Tobacco 7-dehydrocholesterol reductase gene and application thereof
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A technology of dehydrocholesterol 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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[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
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Embodiment 1
[0033] In this example, the cloning of the tobacco NtDHCR7 gene and the construction of the silencing vector are briefly introduced as follows.
[0034] (1) Cloning of tobacco NtDHCR7 gene
[0035] According to the previous research on the tobacco genome and related NtDHCR7 gene, the specific coding sequence was selected as the target fragment, and the primer sequences for PCR amplification were designed as follows:
[0036] NtDHCR7-F: 5'-CGCACTTCAGCTTTTGTTGC-3',
[0037] NtDHCR7-R: 5'-ACCGTGATCAGTGGAAGATGG-3'.
[0038] Using the cDNA of tobacco safflower Dajinyuan leaves (extracting the genome first, then reverse-transcribing it into cDNA) as a template, PCR amplification was performed to obtain the NtDHCR7 gene;
[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 1 minute, and after 34 cycles, complete extension at 72°C for 5 minutes;
[0040] The PC...
Embodiment 2
[0046] On the basis of Example 1, the constructed recombinant TRV2-NtDHCR7 vector was further transformed into tobacco plants using Agrobacterium-mediated VIGS technology, and a verification analysis was performed on the phenotypic changes of related plants. The specific experimental process is 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-NtDHCR7 were transformed into Agrobacterium GV3101 competent cells by electric shock transformation respectively, and were 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) Preparation of bacteria so...
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Abstract
The invention relates to a tobacco 7-dehydrocholesterol reductase gene and application thereof. The nucleotide sequence of the 7-dehydrocholesterol reductase gene is as shown in SEQ ID NO. 1. Based on the important influence of sterol on the safety of tobacco leaves, the tobacco sterol metabolism regulation gene is deeply studied, so that a new tobacco variety is constructed by utilizing genetic engineering, and a good application foundation is laid for improving the tobacco variety. According to the application, preliminary research on specific tobacco sterol 7-dehydrocholesterol reductase shows that the specific tobacco sterol 7-dehydrocholesterol reductase is highly related to the sterol content in tobacco leaves, when the gene is silenced in Bensi tobacco, the stigmasterol content and the campesterol content in the tobacco leaves are reduced, and the total sterol content is reduced by 24.8%. Based on the characteristic, a certain foundation and reference can be provided for tobacco quality regulation and control and new tobacco variety cultivation.
Description
technical field [0001] The invention belongs to the field of tobacco genetic engineering, and in particular relates to a 7-dehydrocholesterol reductase gene NtDHCR7 of tobacco sterol synthesis and metabolism and application thereof. Background technique [0002] Sterols belong to macromolecular alcohols and are a class of steroidal compounds that widely exist in nature. Sterols in nature are divided into animal sterols, mycosterols and phytosterols according to their sources. Phytosterols are important components of biofilm systems, which can regulate plant growth and development and respond to various biotic and abiotic stresses. Sterols account for about 0.1% to 0.3% of the mass of tobacco leaves. It has been confirmed by literature that sterols in tobacco are important precursor compounds of benzopyrene in cigarette smoke. Sterols in tobacco mainly include cholesterol, stigmasterol, campasterol, and β-sitosterol. Therefore, reducing the content of sterols in tobacco le...
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