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Natrinema altunense sp. NaSOD gene transferred in 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-resistant genetic resources

Active Publication Date: 2018-03-27
CHINA JILIANG UNIV
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  • 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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  • Natrinema altunense sp. NaSOD gene transferred in tobacco
  • Natrinema altunense sp. NaSOD gene transferred in tobacco
  • Natrinema altunense sp. NaSOD gene transferred in tobacco

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0022] Cultivate Escherichia coli containing the plasmid pGEX-SOD (patent application 200810163748.8), use the specific primers of the NaSOD gene of the halophilic archaea (respectively adding Pst I and Kpn I restriction sites) to carry out bacterial liquid PCR, and recover the purified PCR product Connected to the intermediate vector pMD19-T, transformed into Escherichia coli DH5α. Positive clones were picked and identified by PCR and sequencing. Shake the correct bacterial solution to extract the plasmid pMD19-NaSOD. Pst I and Kpn I double-digest the plasmid pMD19-NaSOD, then insert it into the multiple cloning site of pCAMBIA1301, and insert the CaMV 35S promoter and NOS terminator before and after the target gene, respectively, to obtain the plant expression vector pCAMBIA1301-NaSOD (such as figure 1 shown). After the recombinant was identified by enzyme digestion, it was introduced into Agrobacterium EHA1...

Embodiment 2

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

[0027] 2.1 Tobacco sterile seedling culture

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

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

[0030] 3) Put the seeds on sterile absorbent paper to absorb trace water, sow on MS0 medium at 25°C (16h / 8h=light / dark), and when the plant grows to 6-8 leaves, it is used to prepare Tobacco transformed explants.

[0031] 2.2 Tobacco Transformation by Leaf Disc Method

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

Embodiment 3

[0039] Identification of embodiment 3 transgenic plants

[0040] Hygromycin screening: After the regenerated seedlings are transplanted into the greenhouse, select new leaves in the same position, take 2-3 cm, and soak them in sterile water containing 50 mg / L hygromycin and 1.0 mg / 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 transgenic tobacco: the total DNA of tobacco leaves was extracted by SDS method, amplified with NaSOD specific primers, and all 14 transgenic samples had a band at 621bp (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 transformed with the NaSOD gene of the extreme halophilic archaea, in particular to the application of the NaSOD gene of the extreme halophilic archaea in reducing the nicotine content in 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 problem 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 saline-alkali land area in the world is about 954 million hm 2 (FAO, 2008). my country has a large area of ​​saline-alkali soil, widely distributed, and various types. The total area of ​​various types of saline-alkali land is 99.133 million hm 2 , of which the modern saline-alkali soil area...

Claims

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Application Information

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