Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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
View PDF0 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Early salt-tolerant genetic engineering mainly focused on scavenging free radicals and increasing osmotic adjustment substances. The salt-tolerant ability of transgenic plants has improved, but the effect is not obvious

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com