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Method for creating low-nicotine tobacco mutant by knocking out NtBBLs by using CRISPR/Cas9 and application of low-nicotine tobacco mutant

A mutant, tobacco technology, applied in the field of plant genetic engineering, can solve the problems of uncultivated utilization value, poor agronomic traits and the like

Pending Publication Date: 2022-04-29
CHINA TOBACCO YUNNAN IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are certain low-nicotine materials in tobacco genetic resources, but these materials generally have poor agronomic traits and have no value for cultivation

Method used

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  • Method for creating low-nicotine tobacco mutant by knocking out NtBBLs by using CRISPR/Cas9 and application of low-nicotine tobacco mutant
  • Method for creating low-nicotine tobacco mutant by knocking out NtBBLs by using CRISPR/Cas9 and application of low-nicotine tobacco mutant
  • Method for creating low-nicotine tobacco mutant by knocking out NtBBLs by using CRISPR/Cas9 and application of low-nicotine tobacco mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 Obtaining of NtBBLs gene

[0033] Using the cultivar Tobacco Safflower Dajin Yuangen as the experimental material, the total RNA of tobacco roots was extracted with an RNA extraction kit, and reverse-transcribed into cDNA for future use:

[0034] Tobacco total RNA was extracted according to the instructions of the plant RNA extraction kit.

[0035] 1 μg of total RNA was extracted from leaves for reverse transcription, and the transcription system was as follows:

[0036] Total RNA 1μg;

[0037] Oligo(dT) (10μM) 1.5μL;

[0038] wxya 2 O up to 15 μL;

[0039] Mix the above system and place it in PCR, keep it warm at 70°C for 5 minutes, put it on ice for 5 minutes immediately after removing it, and then add the following reagents to the system:

[0040]

[0041] The above system was placed in a PCR instrument, kept at 42°C for 65 minutes, 65°C for 10 minutes, kept at 4°C, and then stored in a -20°C refrigerator for use.

[0042] Design NtBBLs gene-spe...

Embodiment 2

[0051] Example 2 Construction of CRISPR / Cas9 gene editing vector

[0052] Using the gene NtBBLs related to nicotine metabolism obtained in Example 1, the present invention further constructed a CRISPR / Cas9 vector.

[0053]Select the NtBBLs gene-specific 23nt nucleotide sequence (SEQ ID No.5) as the guide sequence of CRISPR / Cas9, and connect, transform and PCR-amplify the sequence fragment with the CRISPR / Cas9 vector (provided by Southwest University) , PCR-positive clones were sent to the sequencing company for sequencing confirmation, and finally the CRISPR / Cas9-NtBBLs editing vector was obtained. details as follows:

[0054] (1) Design and synthesis of the sgRNA sequence of the NtBBLs gene:

[0055] Use the online software CRISPR-P 2.0 (http: / / cbi.hzau.edu.cn / crispr / ) to design sgRNA guide sequences, and select guide sequences with higher scores and located at appropriate positions in the NtBBLs gene sequence. The sgRNA sequence selected in this application is: TATGAAATCA...

Embodiment 3

[0079] Example 3 Transformation of Agrobacterium

[0080] Using the CRISPR / Cas9-NtBBLs editing vector plasmid constructed in the previous step, taking Honghua Dajinyuan as an example, carry out genetic transformation and tissue culture, and obtain plants in which the gene NtBBLs related to tobacco nicotine metabolism has been knocked out and edited, and related experimental procedures A brief introduction is as follows.

[0081] Sterilize the surface of tobacco seeds and plant them on MS medium. After they grow to 4 cotyledons (15-20 days), transfer them into culture bottles containing MS solid medium, and place them at 25±1°C under light intensity of 30-50 μmol / ( m2·s), and the light time is 16h / d to continue culturing for 35-40d, and set aside.

[0082] Transform the plasmid with the correct sequence into Agrobacterium, the specific steps are as follows:

[0083] (1) Take out the Agrobacterium competent cells stored at -80°C and freeze-thaw them on ice.

[0084] (2) When ...

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Abstract

The invention discloses a method for creating a low-nicotine tobacco mutant by knocking out NtBBLs by using CRISPR / Cas9 and application. The method comprises the following steps: (1) constructing a CRISPR / Cas9 gene editing carrier; (2) preparing infected agrobacterium; (3) carrying out genetic transformation to obtain regenerated edited plants and homozygous T1-generation plants; and (4) carrying out a detection test on the nicotine content of leaves of the NtBBLs gene homozygous knockout material in the squaring stage by GC-MS (Gas Chromatography-Mass Spectrometer). According to the application, the NtBBLs gene is knocked out by utilizing a CRISPR / Cas9 mediated gene editing technology, so that a tobacco knockout material which keeps original agronomic characters and meanwhile, the nicotine content is greatly reduced can be obtained.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a method and application of using CRISPR / Cas9 to knock out NtBBLs to create low-nicotine tobacco mutants. Background technique [0002] Alkaloids are important chemical components of Nicotiana plants, among which nicotine is the most important alkaloid, accounting for more than 90%. [0003] The nicotine content of tobacco leaves is fundamentally controlled by genetic factors, but strongly influenced by ecological, agronomic and other measures. At present, the cultivation of low-nicotine tobacco varieties includes conventional breeding and genetic engineering. There are certain low-nicotine materials in tobacco genetic resources, but these materials generally have poor agronomic traits and have no value for cultivation. Legg et al. found that alkaloids are controlled by two independent loci, Nic1 and Nic2. Researchers have also bred flue-cured and dark air-cu...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/113C12N15/29A01H5/00A01H6/82G01N30/02
CPCC12N15/8243C12N15/113C07K14/415G01N30/02C12N2310/20
Inventor 张建铎孔维松王晋杨光宇师君丽邓乐乐杨文武许力蒋佳芮高茜向海英曾婉俐李雪梅
Owner CHINA TOBACCO YUNNAN IND
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