Chloroplast locating gene ToxABP1-V and its application

A toxabp1-v, gene technology, applied to the chloroplast localization gene ToxABP1-V and its application fields, can solve the problems of increasing manpower, material resources, environmental pollution and the like

Active Publication Date: 2018-07-31
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Powdery mildew can be controlled by fungicides, but chemical control will inevitably increase manpower and material resources, and will also cause ecological problems such as environmental pollution. Therefore, discovering disease-resistant genes and cultivating disease-resistant varieties are effective measures to prevent and control wheat powdery mildew

Method used

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  • Chloroplast locating gene ToxABP1-V and its application
  • Chloroplast locating gene ToxABP1-V and its application
  • Chloroplast locating gene ToxABP1-V and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Screening of CMPG1-V interacting proteins by yeast two-hybrid and cloning of ToxABP1-V gene

[0024] CMPG1-V is a powdery mildew resistance-related gene located on diploid T. villosa 6V cloned by the Institute of Cytogenetics, Nanjing Agricultural University (references: Zhu, Y., Li, Y., Fei, F., Wang , Z., Wang, W., Cao, A., Liu, Y., Han, S., Xing, L., Wang, H., Chen, W., Tang, S., Huang, X., Shen ,Q.,Xie,Q.,and Wang,X..E3ubiquitin ligase geneCMPG1-V from Haynaldia villosa L.contributes topowdery mildew resistance in common wheat(Triticum aestivum L.).The PlantJournal.2015.84,154-168), with CMPG1-V was used as bait, and the yeast two-hybrid cDNA library was screened by polyethylene glycol-lithium acetate transformation method to obtain an interacting gene of CMPG1-V ( figure 1 ). The interaction gene was sequenced, and the sequence encoded a C-terminal 186 amino acids of ToxinA binding protein1, and homologous cloning primers P1 (CAATAGCCTTTCCTCGCACCTG, SEQ...

Embodiment 2

[0025] Example 2 ToxABP1-V gene is induced by powdery mildew expression characteristics

[0026] The powdery mildew-resistant wheat seeds (references: Qi Lili, Chen Peidu, etc., New source of resistance to powdery mildew in wheat—gene Pm21, Acta Crops, 1995, 21(3):257-262) were sown in a petri dish to germinate , transplanted to pots after dew whitening (isolated with cylindrical transparent plastic sheets around, and closed with filter paper at the top to form an environment without powdery mildew). At the three-leaf stage, shake off the fresh spores of Nanjing local mixed powdery mildew cultured on the susceptible variety Sumai No. 3 on the seedlings of A. After being inoculated with powdery mildew, the wheat tufting continued to be cultured at 16°C. Samples were taken at 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 48h, and 72h after inoculation, and stored in a -70°C refrigerator for later use. TRIZOL (Invitrogen) was used to extract RNA from the leaves of A. villosa induc...

Embodiment 3

[0030] Example 3 Using single cell silencing method to transfer ToxABP1-V gene RNAi vector into wheat leaves

[0031] The single-cell transient silencing method is a reliable and rapid method for identifying gene functions (reference: Schweizer, Pokorny et al. A Transient Assay System for the Functional Assessment of Defense-Related Genes in Wheat Molecular Plant-MicrobeInteractions.1999,12:647 -654). In this study, the single-cell instantaneous silencing method was used to wrap the plasmid DNA on the outer layer of metal particles, and the metal particles were bombarded to the epidermal cells of wheat leaves with the help of a gene gun. It has powdery mildew resistance function.

[0032] The procedure for encapsulating carrier DNA and metal particles is as follows:

[0033] Preparation of tungsten powder: Weigh 30mg of tungsten powder into a 1.5ml eppendorf tube, add 1ml of 70% alcohol, vortex for 3-5min and then let stand for 15min to completely precipitate the gold powder...

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Abstract

The invention discloses a chloroplast locating gene ToxABP1-V and its application. The cDNA sequence of ToxABP1-V is SEQ ID No. 1, and an amino acid sequence coded by ToxABP1-V is SEQ ID No. 3. The gene is derived from diploid Haynaldia villosa, can interact with the powdery mildew resistance-related gene CMPG1-V, and undergoes down-regulated expression in the powdery-mildew-resistant diploid Haynaldia villosa under the induction of powdery mildew. The RNAi silencing vector pWMB006-ToxABP1 of the gene is used for transforming the susceptible wheat variety Yangmai 158 by using single-cell transient silencing techniques, and results show that transient silencing of ToxABP1 can reduce the haustorium index of Yangmai 158. Three positive RNAi transgenic plants are obtained by using transgenic technology, and the plants show moderate and high resistance to powdery mildew of wheat. Therefore, ToxABP1-V is expected to be used for genetic engineering breeding.

Description

technical field [0001] The invention belongs to the field of genetic engineering and discloses a chloroplast positioning gene ToxABP1-V and its application. Background technique [0002] Wheat powdery mildew, caused by the obligate parasitic fungus Blumeria graminis DC f.sp.tritici, is one of the most serious leaf diseases of wheat (Triticum aestivum L., 2n=6x=42, genome AABBDD). Occurs widely in China and many other countries. The disease can occur throughout the growth period of wheat, and can infect all parts of aboveground wheat plants, mainly leaves and leaf sheaths. [0003] With the influence of cultivars, cultivation conditions and climatic conditions, the harm of wheat powdery mildew in my country is increasing. Powdery mildew can be controlled with fungicides, but chemical control will inevitably increase the input of manpower and material resources, and will also cause ecological problems such as environmental pollution. Therefore, discovering disease-resistant ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8218C12N15/8282
Inventor 王秀娥王宗宽李颖波樊安琪张恒赵佳王海燕肖进曹爱忠邢莉萍
Owner NANJING AGRICULTURAL UNIVERSITY
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