Tobacco NtMLO6-1 gene and knockout method and application thereof

A gene and tobacco technology, applied in the field of tobacco NtMLO6-1 gene and its knockout, can solve the problem that the function of tobacco MLO6-1 gene has not been reported

Pending Publication Date: 2022-05-06
CHINA TOBACCO YUNNAN IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The function of the tobacco MLO6-1 gene of the present invention in the regulation of total nitrogen and amino acid content in tobacco leaves has not been reported so far

Method used

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  • Tobacco NtMLO6-1 gene and knockout method and application thereof
  • Tobacco NtMLO6-1 gene and knockout method and application thereof
  • Tobacco NtMLO6-1 gene and knockout method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The acquisition of embodiment 1 NtMLO6-1 gene

[0030] This embodiment mainly focuses on the process of obtaining the tobacco NtMLO6-1 gene, which is briefly introduced as follows.

[0031] Taking the leaves of the cultivar Tobacco safflower Dajinyuan as samples, the total RNA of tobacco leaves was extracted with an RNA extraction kit, and reverse-transcribed into cDNA for future use:

[0032] Tobacco total RNA was extracted according to the instructions of the plant RNA extraction kit.

[0033] 1 μg of total RNA was extracted from leaves for reverse transcription, and the transcription system was as follows:

[0034] Total RNA 1μg;

[0035] Oligo(dT) (10μM) 1.5μL;

[0036] wxya 2 O up to 15 μL.

[0037] Mix the above system and place it in PCR, incubate at 70°C for 5 minutes, immediately place it on ice for 5 minutes after removal, and then add the following reagents to the system:

[0038]

[0039] The above system was put into a PCR instrument, kept at 42°C ...

Embodiment 2

[0048] The construction of embodiment 2 carrier

[0049] Using the tobacco NtMLO6-1 gene obtained in Example 1, the present invention further constructed a CRISPR / Cas9 vector, and transformed it using the leaf disc method to obtain gene-edited plants.

[0050] The more specific 23nt nucleotide sequence (SEQ ID No.5) in the NtMLO6-1 gene was selected as the guide sequence of CRISPR / Cas9, and the sequence fragment was connected with the CRISPR / Cas9 vector (provided by Southwest University), transformed and PCR amplification detection, PCR positive clones were sent to the sequencing company for sequencing confirmation, and finally the CRISPR / Cas9-NtMLO6-1 editing vector was obtained.

Embodiment 3

[0051] Example 3 Transformation of Agrobacterium

[0052] Using the CRISPR / Cas9-NtMLO6-1 editing vector plasmid constructed in the previous step, taking Honghua Dajinyuan as an example, carry out genetic transformation and tissue culture to obtain plants with knockout and edited tobacco NtMLO6-1 genes, and related experimental procedures A brief introduction is as follows.

[0053] 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.

[0054]The LBA4404 electrotransformation competent Agrobacterium cells stored at -80°C were taken out and placed on ice to freeze-thaw. When the competence has just thawed, add 2 μL of the CRISPR / Cas9-NtMLO6-1 editing vector plasmid, mix well, and place on ice. Then transfer th...

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Abstract

The invention discloses a tobacco NtMLO6-1 gene as well as a knockout method and application thereof. The base sequence of the tobacco NtMLO6-1 gene is as shown in SEQ ID No. 1. The method comprises the following steps: knocking out a gene editing plant of an NtMLO6-1 gene by utilizing a CRISPR/Cas9 mediated gene editing technology; the total nitrogen content and the amino acid content in the NtMLO6-1 gene knockout edited plant leaves are remarkably increased, and genetic materials and references are provided for tobacco MLO gene family regulation mechanism research and tobacco leaf total nitrogen and amino acid content regulation.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a tobacco NtMLO6-1 gene and its knockout method and application. Background technique [0002] The Mildew resistance locus O (MLO) gene family is unique to plants and has been isolated and cloned from various plants. The functions of MLO genes are different, and there is a phenomenon of functional redundancy. For example, mutations of one or more specific MLO genes in plants such as tomato, Arabidopsis, tobacco, nightshade, and wheat can cause broad-spectrum resistance to powdery mildew; silencing Tomato SlMLO1 and its homologues SlMLO5 and S1MLO8 have higher levels of resistance to powdery mildew than SlMLO1 mutations, suggesting that the MLO gene has functional redundancy; Arabidopsis AtMLO7 gene mutations lead to reduced plant fertility and excessive growth of pollen tubes in synergid cells. Mutations of AtMLO4 and AtMLO11 genes can lead to abnormal root mor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/12A01H6/82
CPCC07K14/415C12N15/8218C12N15/8251C12N15/8243C12N15/8205
Inventor 杨文武米其利曾婉俐苏杨刘欣高茜蒋佳芮李雪梅李晶张建铎
Owner CHINA TOBACCO YUNNAN IND
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