Tobacco polyphenol oxidase gene NtPPO1 and site-directed mutagenesis method and application thereof

A polyphenol oxidase and site-directed mutagenesis technology, applied in the field of genetic engineering, can solve the problems of reducing the aroma components of tobacco leaves, decreasing the appearance and internal quality of tobacco leaves, and economic losses.

Inactive Publication Date: 2018-02-02
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process is called enzymatic browning reaction mediated by polyphenol oxidase in tobacco. In this process, important tobacco aroma precursors such as chlorogenic acid and rutin are oxidized into light red to dark brown substances; therefore, Significantly reduce the appearance quality of tobacco leaves and reduce the aroma components of tobacco leaves, which will eventually lead to a decline in the appearance and internal quality of tobacco leaves, thus bringing economic losses to tobacco farmers and enterprises

Method used

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  • Tobacco polyphenol oxidase gene NtPPO1 and site-directed mutagenesis method and application thereof
  • Tobacco polyphenol oxidase gene NtPPO1 and site-directed mutagenesis method and application thereof
  • Tobacco polyphenol oxidase gene NtPPO1 and site-directed mutagenesis method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Using the above method, a transgenic NtPPO1 gene NtPPO1-64 was obtained by sequencing. The results after sequencing are compared with the reference sequence as follows figure 1 .shown. The arrow mark shows that after the NtPPO1 gene is at the sgRNA target site, there are double peaks in the sequencing map, indicating that the sequence of the NtPPO1 gene has changed this time.

[0102] The transgenic plant NtPPO1-64 was harvested after selfing, and the NtPPO1 gene of the selfed offspring NtPPO1-64-1 was found to appear as follows figure 2 . Mutations indicated. After the target sgRNA sequence, the NtPPO1 gene is missing -2bp (missing 2 GG bases), resulting in a change in the gene reading frame. After the knockout site of the NtPPO1 gene, the translated protein sequence changed, and finally a TAA stop codon appeared in advance at the original 112th codon that started encoding the amino acid Lys protein, resulting in early termination of translation.

[0103] Determin...

Embodiment 2

[0106] Using the above method, a transgenic NtPPO1 gene NtPPO1-70 was obtained by sequencing. The results after sequencing are compared with the reference sequence as follows Figure 5 .shown. The arrow mark shows that after the NtPPO1 gene is at the sgRNA target site, there are double peaks in the sequencing map, indicating that the sequence of the NtPPO1 gene has changed this time.

[0107] The transgenic plant NtPPO1-70 was harvested after selfing, and it was found by sequencing that the NtPPO1 gene of the selfed offspring NtPPO1-70-1 appeared as follows: Image 6 . Mutations indicated. After the target sgRNA sequence, the NtPPO1 gene is missing -1bp (missing 1 G base), resulting in a change in the gene reading frame. After the knockout site of the NtPPO1 gene, the translated protein sequence changed, and finally a TAA stop codon appeared at the original 193rd start coding amino acid Leu codon frame shift, resulting in premature termination of translation.

[0108] Dete...

Embodiment 3

[0111] Using the above method, a transgenic NtPPO1 gene NtPPO1-72 was obtained by sequencing. The results after sequencing are compared with the reference sequence as follows Figure 9 .shown. The arrow mark shows that after the NtPPO1 gene is at the sgRNA target site, there are double peaks in the sequencing map, indicating that the sequence of the NtPPO1 gene has changed this time.

[0112] The transgenic plant NtPPO1-72 was harvested after selfing, and it was found by sequencing that the NtPPO1 gene of the selfed offspring NtPPO1-72-1 appeared as follows: Figure 10 . Mutations indicated. After the target sgRNA sequence, the NtPPO1 gene inserted +1bp (inserted 1 A base), resulting in a change in the gene reading frame. After the NtPPO1 gene was knocked out, the translated protein sequence changed, and finally a TAA stop codon appeared at the codon of the original 112th start coding amino acid Lys, resulting in premature termination of translation.

[0113] Determination...

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Abstract

The invention discloses a tobacco polyphenol oxidase gene NtPPO1 and a site-directed mutagenesis method and application thereof. The nucleotide sequence of the tobacco polyphenol oxidase gene NtPPO1 is shown in SEQ ID:No.1, the coded amino acid sequence is shown in SEQ ID:No.2. The invention further discloses a clone method of the tobacco polyphenol oxidase gene NtPPO1. The clone method comprisesthe specific steps that firstly, cDNAs of tobacco leaves are synthesized; secondly, PCR amplification of the NtPPO1 gene is conducted; thirdly, a CRISPR/Cas9 carrier of the NtPPO1 gene is constructed;fourthly, sequencing detection of the NtPPO1 gene mutation is conducted. The NtPPO1 gene has a wide application prospect in preventing tobacco browning. The tobacco polyphenol oxidase gene and the encoded protein thereof provide the gene and technology support for crops especially for anti-browning breeding of tobaccos.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a tobacco polyphenol oxidase gene NtPPO1 and its site-directed mutation method and application. Background technique [0002] Polyphenol oxidase (PPO, polyphenol oxidase) is a class of copper-binding enzymes that widely exist in nature, and generally has two activities: oxygenase and dehydrogenase. Polyphenol oxidase is widely found in animals, plants and fungi. Polyphenol oxidase can be divided into monophenol oxidase (tyrosinase, tyrosinase), bisphenol oxidase (catechin monophenol oxidase, catechol oxides) and Laccase (laccase). [0003] Most PPO genes in plants exist in the form of gene families. Among economic crops of Solanaceae, 6 PPO genes were found in eggplant; 7 PPO genes were found in tomato; 2 PPO genes were found in potato. At least 6 PPO genes were found in red clover. Cai et al. found eight different PPO genes in sorghum. But only one P...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/82C12N9/22A01H5/12A01H6/82
CPCC12N9/0059C12N9/22C12N15/8213C12N15/8261C12Y110/03001
Inventor 姚恒杨大海白戈谢贺肖炳光李永平
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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