A Transgenic Hyperhalophilic Archaea nasod Gene Tobacco

A technology of extremely halophilic archaea and tobacco, applied in genetic engineering, plant genetic improvement, recombinant DNA technology, etc., can solve the problems of slow development and no breakthrough in the development and utilization of high-salt-tolerant genetic resources.

Active Publication Date: 2019-10-11
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the 1950s, people have carried out research on its adaptation mechanism, but so far there has been no breakthrough in the development and utilization of high-salt-tolerant gene resources in alpine salt lakes
[0004] Biologists and breeders cultivate salt-tolerant varieties through traditional breeding methods to improve the salt-tolerant ability of plants, thereby reducing the impact of salinity on agricultural and forestry production. Some progress has been made, but the development is slow

Method used

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  • A Transgenic Hyperhalophilic Archaea nasod Gene Tobacco
  • A Transgenic Hyperhalophilic Archaea nasod Gene Tobacco
  • A Transgenic Hyperhalophilic Archaea nasod Gene Tobacco

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Construction of expression vector p1301-NaSOD

[0022] Cultivate Escherichia coli containing plasmid pGEX-SOD (patent application 200810163748.8), use specific primers of halophilic archaea NaSOD gene (add Pst I and Kpn I enzyme cleavage sites respectively) for bacterial liquid PCR, and recover the purified PCR product Linked to the intermediate vector pMD19-T, transformed E. coli DH5α. Pick positive clones, PCR and sequencing identification. Shake the correct bacterial solution to extract the plasmid pMD19-NaSOD. The plasmid pMD19-NaSOD was digested with Pst I and Kpn I, and then inserted into the multiple cloning site of pCAMBIA1301, and the CaMV 35S promoter and NOS terminator were inserted before and after the target gene to obtain the plant expression vector pCAMBIA1301-NaSOD (such as figure 1 Shown). After the recombinant was identified by restriction enzyme digestion, it was introduced into Agrobacterium EHA105 by freeze-thaw method.

[0023] NaSOD specif...

Embodiment 2

[0026] Example 2 Agrobacterium-mediated genetic transformation of tobacco

[0027] 2.1 Tobacco aseptic seedling culture

[0028] 1) On a sterile ultra-clean workbench, put the tobacco seeds into a 1.5mL Eppendorf tube, wash with 75% ethanol for 1 min, and rinse with sterile water for 3 to 4 times;

[0029] 2) Add 10% NaClO and soak for 10 minutes, rinse with sterile water 4 to 5 times to remove trace amounts of NaClO;

[0030] 3) Put the seeds on sterile absorbent paper to absorb trace water, and sown on MS0 medium at 25°C (16h / 8h=light / dark). When the plant grows to 6-8 leaves, it is used for preparation Explants transformed by tobacco.

[0031] 2.2 The leaf disc method transforms tobacco

[0032] 1) Inoculate the Agrobacterium broth containing pCAMBIA1301-NaSOD in LB liquid medium containing 50mg / L Kam (kanamycin) and 40mg / L Rif (rifampicin), shaking at 28°C overnight (200rpm), When the bacteria grow to (OD600=0.5~0.8), centrifuge (5000rpm) at 28℃ for 8min, resuspend the collected ba...

Embodiment 3

[0039] Example 3 Identification of transgenic plants

[0040] Hygromycin screening: After transplanting the regenerated seedlings into the greenhouse, select new leaves in the same position, take 2~3cm, and soak them in sterile water containing 50mg / L hygromycin and 1.0mg / L 6-BA. After 5 days, the leaves of non-transgenic tobacco turned yellow and brown, the leaves of false positive plants also turned yellow, and the leaves of transformed plants remained green.

[0041] PCR detection of genetically modified tobacco: The total DNA of tobacco leaves was extracted by SDS method, and amplified with NaSOD specific primers. All 14 transgenic samples had a band at 621 bp (such as figure 2 Shown).

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Abstract

The invention discloses an application of a Natrinema altunense sp. NaSOD gene for reducing nicotine in tobacco, and a base sequence of the Natrinema altunense sp. NaSOD gene is shown as SEQ ID NO.3.The Natrinema altunense sp. NaSOD gene is transferred in tobacco, and nicotine content in tobacco can be obviously reduced.

Description

Technical field [0001] The invention relates to a tobacco that has transformed the extremely halophilic archaea NaSOD gene, in particular to the application of the extremely halophilic archaea NaSOD gene in reducing the nicotine content in the tobacco. Background technique [0002] With the deterioration of the ecological environment, the development of facility agriculture and the improper use of chemical fertilizers, soil salinization has become a worldwide resource and ecological problem, and the area of ​​saline-alkali land is increasing year by year (Mahajan S, Tuteja N. Cold, salinity and drought stresses:an overview[J].Arch.). According to incomplete statistics, the world's saline-alkali land area is about 954 million hm 2 (FAO, 2008). my country's saline soil has a large area, widely distributed, and diverse types. The total area of ​​various types of saline soil is 9,913 million hectares. 2 , The area of ​​modern saline-alkali soil is 36.93 million hm 2 , The remaining ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/53A01H5/00A01H6/82
Inventor 朱诚丁艳菲
Owner CHINA JILIANG UNIV
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