Ipomoea pescaprae IpASR gene, encoded protein and application thereof

A kind of gene, thick rattan technology, applied in the field of biological genetic engineering

Active Publication Date: 2017-10-27
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the developmen

Method used

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  • Ipomoea pescaprae IpASR gene, encoded protein and application thereof
  • Ipomoea pescaprae IpASR gene, encoded protein and application thereof
  • Ipomoea pescaprae IpASR gene, encoded protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Obtaining the full-length cDNA of the pachyderm abscisic acid / stress / maturation-inducing protein gene IpASR by screening the pachyderm cDNA yeast expression library

[0040] 1.1 Construction of the full-length cDNA expression library of Houteng

[0041] The construction of the thick rattan cDNA library mainly refers to the instructions of the CloneMiner II cDNALibrary Construction Kit, using (Invitrogen) technology. Specifically, the method comprises the following steps: extraction of total RNA, separation of mRNA, construction of a primary cDNA library and construction of a secondary cDNA library. The main steps are summarized as follows:

[0042] (1) Total RNA extraction

[0043] Take 2 g of the same amount of thick vine leaves, leaf buds, vines and young roots, grind them into powder with liquid nitrogen in a pre-cooled mortar, transfer the powder to multiple RNase-free 1.5mL centrifuge tubes, each centrifuge tube Add 1mL Trizol Reagent, shake and mix...

Embodiment 2

[0068] Example 2: Overexpression of IpASR gene in yeast improves salt tolerance and tolerance to oxidative stress of yeast

[0069] 2.1 IpASR-pYES-DEST 52 transformed yeast strain

[0070] Adjust the concentration of the IpASR-pYES-DEST 52 recombinant plasmid verified by the sequencing results to 0.1 μg / μL, and use the lithium acetate method to transform the yeast salt-sensitive mutant strain AXT3 and the corresponding wild-type yeast strain W303, and transform Yeast to H 2 o 2 Sensitive mutants yap1Δ and skn7Δ and corresponding wild-type yeast strain WT. At the same time, the yeast expression vector empty vector pYES2 was used as a control, and the above yeast strains were transformed respectively.

[0071] 2.2 The expression of IpASR gene in yeast salt-sensitive mutant strain AXT3 and wild-type strain W303 can improve the salt tolerance of transgenic yeast

[0072] Pick the single clone of the yeast strain AXT3 transformed into the empty vector pYES2 and the IpASR gene o...

Embodiment 3

[0079] Example 3: Overexpression of IpASR gene in Escherichia coli improves salt tolerance and dehydration tolerance of Escherichia coli

[0080] 3.1 Construction of Escherichia coli recombinant protein expression vector IpASR-pGEX 6p-1

[0081]Using the yeast expression vector pYES-DEST 52 recombinant plasmid containing the cDNA of the abscisic acid / stress / maturation-inducing protein as a template, with SEQ ID NO.3 and SEQ ID NO.4 as primers, high-fidelity Taq enzyme was used to pass PCR The full-length cDNA reading frame of the IpASR gene was amplified. For the PCR system used, refer to the instruction manual of PrimeSTAR HSDNA Polymerase with GC Buffer from TaKaRa Company. The amplified DNA fragments were used according to the instructions of Magen HiPure Gel PureDNA Kits. The recovered fragment was used for insertion into the Escherichia coli recombinant protein expression vector pGEX 6p-1. The pGEX6p-1 plasmid was digested with BamHI, and the linearized plasmid was rec...

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Abstract

The invention relates to the fields of engineering bacteria saccharomyces cerevisiae, escherichia coli and plant molecular biology. The invention provides an ipomoea pescaprae salt stress response related gene IpASR, wherein the coded amino acid sequence thereof is shown as SEQ ID NO.1 and the cDNA reading frame sequence thereof is shown as SEQ ID NO.2. The abscisic acid/stress/mature induced protein IpASR encoded by the IpASR gene provided by the invention is related to the promotion of drought-and-salt tolerance of saccharomyces cerevisiae, escherichia coli and plants. The tolerance of yeast, escherichia coli and Arabidopsis for salt stress and drought stress can be promoted in the manner of over-expressing the IpASR gene in yeast, escherichia coli and Arabidopsis by establishing the yeast, escherichia coli and plant transgenic over-expression vectors of the IpASR gene. The gene can be applied to engineering bacteria and plants for high-drought-and-salt stress genetic engineering breeding and has ultrahigh application value.

Description

technical field [0001] The invention belongs to the field of biogenetic engineering, in particular to IpASR (Ipomoea pes-caprae L.) A bscisic acid-, s tress-, and r ipening-induced gene) gene and its encoded protein and its application in regulating salt and drought tolerance of organisms. Background technique [0002] The full name of ASR protein is abscisic acid / stress / maturation-inducing protein ( A bscisic acid-, s tress-, and r ipening-induced proteins, ASR protein), is a unique family of hydrophilic proteins in plants, usually with transcription factor activity. In recent years, studies have found that they play an important role in plant growth and development, maturation and senescence, and abiotic stress. ASR protein is also a plant LEA protein ( L ate- E mbryogenesis A Bundant Proteins, a subfamily of late embryogenesis rich proteins, has been cloned into this gene in tomato, rice, corn, banana, pine, grape, potato, lily and other higher plants, and ASR ge...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/81C12N15/70C12N15/84A01H5/00C12R1/865
CPCC07K14/415C12N15/70C12N15/81C12N15/8273
Inventor 张美张会简曙光夏快飞
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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