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Ipomoea batatas metallothionein gene (IbMT1) and application thereof in improving salt resistance and drought resistance of plants

A technology of metallothionein and gene, applied in the field of molecular biology and biology, can solve the problems of poor tolerance and limit the extensive growth of plants, so as to improve the resistance to drought, improve the salt tolerance and drought resistance of plants, and improve the salt tolerance of plants Effect

Inactive Publication Date: 2011-08-17
LUDONG UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most plants have poor tolerance to saline-alkali and drought, and can only grow in soil with a sodium chloride content below 0.3%, which greatly limits the extensive growth of plants on saline-alkali flats

Method used

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  • Ipomoea batatas metallothionein gene (IbMT1) and application thereof in improving salt resistance and drought resistance of plants
  • Ipomoea batatas metallothionein gene (IbMT1) and application thereof in improving salt resistance and drought resistance of plants
  • Ipomoea batatas metallothionein gene (IbMT1) and application thereof in improving salt resistance and drought resistance of plants

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specific Embodiment approach

[0018] The specific embodiment: the present invention will be described in further detail below in conjunction with accompanying drawing:

[0019] The methods used in the following examples are conventional methods unless otherwise specified.

[0020] experiment material

[0021] 1. Strains and vectors: Escherichia coli strain DHα and Agrobacterium strain EHA105 were purchased from Huamei Biological Company; expression vector pBI121 was purchased from Clontech Company.

[0022] 2. Main reagent: SMART TM PCR cDNA Synthesis Kit and ClontechPCR-Select cDNASubtraction Kit were purchased from Clontech Company; Taq DNA polymerase was purchased from Beijing Quanshijin Company, DNA standard molecular weight was purchased from Takara Company; restriction endonuclease, T4DNA ligase, M-MLV inversion Recording enzymes were purchased from Promega; RNeasy Plant Mini Kit was purchased from Qiagen; other chemical reagents were of domestic analytical grade.

[0023] 3. The primer sequence ...

Embodiment 1

[0036] Embodiment 1: the acquisition of sweet potato metallothionein gene IbMT1

[0037] 1. Treatment of sweet potato seedlings: Select stem tips or undifferentiated axillary buds of sweet potato plants, after disinfection, cut them into sections and inoculate them on MS solid medium for cultivation. The cultivation temperature is 27±2°C and the light is about 2000 Lux. When the aseptic seedlings grow to a height of about 3 cm, select vigorously growing healthy aseptic seedlings and transfer them to MS solid medium containing 200 mM NaCl for induction and culture for 24 hours.

[0038] 2. Extraction of total RNA: RNeasy Plant Mini Kit (Qiagen Company) was used to extract total RNA of NaCl-treated and untreated sweet potato seedlings, and the specific operation was carried out according to the kit instructions.

[0039] 3. Construction of a salt stress suppression subtractive library: 2 μg of total RNA from NaCl-treated and untreated sweet potato seedlings was reverse-transcrib...

Embodiment 2

[0042] Embodiment 2: Construction of PBI121-IbMT1 plant expression vector

[0043] 1. Amplification of the coding region of IbMT1 cDNA: Design primers IbMT-BamH I and IbMT-Sac I according to the sequences at both ends of the coding region of IbMT1 cDNA, and carry out PCR amplification with the bacterial solution containing IbMT1 as a template. Into the 25μL reaction system, add: 10×buffer (Mg 2+ ) 2.5 μL, dNTP (10 mM) 1 μL, each primer 1 μL (10 μM), template 1 μL, Taq enzyme 0.2 μL, and ddH for the rest 2 O make up.

[0044] The conditions of PCR amplification were as follows: 94° C. for 5 minutes, followed by 1 minute at 94° C., 1 minute at 56° C., and 1 minute at 72° C., for a total of 35 cycles. The results of agarose gel electrophoresis showed that a specific band with a size of about 200 bp was amplified, which was sent to Beijing Huada Gene Research Center for sequencing and confirmed to be the complete sequence of the IbMT1 cDNA coding region.

[0045] 2. Digestion a...

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Abstract

The invention discloses an ipomoea batatas metallothionein gene (IbMT1) and an application thereof in improving salt resistance and drought resistance of plants, belonging to the fields of molecular biology and biotechnology. According to the invention, an ipomoea batatas salt stress suppression subtractive library is constructed, and cloning is randomly picked from the library to perform sequencing and obtain a metallothionein gene which is named as IbMT1. Furthermore, a plant expression vector of the gene is further constructed, and tobacco is converted through an agrobacterium-mediated method. The growth state and physical signs of the transgenic tobacco are obviously superior to those of the wild tobacco in the conditions of high salt, drought stress and the like. Therefore, the gene IbMT1 from ipomoea batatas provided by the invention is effective in improving the resistance to salt, drought and other adversities of the plants, and has very high application value.

Description

Technical field: [0001] The invention relates to a sweet potato metallothionein gene IbMT1 and its application in improving plant salt tolerance and drought resistance, and belongs to the fields of molecular biology and biotechnology. Background technique: [0002] Water shortage and soil salinization are global problems currently restricting agricultural production. About 20% of the world's cultivated land is threatened by salt damage, and 43% of the cultivated land is in arid and semi-arid areas. In recent years, natural disasters have been serious in my country, and there are many unfavorable meteorological factors. The precipitation in the northern region continues to be low, and the trend of drought is serious. Affected by global warming in the future, the situation of drought and water shortage in my country will increase, which may bring many adverse effects on agricultural production and pose a great threat to my country's medium and long-term food security. The imp...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C12N1/21C12N5/10C07K14/415A01H5/00
Inventor 宿红艳王磊张娟韩立亚吴凡林王洋
Owner LUDONG UNIVERSITY
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