can improve the drought tolerance and cold resistance of the arabidopsis thaliana, and the result provides a basis for the research on the abiotic stress response of the chimonanthus praecox and provides a reference for improving the abiotic stress tolerance of ornamental plants.

Description

technical field [0001] The invention belongs to the technical field of plant gene functions, and in particular relates to CpGLIP1, a lipase gene of Wintersweet GDSL and its application. Background technique [0002] Wintersweet [Chimonanthus praecox (L.) Link] is a deciduous tree and shrub of the genus Chimonanthus praecox in the family Cesaceae. Because it blooms in cold winters, it has been endowed with the qualities of not being afraid of severe cold, resolute and strong since ancient times. Wintersweet is resistant to pruning, barrenness, and stress, especially to low temperature and drought. At present, the molecular biology research on the resistance of wintersweet has made some progress, but it is still at the basic stage. The molecular mechanism of wintersweet's resistance to various stresses and the specific pathways of related genes are still unclear, and more related genes need to be discovered Do in-depth research. [0003] GDSL lipase is a family of lipolytic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/82A01H5/00A01H6/20A01H6/00
CPCC12N9/20C12Y301/01003C12N15/8273
Inventor 刘道凤田明杨张艺眭顺照李名扬
Owner SOUTHWEST UNIVERSITY
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