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NtWRKY69 gene affecting tobacco pigment content and application of NtWRKY69 gene

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: 2017-09-19
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 application of NtWRKY69 gene
  • NtWRKY69 gene affecting tobacco pigment content and application of NtWRKY69 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This example mainly affects the content of tobacco pigment 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 4 weeks old cultivated tobacco K326 young leaves as a sample. After being fully ground into powder with liquid nitrogen, about 100 mg of the powdered material is placed in a 1.5 ml centrifuge tube containing 1.0 ml of TRIZOL reagent, and 200 μL is added Chloroform; shake and mix well, after centrifugation, carefully remove the upper aqueous phase and transfer it to another centrifuge tube; add 500 μL isopropanol, precipitate and centrifuge to separate RNA, then wash with 75% alcohol, and dry at room temperature, then add Proper volume of RNase free water, fully dissolved;

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

[0043] Extracted R...

Embodiment 2

[0076] Using the obtained in Example 1 to affect the content of tobacco pigment NtWRKY69 Gene, the inventor further constructed the RNAi interference vector TRV2- for gene silencing NtWRKY69 , The relevant construction process is briefly introduced as follows.

[0077] Select a more specific nucleic acid fragment (the 80-600th nucleotide sequence of SEQ ID NO.1 in the sequence list) of this gene as the VIGS guide sequence, and design primers to obtain this sequence. Then insert this nucleic acid fragment into the VIGS vector to construct TRV2- NtWRKY69 Vector, the specific construction process is:

[0078] (1) Obtain the target fragment, select a more specific nucleic acid fragment (the 80-600th nucleotide sequence of SEQ ID NO. 1 in the sequence table) as the VIGS guide sequence, design primers and obtain the result in implementation 1. The full length of the gene is used as a template for PCR amplification to obtain the target gene fragment. The primer sequence is designed as fol...

Embodiment 3

[0103] Using the VIGS silencing vector TRV2-constructed in Example 2 NtWRKY69 The engineered bacteria, taking Nicotiana benthamiana as an example, the inventors further carried out cultivation experiments to silence the effects of tobacco pigment content in plants NtWRKY69 Genes and related experimental procedures are briefly introduced as follows.

[0104] (1) Sow the tobacco seeds in a nursery bowl to raise seedlings, divide the seedlings two weeks after germination, plant them in a plastic bowl (10cm×10cm), and perform daily fertilizer and water management under 22℃, 16h light / 8h dark conditions Etc.; grow for 4 weeks, select seedlings with consistent growth and group them for later use;

[0105] (2) It will contain TRV1, TRV2, TRV2- NtWRKY69 , TRV2- PDS A single colony of Agrobacterium (refer to the previous construction process for other plasmid construction procedures) was inserted into 5 mL of LB medium (containing 50 mg / L kanamycin, 50 mg / L rifampicin), 28°C, 180 r / min o...

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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 field of bioengineering technology, and specifically relates to a NtWRKY69 Patent applications for genes and their applications. Background technique [0002] Tobacco is a plant of the genus Nicotiana in the Dicotyledon class Pipeflower order Solanaceae. Nicotiana ) There are more than 60 species, two of which are common tobacco (also known as safflower tobacco, Nicotiana tabacum ) Occupies the main area, Nicotiana tabacum ( Nicotiana rustica ) Has a small area. Cultivated tobacco can be divided into six types: flue-cured tobacco, sun-cured tobacco, air-cured tobacco, burley tobacco, oriental tobacco and yellow flower tobacco according to the characteristics of tobacco leaf quality, biological characteristics and cultivation methods. Among them, flue-cured tobacco is the most widely cultivated common tobacco. The planting area and total output of flue-cured tobacco in my country rank first in the world. [0003] As a leaf-use ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00
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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