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3-hydroxy-3-methylglutaryl coenzyme A reductase gene of tobacco and application thereof

A technology of methylglutaryl coenzyme and reductase, applied in the field of tobacco genetic engineering, can solve problems such as few reports

Pending Publication Date: 2021-08-20
CHINA TOBACCO YUNNAN IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the synthesis and metabolism of sterols in plants have been studied, but the genes that regulate sterol synthesis in cultivated tobacco are rarely reported

Method used

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  • 3-hydroxy-3-methylglutaryl coenzyme A reductase gene of tobacco and application thereof
  • 3-hydroxy-3-methylglutaryl coenzyme A reductase gene of tobacco and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] In this example, the cloning of the tobacco NtHMGCR11 gene and the construction of the silencing vector are briefly introduced as follows.

[0034] (1) Cloning of tobacco NtHMGCR11 gene

[0035] According to the previous research on the tobacco genome and related NtHMGCR11 gene, the specific coding sequence was selected as the target fragment, and the primer sequences for PCR amplification were designed as follows:

[0036] NtHMGCR11-F: 5'-ATGGACGTTCGCCGGCGAC-3',

[0037] NtHMGCR11-R: 5'-CAACAGATATATAACAGAAGCGATC-3'.

[0038] The NtHMGCR11 gene was obtained by PCR amplification using the cDNA of tobacco safflower Dajinyuan leaves (extracting the genome first and then reverse-transcribing it into cDNA) as a template;

[0039] The PCR amplification program is: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, extension at 72°C for 2 minutes, and after 34 cycles, complete extension at 72°C for 5 minutes;

[00...

Embodiment 2

[0046] On the basis of Example 1, the constructed recombinant TRV2-NtHMGCR11 vector was further transformed into tobacco plants by using Agrobacterium-mediated VIGS technology, and a verification analysis was performed on the phenotypic changes of related plants. The specific experimental process is briefly introduced as follows.

[0047] (1) Transformation of Agrobacterium

[0048] It should be noted that, referring to the operation of Example 1 and the prior art, a TRV2-GFP recombinant vector was prepared as a control, and the specific transformation process was as follows:

[0049] The positive cloning plasmids of TRV2-GFP (vector control) and TRV2-NtHMGCR11 were transformed into Agrobacterium GV3101 competent cells by electric shock transformation respectively, and cultured and screened on YEB plates containing 50 mg / L Kan and 50 mg / L Rif. After upside-down culture at 28°C for 2 days, colony PCR was used to screen the Agrobacterium with the target gene.

[0050] (2) Prepa...

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Abstract

The invention relates to a 3-hydroxy-3-methylglutaryl coenzyme A reductase gene and an application thereof. The nucleotide sequence of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene is as shown in SEQ ID NO. 1. According to the application, preliminary research on the specific tobacco 3-hydroxy-3-methylglutaryl coenzyme A reductase shows that the 3-hydroxy-3-methylglutaryl coenzyme A reductase is highly related to the content of sterol in tobacco leaves, when the gene is silenced in Bensi tobacco, the content of cholesterol and stigmasterol in the tobacco leaves is obviously reduced, and the content of total sterol is reduced by 35.4%. On the basis of the characteristic, a certain application basis and reference can be provided for tobacco quality regulation and control and new tobacco variety cultivation by utilizing a genetic engineering means.

Description

technical field [0001] The invention belongs to the field of tobacco genetic engineering, in particular to a tobacco 3-hydroxy-3-methylglutaryl-CoA reductase gene and its application. Background technique [0002] Phytosterols are an important class of physiologically active substances that can regulate plant growth and development and respond to various biotic and abiotic stresses. Sterols in tobacco account for about 0.1% to 0.3% of the mass of tobacco leaves. Since the sterol structure contains hydroxyl groups, its parent phenanthrene ring structure can form polycyclic aromatic hydrocarbons during pyrolysis. It has been reported that sterols in tobacco are important precursor compounds of benzopyrene in cigarette smoke. The sterol compounds in tobacco mainly include cholesterol, stigmasterol, campasterol, and β-sitosterol, among which stigmasterol and β-sitosterol are relatively high in content. , followed by campesterol content, and the lowest cholesterol content. Ther...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/04C12N15/82A01H5/00A01H6/82
CPCC12N9/0006C12N15/8243C12Y101/01088
Inventor 曾婉俐李雪梅朱保昆向海英杨光宇刘欣李晶黄海涛蒋佳芮杨文武
Owner CHINA TOBACCO YUNNAN IND
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