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Preparation method of a plastid transgenic plant for controlling peach aphid and its application in controlling peach aphid

A technology for transgenic plants and peach aphid, which is applied in the biological field and achieves the effect of low cost and environmental friendliness

Active Publication Date: 2022-04-08
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 a plastid transgenic plant for controlling peach aphid and its application in controlling peach aphid
  • Preparation method of a plastid transgenic plant for controlling peach aphid and its application in controlling peach aphid
  • Preparation method of a plastid transgenic plant for controlling peach aphid and its application in controlling peach aphid

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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 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. The invention confirms that the green peach aphid feeds on plant plastids, and after screening and obtaining the deadly gene MpDhc64C of the green peach aphid RNAi, constructs a tobacco plastid transformation vector expressing dsMpDhc64C, and transforms and obtains plastid transgenic tobacco. After the peach aphid ate the transgenic plants, the expression level of the target gene was significantly down-regulated, the survival rate and the number of offspring were significantly reduced, and the weight of the surviving peach aphid was significantly reduced, indicating that the dsRNA expressed by the plant plastid successfully entered the body of the peach aphid and triggered the RNAi effect, which in turn led to insect target. The down-regulation of the gene causes a variety of adverse symptoms against the green peach aphid, and the invention significantly improves the resistance of the plant to the green peach aphid, and provides a new technical solution for the control of the green peach aphid.

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 Patents(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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