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Leymus chinensis aquaporin and encoding gene and application thereof

An aquaporin and Leymus chinensis technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as rare reports in molecular biology research, and achieve the effects of improving plant varieties, maintaining water balance, and enhancing drought resistance.

Inactive Publication Date: 2010-07-28
HARBIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the research on Leymus chinensis is limited to human and livestock consumption and component analysis, and there are few reports on its molecular biology as a drought-tolerant plant

Method used

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  • Leymus chinensis aquaporin and encoding gene and application thereof
  • Leymus chinensis aquaporin and encoding gene and application thereof
  • Leymus chinensis aquaporin and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Cloning of Aquaporin LcPIP1 Gene of Leymus chinensis

[0045] 1. Extraction of total RNA:

[0046]When the Leymus chinensis seedlings grew to the 3-leaf stage, they were subjected to rhizosphere stress with 20% PEG6000, and the leaves were cut after 3 hours, ground into powder with liquid nitrogen, and total RNA was extracted with RNAiso Reagent (TaKaRa).

[0047] 2. Cloning of full-length cDNA of Leymus chinensis aquaporin LcPIP1 gene:

[0048] (1) Synthesis of the first strand of cDNA

[0049] The first strand of cDNA was obtained by reverse transcription with Invitrogen's SuperScriptTM First-Strand Synthesis System for RT-PCR kit. The reaction system is as follows, cDNA Synthesis Mix: 5×cDNA Synthesis Buffer 4μl, 0.1M DTT 1μl, DEPC-treated Water 1μl, RNaseOUT (40U / μl) 1μl, ThermoScript RT (15U / μl) 1μl, total volume 8μl. Mix on ice: RNA 5 μl (<5 μg), Primer Oligo (dT) 20 (50 μM) 1 μl, dNTPs (10 mM) 2 μl, DEPC-treated Water 4 μl, total volume 12 μl. Put the RNA+Pri...

Embodiment 2

[0067] LcPIP1 Gene Sequence Analysis

[0068] The full-length cDNA of Leymus chinensis aquaporin gene is 1116bp, including an open reading frame (Open Reading Frame, ORF) with a length of 867bp from position 9 to position 875 of the 5' end, and a 5' untranslated region (NonTranslated region) of 8 bases. Region, NTR) and the 3' untranslated region of 241 bases, as shown in the sequence listing SEQ ID No: 2. This cDNA encodes a polypeptide consisting of 288 amino acids, such as SEQ ID No: 1 in the sequence listing, with a predicted molecular weight of 30.8KD.

[0069] Using DNAMAN4.0 to compare the amino acid sequences of different aquaporins, the amino acid sequence of LcPIP1 has high homology with other aquaporins that have been cloned (such as figure 1 shown), the GenBank accession numbers of the aquaporins of each species in the figure are: barley (BAF41978), wheat (ABI96812), rice (NP_001047672), corn (AA086706), sugarcane (ACC59097), tobacco (BAA20076) , Arabidopsis (BAA...

Embodiment 3

[0071] Construction of LcPIP1 Gene Plant Expression Vector

[0072] The pBIN437 plasmid was digested with BamHI and KpnI, and the vector was recovered; at the same time, the LcPIP1 gene was connected to the pMD18-T vector, named pT-LcPIP1, pT-LcPIP1 was digested with BamHI and KpnI, and a small fragment of about 0.9 kb was recovered , the vector was connected with the 0.9kb target fragment with T4DNAligase, transformed into DH5α and identified by PCR and enzyme digestion to obtain the expression vector pBPIP1 (such as Figure 4 shown). The promoter of this expression vector is 35S, the terminator is Tnos, and the selection marker gene is NPT Ⅱ.

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Abstract

The invention relates to a Leymus chinensis aquaporin and an encoding gene and application thereof. The current researches of Leymus chinensis are only to perform eating and composition analysis for human or livestock, but the researches in molecular biology aspect, regarding Leymus chinensis as drought tolerant crop are less. The Leymus chinensis aquaporin is the protein with the following amino acid sequences: (1) the amino acid sequence in SEQ ID No:1 of the sequence table; and (2) the protein derived from the protein with the amino acid sequence in SEQ ID No:1, with the same activity of the amino acid sequence in SEQ ID No:1 of the sequence table by replacing, deleting or adding one or more of amino acid residues to amino acid sequence in SEQ ID No:1 of the sequence table. The encoding gene of the Leymus chinensis aquaporin of the invention can be transferred in other plants to increase the drought-resisting property of transgenic plants. The Leymus chinensis aquaporin and encoding gene of the invention are used in plant gene engineering field.

Description

Technical field: [0001] The invention relates to an aquaporin and its encoding gene and application, in particular to the aquaporin LcPIP1 derived from Leymus chinensis and its encoding gene and application. Background technique: [0002] Plants are often damaged by water stress during their growth. In order to maintain water homeostasis under water stress, plants have formed a set of water transport regulation mechanisms to adapt to water stress. [0003] Studies have shown that the cell-cell pathway in plants is an important pathway to adapt to water stress, and in this pathway, the water transport pathway through aquaporin is the most important pathway. Plant aquaporin (Aquaporin, AQP) is a specific and efficient water transmembrane bidirectional transport channel on the plasma membrane and tonoplast membrane. It belongs to the MIP (Majorintrinsic protein) family member. Classes, that is, Plasma membrane intrinsic proteins (PIPs) located on the plasma membrane, Tonoplas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N5/10C12N1/00C12N15/09A01H5/00
Inventor 李新玲曲敏徐静徐香玲
Owner HARBIN NORMAL UNIVERSITY
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