Guinea grass Na+/H+ reversal transportation protein gene sequence and its clone and application

A technology of antiporter and gene, applied in the field of molecular biology and biology, can solve problems such as less obvious effects

Inactive Publication Date: 2008-09-10
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Early salt-tolerant genetic engineering mainly focused on scavenging free radicals and increasing osmotic ...

Method used

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  • Guinea grass Na+/H+ reversal transportation protein gene sequence and its clone and application
  • Guinea grass Na+/H+ reversal transportation protein gene sequence and its clone and application
  • Guinea grass Na+/H+ reversal transportation protein gene sequence and its clone and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1: Leymus chinensis Na + / H + Cloning method of antiporter gene

[0086] 1. Extraction of total RNA: Total RNA was extracted using RNAeasy mini kit (product of Promoga).

[0087] 2. Synthesis of the first strand of cDNA: take 2 micrograms of total RNA, add 4 μl of 5× reaction buffer, 2 μl of 10 mM deoxyribonucleic acid (dNTP), 0.5 μl of ribonuclease inhibitor (40-200u / μl), primer T26 (10 pmol / μl) 1 μl, reverse transcriptase (10u / μl) 2 μl, react at 42°C for 60 minutes, and stop the reaction at 85°C for 10 minutes.

[0088] 3. PCR reaction: polymerase chain reaction (PCR) reagents and conditions are:

[0089] First mix the following reagents together:

[0090] 10×Reaction Buffer 5μl

[0091] 10mM deoxynucleotide mixture (dNTP) 1μl

[0092] Forward primer (10μM) 2μl

[0093] Reverse primer (10(M) 2μl

[0094] Template cDNA 1μl

[0095] Taq DNA polymerase 0.5μl

[0096] Total volume 50μl

[0097] The PCR reaction conditions were: 94°C for 5 minutes, an...

Embodiment 2

[0103] Embodiment 2: Leymus chinensis Na + / H + The sequence determination of the antiporter gene AcNHX was carried out at Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0104] 7. Separation of 3' and 5' sequences: according to the instructions of SMART RACE cDNA Amplification Kit of Clontech Company

[0105] Leymus chinensis + / H + The amino acid sequence of the antiporter gene AcNHX1 and its encoded protein is as follows:

[0106] (1) Information on SEQ ID NO 1

[0107] (a) Sequential features

[0108] * Length: 2022 bp

[0109] *Type: nucleic acid

[0110] * Chain type: double chain

[0111] *Topology: Linear

[0112] (b) Molecular type: cDNA

[0113] (c) Assumption: No

[0114] (d) Antisense: no

[0115] (e) Original source: Leymus chinensis

[0116] (f) Sequence description: SEQ IN NO.1

[0117] ATGGGGTTCGAAATAGTGACGGCGCAGCTGGCGCGGCTGAGCGGCGCGCTGGGCACCTCG----60

[0118] GACCACGCGTCCGTGGTCTCCATCAACCTCTTCGTGGCGCTGCTCTGCGCCTGCATCGTC---120

[01...

Embodiment 3

[0167] Embodiment 3: Construction of expression vector

[0168] 1. According to the isolated Leymus chinensis Na + / H + Nucleotide sequence of antiporter gene NHX1, designed primers:

[0169] Forward primer: 5′-CGGTCTAGAATGGGGTTCGAAATAGTGAC-3′

[0170] Reverse primer: 5'-TATGAGCTCTCATCCCACGTGTACATTCG-3'

[0171] The polymerase chain reaction was performed using the cDNA reverse-transcribed from the total RNA of leaves as a template.

[0172] 2. Take 2 μl of PCR and connect it with the pMD18-T vector, and the operation steps are carried out according to the instructions of the product pMD18-TVector system of Promega Company. Then transform E. coli DH5α strain and grow overnight on LB plates coated with 5-bromo-4-chloro-3-indole-β-D-galactoside and X-gal containing ampicillin (100 μg / ml) . Pick white colonies and culture them overnight in LB liquid medium. Plasmid DNA was extracted by alkaline method and sequenced.

[0173] 3. The gene was excised from the pMD18-T vector...

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Abstract

The invention discloses a gene sequence of Aneurolepidiumchinense Na+/H+ antiport protein, a clone and application thereof, belonging to the molecular biology field. A coded gene of vacuole type Na+/H+ antiport protein is separated from the Aneurolepidiumchinense for the first time with RT-PCR through designing a primer according to an amino acid sequence of the Na+/H+ antipor protein published in other plants. With the transfer of the gene, the anti-salt property of a salt-sensitive yeast mutant can be recovered to a certain degree. A transgenic plant has higher salt tolerance which can reach 200mM through further constructing a sense expression vector and transforming tobacco. The gene can be used in genetic transformation of a plant to improve the salt tolerance of the plant.

Description

(1) Technical field [0001] The present invention relates to Na in Leymus chinense (Aneurolepidium chinense) + / H + The cloning, recombination and salt tolerance function analysis and application of the antiporter gene AcNHX1 belong to the field of molecular biology and biotechnology. (2) Background technology [0002] High concentrations of salt cause ion imbalance and hyperosmotic stress in plants, resulting in poor plant development, slow growth and especially reduced crop yields. Severe salt stress or osmotic stress will cause the second stress in plants - oxidative stress, and even cause plant death. Therefore, people pay more and more attention to salt stress, and people pay more and more attention to improving the salt tolerance of crops. Under high salinity, plants mitigate the toxicity caused by salt stress by producing stress proteins and soluble osmoregulatory substances. Early salt-tolerant genetic engineering mainly focused on scavenging free radicals and inc...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/10C12N15/82C12N15/81C07K14/415A01H1/00A01H5/00
Inventor 李海燕郭嘉李校堃王鑫许越魏峰江源
Owner JILIN AGRICULTURAL UNIV
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