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

Novel plant strong-salt resistance gene NHXFS1 and encoding protein

A salt-tolerant gene, plant technology, applied in the field of plant genetic engineering

Inactive Publication Date: 2011-02-02
EAST CHINA NORMAL UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of this technology to improve the performance of the Na+ / H+ antiporter gene and its protein has not been reported at home and abroad.

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
  • Novel plant strong-salt resistance gene NHXFS1 and encoding protein
  • Novel plant strong-salt resistance gene NHXFS1 and encoding protein
  • Novel plant strong-salt resistance gene NHXFS1 and encoding protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1. Arabidopsis, rice, chrysanthemum Na + / H + Cloning of the antiporter gene

[0034] Total RNA was extracted from the young leaves of Arabidopsis thaliana, rice and chrysanthemum with TRizol reagent, and reverse transcription was performed using the total RNA as a template to synthesize the first strand of cDNA. Using the synthesized cDNA as a template and referring to the cDNA sequences of AtNHX1, OsNHX1, and DmNHX1, the following primers were designed (the 5' end contains SmaI and SalI restriction sites respectively):

[0035] AtR(5'-GC GTC GAC TCAAGCCTTACTAAGATCAGGAGG-3')

[0036] AtF(5'-TCC CCCGGG ATGTTGGA TTCTCTAGTG-3')

[0037] OsR(5'-ACGC GTC GAC TCATCTTCCTCCATGGC-3')

[0038] OsF(5'-TCC CCCGGG ATGGGGATGGAGGTGGCG-3')

[0039] DmR (5'-GC GTC GAC TTAGTTTCTTTCTTTCATCTTC-3')

[0040] DmF(5'-TC CCCCGG GATGGTGTTCGATTC-3')

[0041] Arabidopsis Na + / H + Antiporter gene AtNHX1, rice Na + / H + Antiporter genes OsNHX1 and chrysanthemum Na...

Embodiment 2

[0042] Example 2. DNA shuffling of AtNHX1, OsNHX1, DmNHX1 genes

[0043] 1) Preparation of starting materials Using pGM-T-AtNHX1, pGM-T-OsNHX1, and pGM-T-DmNHX1 as templates, use PfuDNA polymerase to PCR amplify the three genes, and use them as starting materials for DNA shuffling after purification .

[0044] 2) Random enzyme digestion with DNase I Determine the concentration of purified DNA by ultraviolet absorption method, mix AtNHX1, OsNHX1, and DmNHX1 with a content of 9ug, so that the ratio of the three gene substances is 1:1:1. Take 40ul of mixed AtNHX1, OsNHX1, DmNHX1 and add it to 50μl enzyme digestion reaction system (10mM Tris-HCl, pH7.5, 50mM MnCl 2 ), react at 15°C for 10 min. Add DNase I 0.15U, mix well, 15°C, 1min16s, 90°C, 10min. The product goes through 2.5% agarose gel electrophoresis, and the gel containing 100-200bp fragments is excised and recovered.

[0045] 3) PCR without primers Take the recovered small fragments of 1 μm and add them to a 50 μl react...

Embodiment 3

[0048] Example 3. Construction of expression shuffling library and resistance to high salt Na + / H + Screening of antiporter gene NHXFS1

[0049] The recombinant product obtained in Example 2 was digested with Sal I and Sma I, purified and connected to the same digested yeast expression vector pYPGE15, and the constructed expression shuffling library was introduced into the yeast mutant strain W303-1BΔena1- 4::HIS3 Δnhx1::TRP1, coated on APG selection medium without uracil, cultured at 30°C for 2 days, selected to contain Na + / H + The yeast strain of the antiporter gene was further screened by increasing the NaCl concentration, and finally a normal growth yeast mutant was obtained on the selection medium with a concentration of 200mM NaCl, which contained a new plant Strong salt tolerance gene NHXFS1.

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 relates to the field of plant genetic engineering and provides a novel plant strong salt-tolerant gene NHXS1 obtained by a restructuring technology. A nucleotide sequence of the plant strong salt-tolerant gene NHXS1 is SEQ ID NO: 1, or a DNA sequence which has 70 to 100 percent homology with the SEQ ID NO: 1 nucleotide sequence or a DNA sequence for coding a protein sequence of SEQ ID NO: 2. Na + / H + antiporter NHXS1 colded by the gene has stronger ion transport activity than the wild-type Na + / H + antiporter AtNHX1. The invention also provides methods for construction of recombinant vectors and transgenic plants to apply the genes and proteins, and can culture a novel breed of transgenic plant with stronger salt-tolerant property or other improved biological characteristics.

Description

technical field [0001] The present invention relates to the field of plant genetic engineering, in particular to the use of DNA family shuffling technology to obtain significantly improved plant Na + / H + The antiporter gene, the invention also relates to the use of the gene to breed transgenic salt-tolerant plants. Background technique [0002] Soil salinization is an important abiotic stress factor affecting crop production and ecological environment. At present, about 20% of the cultivated land and nearly 50% of the irrigated land in the world are seriously harmed by salinity (Flowler T J, Yeo A R. Breeding for salinity resistance in crop plants. Where next? Aust.J.Plant Physiol.1995 , 22: 875-884). There are about 500 million mu of saline-alkali land in my country, and its area has a tendency to increase continuously. Cultivating salt-tolerant plants is an effective way to promote the development and utilization of saline-alkali land, improve soil and ecological man...

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
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/63C12N5/10C07K14/415A01H1/00
CPCC12N15/8273C07K14/705
Inventor 夏涛张辉徐凯
Owner EAST CHINA NORMAL 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