Apricot ttasr gene and its encoded protein and application

An apricot and gene technology, which is applied in the field of biological genetic engineering to achieve the effects of improving tolerance, salt tolerance and drought tolerance

Active Publication Date: 2022-05-20
GUANGZHOU UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the development and utilization of ASR gene resources in Apricot apricot

Method used

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  • Apricot ttasr gene and its encoded protein and application
  • Apricot ttasr gene and its encoded protein and application
  • Apricot ttasr gene and its encoded protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Obtaining the full-length cDNA of the apricot abscisic acid / stress / maturation-inducing protein gene TtASR by monoclonal sequencing of the apricot cDNA yeast expression library

[0037] 1.1 Construction of the full-length cDNA expression library of Apricot apricot

[0038] The construction of the apricot cDNA library mainly refers to the instruction manual of CloneMiner II cDNA Library 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:

[0039] (1) Total RNA extraction

[0040] Take 2g of the same amount of apricot 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, ...

Embodiment 2

[0064] Example 2: Overexpression of TtASR gene in yeast improves salt tolerance and tolerance to oxidative stress of yeast

[0065] 2.1 Transformation of yeast strains with TtASR-pYES-DEST 52

[0066] Adjust the concentration of the TtASR-pYES-DEST 52 recombinant plasmid verified by the sequencing results to 0.1 μg / μL, and use the lithium acetate method to transform the wild-type yeast strain W303 and the yeast pair 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.

[0067] 2.2 The expression of TtASR gene in yeast wild-type strain W303 can improve the salt tolerance of transgenic yeast

[0068] Pick the yeast strain W303 to transform the single clone of the empty vector pYES2 and the TtASR gene overexpression vector TtASR-pYES-DEST52, inoculate in 2ml yeast limit liquid medium with galactose ...

Embodiment 3

[0073] Example 3: Overexpression of TtASR gene in Escherichia coli improves salt tolerance and hyperosmotic pressure tolerance of Escherichia coli

[0074] 3.1 Construction of Escherichia coli recombinant protein expression vector TtASR-pGEX 6p-1

[0075] With the yeast expression vector pYES-DEST 52 recombinant plasmid containing abscisic acid / stress / maturation-inducing protein gene cDNA as a template, with SEQ ID NO.3 (5'-GGGGCCCCTGGGATCCATGGCCGAAAGCCGTGACAG-3') and SEQ ID NO.4 (5' -GGAATTCCGGGGATCCTTAAAAGAAGTGGTGCTTCT-3') was used as a primer, and the full-length cDNA reading frame of the TtASR gene was amplified by PCR using high-fidelity Taq enzyme. 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 expressio...

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Abstract

The present invention relates to the field of molecular biology. The present invention provides the gene TtASR related to salt stress response of apricot, whose coded amino acid sequence is shown in SEQ ID NO.1, and its full-length cDNA sequence is shown in SEQ ID NO.2. The abscisic acid / stress / maturation-inducing protein TtASR encoded by the TtASR gene of the present invention is related to improving the salt and drought tolerance of Saccharomyces cerevisiae, Escherichia coli and plants. By constructing yeast, E. coli and plant transgenic overexpression vectors of TtASR gene, overexpressing TtASR gene in yeast, E. coli and Arabidopsis can improve the tolerance of yeast, E. coli and Arabidopsis to salt stress and drought stress sex. The gene can be applied to genetic engineering breeding of engineering bacteria and plants against high-salt and drought stress, and has great application value.

Description

technical field [0001] The invention belongs to the field of biological genetic engineering, in particular to TtASR (TtASR) in Tetragonia tetragonioides 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] Apricot (Tetragonia tetragonioides L.), also known as New Zealand spinach, wild spinach, sea spinach, and amaranth, is an annual or biennial semi-climbing herb of the Apricot family. It is widely distributed in coastal salt-rich areas in subtropical and temperate regions. , is a saline vegetable with economic and ecological value. In addition, apricot has high nutritional value, rich in minerals and multivitamins, rich in nutrition, fresh and tender fleshy stems and leaves, and refreshing taste. The whole plant of apricot can be used as medicine, which has the effects of clearing away heat and detoxifying, and has certain relieving ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/81C12N15/70C12N1/19C12N1/21C07K14/415A01H5/00A01H6/20C12R1/865C12R1/19
CPCC12N15/70C12N15/81C12N15/8273C07K14/415
Inventor 杨礼香叶玉妍陈红锋罗鸣张美
Owner GUANGZHOU UNIVERSITY
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