Rice aquaporin gene OsPIP2;7 plant expression vector and application thereof

A plant expression vector and expression vector technology, applied in the field of rice water porin gene OsPIP2, can solve the problem of inaccurate expression, achieve accurate detection results, high degree of commercialization, and a single target band

Inactive Publication Date: 2018-02-02
天津市农作物研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The construction of this vector solves the problem that a large number of homologous proteins can be detected in the protein expression level experiment of OsPIP2;7 using an aquaporin antibody, resulting in inaccurate detected expression levels, and provides a basis for exploring new functions of OsPIP2;7 Experimental basis

Method used

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  • Rice aquaporin gene OsPIP2;7 plant expression vector and application thereof
  • Rice aquaporin gene OsPIP2;7 plant expression vector and application thereof
  • Rice aquaporin gene OsPIP2;7 plant expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. Cloning of OsPIP2;7-3× HA gene

[0028] (1) Extract total RNA from rice leaves: Take the material, add liquid nitrogen and grind to powder, add 1mL TRIZOL (Invitrogen) for every 50~100 mg material, shake and mix well. The suspension was left at room temperature for 5 min, centrifuged at 12000 rpm, 4°C for 10 min. Take the supernatant and add it to a new 1.5 mL Eppendorf tube, add 0.2 mL chloroform, shake vigorously for 15 s, place at room temperature for 2-3 min, and centrifuge at 12,000 rpm at 4°C for 15 min after stratification. Carefully pipette the colorless supernatant, add 0.5 mL of isopropanol, and place at room temperature for 10 min. Centrifuge at 12000 rpm, 4°C for 10 min. After removing the supernatant, wash the precipitate with 1 mL of 75% ethanol, centrifuge at 7500 rpm, 4°C for 5 min. Remove the supernatant and dry at room temperature (usually 5-15 min), add 40 μl nuclease-free deionized water, and place in a 65°C water bath for 10 min. Centr...

Embodiment 2

[0045] Application example of plant expression vector pHB-OsPIP2;7-3×HA in accurately detecting the protein expression level of OsPIP2;7 in plants

[0046] 1. Extraction of Membrane Proteins

[0047] (1) The rice variety Zhonghua 11 was transformed by the mature embryo method to obtain the transgenic rice with OsPIP2;7 overexpression, and the membrane proteins were extracted from the leaves of 5 lines and the control variety Zhonghua 11.

[0048] (2) Leaf tissues were cryopreserved in liquid nitrogen and ground into powder, poured into a centrifuge tube added with 0.5 mL Lysis buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1% v / v 10 mM PMSF).

[0049] (3) Put the centrifuge tube in an ice-water bath, add an equal volume of pre-cooled Dilution buffer (300 mM NaCl, 20mM Tris-HCl, pH 7.4, 4% v / v Triton X-114), the total volume is 1 mL. Oscillate 3 times, each time for 30 s, and place the centrifuge tube in an ice-water bath between oscillations. After shaking, the centrifuge tube...

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Abstract

The invention relates to a rice aquaporin gene OsPIP2;7 plant expression vector and application thereof. The plant expression vector is pHB-OsPIP2;7-3*HA. The nucleotide sequence of the plant expression vector is as shown in SEQ ID NO.1, and the coded amino acid sequence is as shown in SEQ ID NO.2. The terminator sequence of a gene OsPIP2;7 is replaced with a base sequence of a 3*HA tag, the modified OsPIP2;7-3*HA gene sequence is inserted into a cloning vector pMD19-T simple vector and is bonded to Hind III and Xba I loci of a binary vector pHB after double enzyme digestion of Hind III and Xba I, and the plant expression vector is obtained. After the plant expression vector is used for transforming plants, a target protein OsPIP2;7 is greatly expressed in the plants, introduction of the 3*HA tag solves the problem that in the experiment process that an aquaporin antibody is used for detecting the expression quantity of the target protein, a large quantity of homologous proteins are detected and accordingly the detection quantity of OsPIP2;7 is not accurately, and experiment bases are provided for research of new functions of OsPIP2;7.

Description

technical field [0001] The invention belongs to the technical field of rice genetic engineering detection, and relates to a rice water porin gene OsPIP2;7 plant expression vector and application thereof. Background technique [0002] Water is an essential substance for plant growth and development. Aquaporins (AQPs) are a class of membrane-intrinsic proteins that can permeate water and other small neutral molecules, and play an important role in regulating water balance in organisms. According to the comparison of protein homology, in most plant species, aquaporins are divided into four categories, namely plasma membrane intrinsic protein (PIP), tonoplastintrinsic protein (TIP), NOD26 similar Intrinsic protein (NOD26-like intrinsic protein, NIP) and small molecular weight basic intrinsic protein SIP (small and basic intrinsic protein, SIP). [0003] In plants, the first aquaporins to be discovered were tonoporin aquaporins gamma -TIP, and was shown to be an aquaporin by th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C07K14/415A01H5/00G01N33/68A01H6/46
CPCC07K14/415C12N15/8251G01N33/68G01N2333/415
Inventor 王晓静张新建孙林静闫双勇孙玥张融雪李军玲刘学军
Owner 天津市农作物研究所
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