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Plant disease resistance gene NtWRKY50 and application thereof in bacterial wilt resistance of tobacco

A disease-resistant gene and plant technology, applied in the direction of plant gene improvement, application, plant peptides, etc., can solve the problems of lack of effective eradication measures, difficult disease control, and pathogenic bacteria resistance.

Pending Publication Date: 2018-01-23
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disease has been difficult to control due to the lack of effective eradication measures, continuous production on contaminated land, and the ability of the pathogen to survive for many years on wet soils, ponds, plant debris, or asymptomatic weed hosts
At present, the main method of preventing bacterial wilt is to use chemical pesticides and agricultural measures, but this brings many problems, such as polluting the environment, inducing pathogenic bacteria to develop resistance, etc.

Method used

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  • Plant disease resistance gene NtWRKY50 and application thereof in bacterial wilt resistance of tobacco
  • Plant disease resistance gene NtWRKY50 and application thereof in bacterial wilt resistance of tobacco
  • Plant disease resistance gene NtWRKY50 and application thereof in bacterial wilt resistance of tobacco

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Embodiment one: preparation of aseptic seedlings

[0035] In this embodiment, the wild-type tobacco cloud smoke seeds from Chongqing Tobacco Company are used, soaked in 75% alcohol solution for 1 minute, taken out and soaked in 10% sodium hypochlorite for 10 minutes, and then washed with sterilized water for 5 minutes. The second time, the seeds were sown on MS solid medium provided by Qingdao Haibo Biotechnology Co., Ltd., placed in an artificial climate box at 25-28°C, and cultivated in a climate chamber with a photoperiod of 16h light / 8h dark.

Embodiment 2

[0036] Example 2: Construction of NtWRKY50 gene overexpression vector and RNAi interference vector

[0037] Fluorescent real-time quantitative PCR: Extract RNA with the plant total RNA extraction kit provided by Tiangen Biochemical Technology Co., Ltd., synthesize cDNA with BIO-RAD cDNA Synthesis Kit, and use BIO-RAD, C1000Touch for fluorescent quantitative PCR (QRT-PCR) TM Thermal Cycler, SsoFAST TM Eva Green Supermix, the detection system is CFX96real-time system. The PCR amplification reaction system is 20 μl, containing 10 μl SYBR mix, 1 μl cDNA, 1 μl forward primer, 1 μl reverse primer and 7 μl H2O. Reaction conditions: 95°C for 3min, 40 cycles: 95°C for 10s, 55°C for 30s. Each PCR reaction was performed in three biological replicates and passed through 2 -ΔΔCt Methods to calculate gene expression. Internal reference genes were UBI3 and NtEF1α.

[0038] The expression vector used in the construction of the overexpression vector in this example is the pVCT2024 vecto...

Embodiment 3

[0040] Embodiment three: cultivating transgenic tobacco plants

[0041] Step 1: Streak the EHA105 bacterial solution containing the constructed plasmid of the NtWRKY50 gene on the YEB+Kan plate, culture it at 20°C for 48 hours, pick a single clone into 5mL YEB medium and culture it at 280rpm and 28°C for 48 hours, according to the ratio of 1:100 The ratio of ~1:50 was expanded to 50mL overnight, until the optical density OD=0.6, and then the bacteria were collected by centrifugation, suspended in 50mL MS liquid medium provided by Qingdao Haibo Biotechnology Co., Ltd. to obtain the infecting bacteria solution, and set aside.

[0042] Step 2: Transfer about 10 mL of the prepared Agrobacterium liquid to an empty sterile petri dish, remove the midrib and edge of the tobacco leaves, and cut them into uniform small pieces of 0.5-0.8 cm×0.5-0.8 cm; Transfer the leaves to the infecting bacteria solution, gently shake and dip for 5-8 minutes; clamp the leaf disk to the sterilized filte...

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Abstract

The invention relates to the technical field of crop disease control, in particular to a plant disease resistance gene NtWRKY50, and application of the plant disease resistance gene NtWRKY50 in bacterial wilt resistance of tobacco. A nucleotide sequence of the plant disease resistance gene NtWRKY50 is shown as SEQ ID NO. (sequence identifier number) 1. The gene NtWRKY50 is further transferred intothe tobacco for functional verification; a result shows that NtWRKY50 is a nuclear localization protein; expression of the gene is induced by pathogenic bacterium infection of plant bacterial wilt; overexpression of NtWRKY50 can improve the resistance of the tobacco to the bacterial wilt; silence of the gene NtWRKY50 has no significant influence on the plant disease resistance; NtWRKY50 can be induced by various biological and non-biological stresses; and the overexpression of NtWRKY50 significantly raises an SA level but inhibits JA accumulation induced by a pathogen. Data show that the overexpression of NtWRKY50 results in changes of SA and JA contents; the expression of defense related genes is increased; and the ralstonia solanacearum resistance of a plant is improved. A gene resourceis provided for cultivating a novel bacterial wilt resistance tobacco variety by a gene engineering manner, and the gene has an important application prospect.

Description

technical field [0001] The invention relates to the technical field of crop disease prevention and control, in particular to a plant disease resistance gene NtWRKY50 and its application in tobacco resistance to bacterial wilt. Background technique [0002] Ralstonia solanacearum is a soil-borne vascular disease caused by Ralstonia solanacearum (Ralstonia solanacearum). Ralstonia solanacearum is one of the most destructive plant pathogens in the world, ranking second among the top ten plant pathogenic bacteria. R. solanacearum infects more than 50 families and more than 450 species of plants, including Solanaceae crops, bananas, peanuts, ginger, ornamental plants and forest trees, among which Solanaceae crops are mainly represented by tomato, potato, tobacco, pepper, and new Varieties and new hosts are constantly being reported. Due to its wide range of hosts and geographical distribution, it causes serious economic losses (15%-95%) worldwide, especially in tropical develop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C12N15/66C07K14/415A01H5/00A01H6/82
Inventor 丁伟刘秋萍刘颖唐元满
Owner SOUTHWEST UNIVERSITY
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