Preparation method of plastid transgenic plant for preventing and treating Myzus persicae (Sulzer), and application of plastid transgenic plant in prevention and treatment of Myzus persicae (Sulzer)

A technology of transgenic plants, Myzus persicae, applied in the field of biology

Active Publication Date: 2021-09-10
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although nuclear transfer plant nuclear-mediated RNAi technology has made great progress in the control of non-chewing pests of Hemiptera such as green peach aphid, non-chewing pests of Hemiptera

Method used

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  • Preparation method of plastid transgenic plant for preventing and treating Myzus persicae (Sulzer), and application of plastid transgenic plant in prevention and treatment of Myzus persicae (Sulzer)
  • Preparation method of plastid transgenic plant for preventing and treating Myzus persicae (Sulzer), and application of plastid transgenic plant in prevention and treatment of Myzus persicae (Sulzer)
  • Preparation method of plastid transgenic plant for preventing and treating Myzus persicae (Sulzer), and application of plastid transgenic plant in prevention and treatment of Myzus persicae (Sulzer)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The acquisition of embodiment 1 Dhc64C gene

[0036] 1. Green peach aphids are raised in an artificial environment at 25±1°C, 75-80% relative humidity, and 16L:10D photoperiod. The cDNA sequence of the green peach aphid Dhc64C gene used for dsRNA synthesis is shown in SEQ ID NO: 1; the CDS interval sequence of the green peach aphid Dhc64C gene is shown in SEQ ID NO: 2.

[0037] The sequence of specific primers for Dhc64C gene of green peach aphid is as follows:

[0038] Upstream primer F: 5'-CTCATGTAATTGATCCAAAAGCCAT-3', SEQ ID NO.3;

[0039] Downstream primer R: 5'-TCGAACATTGGGTGGAAGAGAT-3', SEQ ID NO.4.

[0040] 2. The total RNA of peach aphid was extracted with RNAiso Plus reagent from Takara Company.

[0041] 3. Reverse transcription reaction

[0042] Residual Genomic DNA Removal

[0043] Prepare the following mixture in an RNase free centrifuge tube, mix well by microcentrifugation, and incubate at 42°C for 2 minutes.

[0044]

[0045] Reverse transcriptio...

Embodiment 2

[0063] The construction of embodiment 2 plant expression vectors

[0064] 1. pUC-Dhc64C 1st intermediate vector construction

[0065] 1.1 Double digestion of vector fragments

[0066] The pUC-RNAi intermediate vector was used to introduce the intron structure. pUC-RNAi is the previous scientific research achievement of our laboratory, which was published in the article Full crop protection from an insect pest by expression of long double-stranded RNAs in plastids in the journal Science in 2015. Dhc64C-1 st The (XhoI / BglII) fragment and the intermediate vector pUC-RNAi vector were subjected to XhoI / BglII double enzyme digestion, and the reaction was carried out for 3 hours. The system was as follows:

[0067]

[0068] Carry out sample loading and electrophoresis for the enzyme digestion system of the intermediate carrier, observe the target bands in the UV gel imaging system, and perform gel recovery of the target fragments, the method is the same as above. Perform solu...

Embodiment 3

[0100] The preparation of the plant of embodiment 3 plastid transformation

[0101] 1. Plant material preparation:

[0102] Tobacco seeds were cultivated in a sterile environment, temperature: 25°C / 20°C, photoperiod: 16h light / 8h dark, light intensity: 25μE. When the plant reaches 1 / 2 to 1 / 3 of the height of the bottle, take 2-4 pieces of the youngest leaves and spread them on RMOP (antibiotic-free) plastic plates with the stomata facing up (the plates should be thinner and contain about 30 mL of culture) base).

[0103] 2. Preparation for gene gun bombardment: the main steps are:

[0104] 1) Turn on the vacuum pump of the particle gun half an hour before use (adjust the air pressure to 1350psi);

[0105] 2) Sterilized large glass plate; absolute ethanol; each gun: 1 cleavable membrane (1100psi) and 7 micro slides; 1 blocking net for each plasmid;

[0106] 3) Wash the splittable membrane (less than 1 min), micro slide (5 min), blocking net and gene gun parts (5 min) with a...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation method of a plastid transgenic plant for preventing and treating Myzus persicae (Sulzer), and application of the plastid transgenic plant in prevention and treatment of the Myzus persicae (Sulzer). The invention confirms that the Myzus persicae (Sulzer) eats plant plastids, after a Myzus persicae (Sulzer) RNAi lethal gene MpDhc64C is obtained through screening, a tobacco plastid transformation vector for expressing dsMpDhc64C is constructed, and plastid transgenic tobacco is obtained through transformation. After the Myzus persicae (Sulzer) eats the transgenic plant, the expression level of a target gene is obviously reduced, the survival rate and the number of later generations are obviously reduced, and the weight of the survived Myzus persicae (Sulzer) is obviously reduced so as to indicate that dsRNA expressed by plant plastid successfully enters the Myzus persicae (Sulzer) body and triggers the RNAi effect, so that the target gene of the insect is down-regulated, and various adverse symptoms aiming at the Myzus persicae (Sulzer) are triggered. The Myzus persicae (Sulzer) resistance of the plant is remarkably improved, and a new technical scheme is provided for Myzus persicae (Sulzer) prevention and treatment.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing a plastid transgenic plant for controlling peach aphid and its application in preventing and controlling peach aphid. Background technique [0002] Insect pests can be divided into chewing pests and non-chewing pests according to the type of mouthparts. Many non-chewing pests belong to Hemiptera, such as aphids, whiteflies, brown planthoppers, mirid bugs, etc. Most of them are important agricultural pests and cause great harm to agricultural production. [0003] Green peach aphid is a typical piercing-sucking pest belonging to the Hemiptera Aphididae, and it is also one of the most harmful agricultural pests in the world. It hosts more than 400 species of plants, including peaches, plums, cherries, potatoes, cabbage, Cabbage, radish, sweet pepper, brassica, tobacco and many other important economic crops. Green peach aphids will carry out asexual ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/82
CPCC12N15/8218C12N15/8286C12N15/113C12N2310/14
Inventor 张江董毅张琪杨勇王子灿常玲
Owner HUBEI UNIV
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