Nitrariatangutoruntonoplast H<+>PPase protein gene NtVP1, encoding protein thereof, and cloning method

A technology of tonoplast membrane and protein, applied in the field of molecular biology, can solve the problem of no tongut white thorn membrane and other problems

Inactive Publication Date: 2017-07-07
CHINESE ACAD OF FORESTRY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But there is no Nitraria tangutica tonoplast membrane H i

Method used

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  • Nitrariatangutoruntonoplast H&lt;+&gt;PPase protein gene NtVP1, encoding protein thereof, and cloning method
  • Nitrariatangutoruntonoplast H&lt;+&gt;PPase protein gene NtVP1, encoding protein thereof, and cloning method
  • Nitrariatangutoruntonoplast H&lt;+&gt;PPase protein gene NtVP1, encoding protein thereof, and cloning method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Nitraria tangutica Tonoplast Membrane H + - Cloning of PPase protein gene (NtVP1)

[0038] According to the cloned 10 species (Gossypium hirsutum ADN96173.1), grape (Vitis vinifera XP_002273207.1), poplar (Populus trichocarpa XP_006381091.1), Arabidopsis (NP_173021.1), Grass (AEI17666.1), Kalidium foliatum ABK91685.1, Rice (BAA31523.1), Suaeda corniculata ADQ00196.1, Halostachyscaspica ABO45933.1, Overlord (ABU92563 .1)) The gene was subjected to multiple sequence alignment, and a pair of degenerate primers NtVP1-F1 (SEQ ID NO: 1) and NtVP1-R1 (SEQ ID NO: 2) were designed in the amino acid conserved region. Extract the total RNA of Nitraria tangutica leaves (RNAiso Plus from Takara Company), and amplify the conserved region cDNA fragment (1156bp) (SEQ ID NO: 3) from the total RNA of Nitraria tangutica by RT-PCR. The method of the RT-PCR is as follows:

[0039] Take 8 μl of total RNA, 1 μl of 50 μM oligo(Dt) 20 , 1μl 10mM dNTP mixture (both carried out on ...

Embodiment 2

[0045] Example 2: Tonoplast Membrane H of Different Species + - PPase protein sequence comparison

[0046] Select 10 tonoplast H from different plants from GenBank + -Amino acid sequences of PPase proteins, respectively upland cotton (Gossypium hirsutum ADN96173.1), grape (Vitis vinifera XP_002273207.1), poplar trichocarpa (Populus trichocarpa XP_006381091.1), Arabidopsis thaliana (NP_173021.1), salt horn Grass (AEI17666.1), Kalidium foliatum ABK91685.1, Rice (BAA31523.1), Suaeda corniculata ADQ00196.1, Halostachys caspica ABO45933.1), Overlord ( ABU92563.1), using DNAMAN software, these 10 proteins were compared with the NtVP1 protein of the present invention. The results showed that the amino acid sequence of NtVP1 was consistent with that of the cloned upland cotton H + -PPase protein homology up to 93%, and Arabidopsis tonoplast H + -PPase protein AtVP1 homology is 89.29%, and Arabidopsis tonoplast H + -The homology of PPase protein AtVP2 is only 33.53% (AAF31163.1). ...

Embodiment 3

[0047] Example Three: Tonoplast Membrane H from Different Species + -PPase protein phylogenetic tree

[0048] in the tonoplast H + -Based on the alignment of PPase protein sequences, the above 10 tonoplast H + -PPase protein (respectively upland cotton (Gossypium hirsutum ADN96173.1), grape (Vitis vinifera XP_002273207.1) and (CAO41672.1), poplar (Populus trichocarpaXP_006381091.1), Arabidopsis (NP_173021.1) and (AAF31163.1), salt hornwort (AEI17666.1), salt claw (Kalidium foliatum ABK91685.1), rice (BAA31523.1), Suaeda corniculata (Suaeda corniculataADQ00196.1), salt ear wood (Halostachys caspica ABO45933.1), Overlord (ABU92563.1)) and the amino acid sequences of the NtVP1 protein of the present invention are subjected to multiple sequence matching arrangement, and then MEGA5.1 software is used to construct a phylogenetic tree. The method is based on the Neighbor-joining method, by The Poisson distance method has been analyzed by 1000 bootstrap. result( figure 2 ) showe...

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Abstract

The invention belongs to the field of molecular biology, provides a nitrariatangutoruntonoplast H<+>PPase protein gene NtVP1 and encoding protein thereof, and provides a method for cloning the gene and constructing a vector of the gene. The gene and the encoded protein thereof have important effect in salt stress resistance of thenitrariatangutoruntonoplast, thereby providing a new choice for cultivating plant new varieties.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to gene cloning and carrier construction. Background technique [0002] tonoplast H + Transport inorganic pyrophosphatase (V-H + -PPase) is a unique acidifying vacuolar proton pump that exists in terrestrial plants and a few algae, protozoa and bacteria. The main functions of the enzyme are as follows: (1) Proton pump function: it can hydrolyze the free energy and H produced by inorganic pyrophosphate (PPi) + The proton transmembrane transporter is coupled to hydrolyze PPi into 2 Pi, and H in the cytoplasm + Pumped into the vacuole through the tonoplast membrane, it acts as a proton pump, the same as the tonoplast H + -ATPase together to form H + The electrochemical gradient across the tonoplast membrane provides the driving force for the active transport of molecules of various solutes (amino acids, sugars, cations and anions, etc.) across the tonoplast membrane. (2) Ion compart...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/55C12N15/10C12N15/82A01H5/00
CPCC12N9/14C12N15/8205C12N15/8273C12Y306/01001
Inventor 杨秀艳唐欣李焕勇刘正祥张华新朱建峰成铁龙常二梅段晓波王文军
Owner CHINESE ACAD OF FORESTRY
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