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Ipomoea pes-caprae IpLEA gene, coding protein and application thereof

A gene and protein technology, applied in the field of biological genetic engineering, can solve problems that need to be developed

Active Publication Date: 2017-10-20
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the role of the LEA protein and its gene in Austenitic austenifolia has yet to be explored.

Method used

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  • Ipomoea pes-caprae IpLEA gene, coding protein and application thereof
  • Ipomoea pes-caprae IpLEA gene, coding protein and application thereof
  • Ipomoea pes-caprae IpLEA gene, coding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Obtaining the full-length cDNA of the abundant protein gene IpLEA in the late embryogenesis stage of the Achilles vine cDNA through screening the 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 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:

[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, shak...

Embodiment 2

[0061] Example 2: Overexpression of IpLEA gene in yeast improves salt tolerance and tolerance to oxidative stress of yeast

[0062] 2.1 Yeast strain transformed with IpLEA-pYES-DEST 52

[0063] Adjust the concentration of the IpLEA-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.

[0064] 2.2 The expression of IpLEA gene in yeast salt-sensitive mutant strain AXT3 and wild-type strain W303 can improve the salt tolerance of transgenic yeast

[0065] Pick the single clone of the yeast strain AXT3 transformed into the empty vector pYES2 and the IpLEA g...

Embodiment 3

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

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

[0081] Using the yeast expression vector pYES-DEST 52 recombinant plasmid containing the cDNA of the abundant protein gene cDNA of the late embryogenesis of A. spp. The full-length cDNA reading frame of the gene. For the PCR system used, refer to the instruction manual of PrimeSTAR HS DNA Polymerase with GC Buffer from TaKaRa Company. The amplified DNA fragments were used according to the instructions of HiPure Gel Pure DNA Kits from Magen Company. The recovered fragment was used for insertion into the Escherichia coli recombinant protein expression vector pGEX 6p-1. The pGEX 6p-1 plasmid was digested with BamHI, and the linearized plasmid was recovered. The cDNA reading frame PCR fragment of recovered IpLEA and the linearized pGEX6p-...

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Abstract

The invention provides an ipomoea pes-caprae salt stress response related gene IpLEA, which has a coded amino acid sequence shown as SEQ ID NO.1, and a cDNA reading frame sequence shown as SEQ ID NO.2. The IpLEA gene coded late embryogenesis abundant protein IpLEA has correlation with the salt tolerance and drought resistance improvement of saccharomyces cerevisiae, escherichia coli and plants. By constructing the yeast, escherichia coli and plant transgenic over-expression vector of the IpLEA gene, over-expression vector of the IpLEA gene in yeast, escherichia coli and Arabidopis thaliana can be achieved, and the tolerance of yeast, escherichia coli and Arabidopis thaliana to salt stress and drought stress can be improved. The gene can be applied to genetic engineering breeding of engineering bacteria and plants directed against high salinity and drought stress, and has great application value.

Description

technical field [0001] The invention belongs to the field of biological genetic engineering, and in particular relates to a new salt-tolerant and drought-resistant gene IpLEA in Ipomoea pes-caprae L., which encodes a late embryogenesis rich protein (Ipomoea pes-caprae L.) L ate- E mbryogenesis A Bundant Proteins, LEA protein), the application in regulating the salt tolerance and drought resistance of organisms. Background technique [0002] Ipomoea pes-caprae L. is a halophyte with high salt tolerance and drought resistance. It is distributed in tropical and subtropical coastal and island regions all over the world, and is one of the typical halophytes. Due to the high stress resistance of A. sarachia, the exploration of its molecular mechanism of stress resistance has important theoretical significance, and the exploration of its stress resistance genetic resources and in-depth research on stress resistance functional genes have broad application prospects. [0003] Duri...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/81C12N1/19C12N1/21C12N15/70A01H5/00
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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