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A kind of Trichoderma hydrophobin thfb6 and its strain, plant ISR elicitor and excitation method

A hydrophobin, Trichoderma technology, applied in the field of molecular biology, can solve the problem of poor accumulation of signal transmission research

Active Publication Date: 2022-04-19
新疆西研生物防控科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The accumulation of research on signal transduction of plant ISR induced by Trichoderma elicitors is relatively poor

Method used

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  • A kind of Trichoderma hydrophobin thfb6 and its strain, plant ISR elicitor and excitation method
  • A kind of Trichoderma hydrophobin thfb6 and its strain, plant ISR elicitor and excitation method
  • A kind of Trichoderma hydrophobin thfb6 and its strain, plant ISR elicitor and excitation method

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0094] Experimental example 1. Cloning, heterologous expression and purification of hydrophobin Thfb6 gene cDNA

[0095] 1. Extraction of total RNA from Trichoderma Harzianum NM11 and synthesis of cDNA

[0096] Inoculate Harzwood wild fungus NM11 on PDA medium, culture at a constant temperature of 28°C for 3 days, use a puncher with a diameter of 0.5cm to take out the fungus cake, and inoculate 3 to 5 fungus cakes into a conical flask containing 100mL PD liquid medium cultured at 28°C for 48 h with constant temperature and shaking, the mycelia were collected, and the total RNA was extracted using an RNA extraction kit (TRNzol Universal Reagent), and the quality of the RNA was detected by 1% agarose gel electrophoresis ( figure 1 ). The first strand of cDNA was synthesized using Tiangen FastQuant RT Kit (With gDNAase).

[0097] 2. Cloning, sequencing and analysis of hydrophobin gene Thfb6 cDNA

[0098] According to the previously obtained Thfb6 gene sequence, design hydropho...

experiment example 2

[0104] Experimental example 2, hydrophobin induces tobacco early defense response

[0105] 1. Hydrophobin induces cell necrosis in tobacco leaves

[0106] Three weeks after sowing tobacco (N.tabacum cv.Samsun NN), a single plant was transplanted into a single small pot, and cultivated in a light incubator at a constant temperature of 25°C. After growing for 6 weeks, healthy tobacco seedlings were selected, and the above-mentioned Thfb6 recombinant protein solution (i.e. pH 8.0, 20mM Tris-HCl solution containing Thfb6 recombinant protein with a concentration of 20 μmol / L) was used as the experimental group, and was gently injected into the blade at the back of the tobacco leaf with a 1mL needle-free disposable syringe. To observe the response of tobacco leaves, the control group was an equal amount of buffer solution (ie, pH 8.0, 20 mM Tris-HCl solution). Observation after 24h, Thfb6 can cause necrosis of tobacco leaf cells ( Figure 5 ).

[0107] Preparation of buffer solut...

experiment example 3

[0119] Experimental example 3, Thfb6 induces stress resistance enzyme activity of tobacco leaves

[0120] 1. Thfb6 induces increased activity of tobacco phenylalanine ammonia-lyase

[0121] Phenylalanine ammonia-lyase (phenylalaninammo-nialyase, PAL) is a key enzyme in the phenylpropane metabolic pathway in plants, and its activity controls various secondary phenolic compounds, flavonoid phytoalexin, lignin and other antibacterial substances in plants Therefore, PAL plays an important role in plant secondary metabolism and disease resistance metabolism, and is a key enzyme in plant defense response. Studies have found that when many plants suffer from external damage, the plant's defense system, especially the metabolism of phenylpropanoids, is activated, and the activity of PAL rises rapidly. Therefore, PAL activity can be used as a physiological indicator of plant resistance and defense.

[0122] 2.5-month-old tobacco plants were selected, and the tobacco leaves were treate...

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Abstract

The present invention "a trichoderma hydrophobin Thfb6 and its strain, plant ISR elicitor and excitation method" belongs to the field of molecular biology. The amino acid sequence of the Trichoderma hydrophobin Thfb6 is shown in SEQ ID NO.1. Based on the Trichoderma hydrophobin Thfb6, the present invention also provides a plant ISR elicitor and its stimulating method, a TMV resistance inducer and its inducing method. The trichoderma hydrophobin Thfb6 of the present invention can cause active oxygen bursts in plant leaves, induce cell extracellular alkalization, callose and phenolic substance accumulation and other early defense reactions, and improve stress resistance enzyme activities such as PAL, POD and PPO in leaves Improve and induce the expression of disease process-related protein gene PR‑1b, systemic resistance marker gene NPR1 and defense response key enzyme gene PAL, and induce tobacco to improve disease resistance to tobacco mosaic disease, and at the same time induce cucumber resistance to Fusarium wilt sex.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a Trichoderma hydrophobin Thfb6 and its strain, a plant ISR elicitor and an eliciting method. Background technique [0002] Trichoderma spp. is a widely studied and applied plant disease biocontrol fungus. It is easy to colonize the plant rhizosphere and establish beneficial interactions with plants. Inducing plant disease resistance is one of the main disease control mechanisms of Trichoderma. As early as 1997, Bigirimana et al. first proved that Trichoderma can induce plant disease resistance, and later found that a variety of Trichoderma can induce local or systemic resistance of plants to pathogens. Its disease resistance signal transmission pathway is very complex, with multiple signaling pathways involved, and the signal regulatory factors in each pathway interact to form a complex signaling network. [0003] Substances of biotic and abiotic origin capable of i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/37C12N15/31C12N1/14A01N47/44A01P1/00A01P3/00A01P21/00C12R1/885
CPCC07K14/37A01N47/44C12R2001/885C12N1/145
Inventor 李梅蒋细良黄佩梅杰田莹
Owner 新疆西研生物防控科技有限公司
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