ntwrky69 Gene Affecting Tobacco Pigment Content and Its Application

A technology of pigment content and tobacco, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of tobacco leaf green miscellaneous gas, unfavorable chemical components, etc.

Active Publication Date: 2020-11-03
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally speaking, fully degraded chlorophyll is beneficial to tobacco quality, but if it cannot be degraded completely, chlorophyll will become an unfavorable chemical component in the dried tobacco leaves, which will make the tobacco leaves have obvious green odor.

Method used

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  • ntwrky69 Gene Affecting Tobacco Pigment Content and Its Application
  • ntwrky69 Gene Affecting Tobacco Pigment Content and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This embodiment mainly affects the tobacco pigment content NtWRKY69 The process of gene acquisition is briefly introduced as follows.

[0039] (1) Tobacco RNA extraction and cDNA synthesis

[0040] For RNA extraction, please refer to the following steps:

[0041] Take the 4-week-old young leaves of cultivated tobacco K326 as samples, and after being fully ground into powder with liquid nitrogen, take about 100 mg of powdered material and place it in a 1.5 ml centrifuge tube filled with 1.0 ml of TRIZOL reagent, add 200 μL of chloroform; oscillate to mix, and after centrifugation, carefully remove the upper aqueous phase and transfer to another centrifuge tube; add 500 μL of isopropanol, precipitate, centrifuge to separate RNA, wash with 75% alcohol, and dry at room temperature. Add an appropriate volume of RNase free water to fully dissolve;

[0042]The extracted total RNA was treated with DNase I, and the 10 μL digestion reaction system was designed as follows:

[...

Embodiment 2

[0076] Utilize the obtained in embodiment 1 to influence tobacco pigment content NtWRKY69 gene, the inventor further constructed the gene silencing RNAi interference vector TRV2- NtWRKY69 , the relevant construction process is briefly introduced as follows.

[0077] A relatively specific nucleic acid fragment in this gene (the 80th-600th nucleotide sequence of SEQ ID NO.1 in the sequence table) is selected as the guide sequence of VIGS, and primers are designed to obtain this sequence. This nucleic acid fragment is then inserted into the VIGS vector to construct TRV2- NtWRKY69 Carrier, the specific construction process is:

[0078] (1) Obtain the target fragment, select a more specific nucleic acid fragment in this gene (the 80th-600th nucleotide sequence of SEQ ID NO.1 in the sequence table) as the guide sequence of VIGS, design primers and use the method obtained in Implementation 1 The full length of the gene was used as the template for PCR amplification to obtain the t...

Embodiment 3

[0103] Using the VIGS silencing vector TRV2- constructed in Example 2 NtWRKY69 Taking Nicotiana benthamiana as an example, the inventor further carried out a cultivation test to silence the factors that affect the tobacco pigment content in the plant. NtWRKY69 Genes and related experimental procedures are briefly introduced as follows.

[0104] (1) Tobacco seeds were sown in seedling pots to grow seedlings, and seedlings were divided two weeks after germination, planted in plastic pots (10cm×10cm), and daily fertilizer and water management was carried out at 22°C under 16 h light / 8 h dark conditions etc.; grow for 4 weeks, select seedlings with consistent growth and group into groups for subsequent use;

[0105](2) will contain TRV1, TRV2, TRV2- NtWRKY69 , TRV2- PDS Agrobacterium single colony (other plasmid construction process can refer to the above construction process) into 5 mL of LB medium (containing 50 mg / L kanamycin, 50 mg / L rifampicin), 28 ℃, 180 r / min Overnig...

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Abstract

The invention belongs to the technical field of biology engineering and particularly relates to an NtWRKY69 gene affecting tobacco pigment content and an application of the NtWRKY69 gene. The gene comprises a 918bp base with the base sequence shown in SEQ ID NO.1, wherein 80th-600th nucleotides are specific core fragments. Through cloning of the NtWRKY69 gene of tobacco and analysis of corresponding protein, the gene is found to play important roles in regulating tobacco pigment synthesis ways and be highly correlated with the content of pigments in plant leaves. Further, by the aid of a virus mediated gene silencing technology, after the NtWRKY69 gene is silent, the content of the pigments in new transgenic plants is obviously reduced. By means of the characteristic, a new strategy and path can be provided for gene engineering breeding of the tobacco or breeding of other new plant varieties.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a method for affecting tobacco pigment content. NtWRKY69 Patent applications for genes and their applications. Background technique [0002] Tobacco is a plant of the genus Nicotiana in the genus Nicotiana ( Nicotiana ) There are more than 60 species, of which the common tobacco (also known as safflower tobacco, Nicotiana tabacum ) occupies the main area, Nicotiana japonica ( Nicotiana rustica ) is very small. Cultivated tobacco can be divided into six types: flue-cured tobacco, sun-cured tobacco, air-dried tobacco, burley tobacco, oriental tobacco and yellow flower tobacco according to the quality characteristics of tobacco leaves, biological characteristics and cultivation methods, among which flue-cured tobacco is the most widely cultivated common tobacco. my country's flue-cured tobacco planting area and total output both rank first in the world. [...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/82
CPCC07K14/415C12N15/825
Inventor 徐国云周会娜张慧刘萍萍陈千思翟妞郑庆霞金立锋陈霞申晓晔
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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