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Ipomoea pescaprae dehydrin gene IpDHN as well as coding protein and application thereof

A dehydrin protein and gene technology, applied in the regulation of salt tolerance and drought resistance of organisms, in the field of salt and drought resistance gene IpDHN, can solve the problem that the actual function of the dehydrin gene is not very clear

Inactive Publication Date: 2017-11-24
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

Different types of dehydrin genes have different functions, and the actual functions of dehydrin genes in the body are still not very clear, so there is still a lot of work to be done in the study of dehydrin genes

Method used

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  • Ipomoea pescaprae dehydrin gene IpDHN as well as coding protein and application thereof
  • Ipomoea pescaprae dehydrin gene IpDHN as well as coding protein and application thereof
  • Ipomoea pescaprae dehydrin gene IpDHN as well as coding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Obtaining the full-length cDNA of the pachyderm dehydrin gene IpDHN by screening the pachyderm cDNA yeast expression library

[0039] 1.1 Construction of the full-length cDNA expression library of Houteng

[0040] 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:

[0041] (1) Total RNA extraction

[0042] 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 quickly, and operate according to th...

Embodiment 2

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

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

[0070] Adjust the concentration of the IpDHN-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 IpDHN 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 IpDHN gene o...

Embodiment 3

[0079] Example 3: Overexpression of IpDHN 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 IpDHN-pGEX 6p-1

[0081] Using the yeast expression vector pYES-DEST 52 recombinant plasmid containing the cDNA of the pachyderm dehydrin gene as a template, and using SEQ ID NO.3 and SEQ ID NO.4 as primers, the cDNA of the IpDHN gene was amplified by PCR with high-fidelity Taq enzyme The full length of the reading frame. For the PCR system used, refer to the instruction manual of PrimeSTAR HS DNA Polymerase with GCBuffer 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 r...

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Abstract

The invention provides an ipomoea pescaprae salt-responsive gene IpDHN, a sequence of an amino acid coded by the gene IpDHN is as shown by SEQ ID NO.1, and a cDNA reading frame sequence is as shown by SEQ ID NO. 2. Proteins IpDHN coded by the IpDHN gene is related to salt and drought resistance of brewing yeast, escherichia coli and plants. By building the yeast, escherichia coli and plant transgenic over-expression vector coded by the IpDHN gene, the IpDHN gene is over-expressed in the yeast, eschirichia coli and arabidopsis thaliana, so that the resistance of the yeast, the eschirichia coli and arabidopsis thaliana to the salt stress and drought stress can be improved. The gene can be used for the genetic engineering seed breeding of the engineering bacteria and plants for the high salt and drought stress, and is high in application value.

Description

technical field [0001] The invention belongs to the field of biological genetic engineering, and specifically relates to a new salt-tolerant and drought-resistant gene IpDHN in Ipomoea pes-caprae L., the gene encodes Ipomoea pes-caprae dehydrin protein, and its application in regulating the salt-tolerance and drought-resistance of organisms . Background technique [0002] During their growth cycle, plants are usually subjected to a variety of adversity stresses, including salinity, drought, flooding, freezing, pests and diseases, etc. Among them, abiotic stress is one of the main reasons that affect plant growth and cause crop yield reduction. Among various abiotic stresses, high salinity / drought is the most common dehydration stress, which mainly causes the loss of water in plant cells by limiting the absorption of water by plants, thus affecting the normal physiological activities of plants and further inhibiting the growth of plants. grow. With the global climate deteri...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/81C12N1/21C12N1/19C12N15/70C12N15/84A01H5/00C12R1/865
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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