Application of autophagy gene of tomatoes in improvement on root-knot nematode resistance of plants

A technology of autophagy genes and root-knot nematodes, applied in the fields of application, plant peptides, plant products, etc., can solve the problems of lack and less regulation of plant autophagy

Active Publication Date: 2021-02-05
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the research on plant autophagy is mainly in the model plant Arabidopsis thaliana, and focuses on the identification and functional analysis of autoph

Method used

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  • Application of autophagy gene of tomatoes in improvement on root-knot nematode resistance of plants
  • Application of autophagy gene of tomatoes in improvement on root-knot nematode resistance of plants
  • Application of autophagy gene of tomatoes in improvement on root-knot nematode resistance of plants

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] RKN treatment induces autophagy in wild-type tomato

[0091] RKN treatment of wild-type tomato, the specific method is as follows: when the tomato grows to five leaves and one heart, inoculate nematode treatment, each plant is inoculated with about 1000 J2 stage nematodes, and water normally during this period.

[0092] a. Take tomato root samples at 0h, 24h, 36h, 48h, and 72h after inoculation, stain with acid fuchsin, and count the phenotype and invasion number of each root. figure 1 A is the RKN invasion in each root tip after staining with acid fuchsin Bar=100 μm; figure 1 B is the number of RKN invaded in each root.

[0093] b. After inoculation 0h, 24h, 36h, 48h, 72h, take tomato root samples, extract protein, and perform Western Blot. The specific method is as follows:

[0094] 1) Western Blot

[0095] Take 0.3g protein sample in a 1.5mL centrifuge tube, put steel balls into it, grind it into powder in a sample mill, add an appropriate amount of protein extrac...

Embodiment 2

[0103] Construction and Detection of Tomato ATG10 Overexpression Plants

[0104] 1. Tomato Total RNA Extraction

[0105] Adopt Tiangen Plant total RNA extraction kit to extract the total RNA of tomato tender root, and its steps are:

[0106] (1) Take 0.1g tomato root sample and grind it in liquid nitrogen, add 1mL lysate RZ, and vortex to mix;

[0107] (2) Place at room temperature for 5 minutes to completely separate the nucleic acid-protein complex;

[0108] (3) Centrifuge at 12,000 rpm for 5 minutes at 4°C, remove the supernatant, and transfer to a new RNase-free centrifuge tube;

[0109] (4) Add 200 μL of chloroform, cover the tube cap, shake vigorously for 15 seconds, and place at room temperature for 3 minutes;

[0110] (5) 4°C, 12000rpm centrifuge for 10min, the sample will be divided into three layers: yellow organic phase, middle layer and colorless aqueous phase, RNA is mainly in the upper aqueous phase, the volume of the aqueous phase is about the lysate RZ reage...

Embodiment 3

[0152] Construction and detection of tomato atg10, atg4, atg6, atg7 mutant plants

[0153] 1. Tomato Total RNA Extraction

[0154] The total RNA of tomato tender roots was extracted using Tiangen Plant total RNA extraction kit, and the steps were the same as the above-mentioned total RNA extraction method.

[0155] 2. Gene cloning and construction of Agrobacterium tumefaciens engineering bacteria

[0156]Design the target sequences of tomato ATG10, ATG4, ATG6, and ATG7 genes on the CRISPR-P website (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR), and design primers to delete and edit large fragments of genes sgRNA-atg10-F1 (SEQ ID NO: 11), sgRNA-atg10-R1 (SEQ ID NO: 12), sgRNA-atg10-F2 (SEQ ID NO: 13), sgRNA-atg10-R2 (SEQ ID NO: 14 ). The remaining ATG4, ATG6, and ATG7 were designed in the same way, and the primer sequences were: sgRNA-atg4-F1, sgRNA-atg4-R1, sgRNA-atg4-F2, sgRNA-atg4-R2 (SEQ ID NO: 15-18) ; sgRNA-atg6-F1, sgRNA-atg6-R1, sgRNA-atg6-F2, sgRNA-atg6-R2 (SEQ ID NO: 19...

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Abstract

The invention discloses application of an autophagy gene ATGs of tomatoes in improvement on root-knot nematode resistance of plants. The application comprises the steps: (1) constructing a root-canceragrobacterium tumefaciens engineering strain A containing a tomato ATG10 gene overexpression vector; (2) mediating the root-cancer agrobacterium tumefaciens engineering strain A to convert an explantof a target plant, so as to obtain an ATGs-gene-overexpressed plant; and (3) subjecting the transgenic plant to root-knot-nematode-inoculated stress treatment, and observing autophagy changes, a root-knot quantity and a phenotype. Researches of the invention discover that Southern root-knot nematode infections can rapidly induce expression of autophagy genes in tomato root systems, the sensitivity to the root-knot nematodes is improved by mutants, and the sensitivity is lowered due to ATG10 overexpression. Namely, autophagy plays a role in root-knot nematode resistance of the tomatoes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of a tomato autophagy gene in improving plant root-knot nematode resistance. Background technique [0002] In recent years, with the steady development of my country's economy, modern agriculture has been paid more and more attention. The facility horticulture has developed rapidly, the planting area of ​​facility vegetables has increased rapidly, the multiple cropping index has continued to increase, the phenomenon of repeated cropping of vegetables has become more and more common, and the problem of continuous cropping in soil is serious. Continuous cropping obstacles are very harmful, which will lead to aggravation of pests and diseases, soil diseases, allelopathy, etc., especially the root-knot nematodes (Root-knot nematodes; RKN; Meloidogyne spp.), which is extremely hidden and dangerous, is becoming more and more serious. Technical bottlenecks in the development o...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/84C12N15/66C12N15/29A01H5/00A01H6/82
CPCC12N15/8239C12N15/8285C12N15/66C07K14/415
Inventor 周杰邹金萍喻景权
Owner ZHEJIANG UNIV
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