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Application of rice genes cyp71a1 and 5-HT in regulating insect resistance in rice plants

A CYP71A1, insect resistance technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problem of unclear resistance mechanism, and achieve the effect of improving plant insect resistance and high insect resistance

Active Publication Date: 2019-07-09
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although more QTL positions have been discovered, judging from the number of cloned genes, it is still relatively small, and the resistance mechanism behind it is not clear

Method used

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  • Application of rice genes cyp71a1 and 5-HT in regulating insect resistance in rice plants
  • Application of rice genes cyp71a1 and 5-HT in regulating insect resistance in rice plants
  • Application of rice genes cyp71a1 and 5-HT in regulating insect resistance in rice plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 selectivity experiment

[0036] In this example, the rice mutant Jiazhe LM (Jiazhe lesion mimic) and the wild type Jiazhe B were used as materials for experiments; wherein, the mutant Jiazhe LM was obtained by using 350Gy 60 The wild-type rice Jiazhe B was obtained by Co irradiation mutagenesis; after detection, there was a G deletion in the gene CYP71A1 in the mutant genome.

[0037] 1. Resistance experiment of brown planthopper

[0038] a) Feeding selectivity and egg production: the wild-type rice and mutants were hydrocultured in the same culture container for the brown planthopper to feed freely (the results were as follows: figure 1 shown in a). The wild-type rice and the mutant were hydroponically cultured in the same culture container, and the stalks of the rice were surrounded by a gas-permeable glass cover (4 cm in diameter, 8 cm in height) (the two plants were in the same glass cover), and ventilated. 15 female brown planthoppers with eggs were...

Embodiment 2

[0054] Example 2 Effect of 5-hydroxytryptamine on insect resistance of rice

[0055] In order to explore whether the resistance of the mutants to N. lugens is related to the lack of 5-HT, the 5-HT content of rice materials known to carry resistance genes and BPH-sensitive materials was further determined.

[0056] The assay method of 5-hydroxytryptamine content in the plant body in the present embodiment is as follows:

[0057] (1) Sample pretreatment: Grind five grams of leaves, stems or root tissues in liquid nitrogen to powder, add 9mL of methanol, mix well at 10°C, shaker at 180rpm, fully extract for 30min, 13,500×g, 10°C After centrifuging for 15 minutes, 8 mL of supernatant can be drawn out, and 2 mL of distilled water is added according to the ratio of 1:4, and mixed. C18 SPE cartridges were activated with three bed volumes of methanol and rinsed with distilled water. Pass the water-added sample through the small column, and recover the effluent; then rinse it with 10...

Embodiment 3

[0068] In order to further verify the conclusions obtained in Example 1, we further used CRISPR technology to perform site-directed mutations on the gene CYP71A1 in the rice variety "Xindao No. 1" to obtain gene CYP71A1 silenced plants, namely rice plants CYP71A1-KO.

[0069] Specific steps are as follows:

[0070] According to the gene sequence, the target sequence was designed as 5'-TGGTCGCGTTGAGGAGGA GC-3', connected with the CRISPR vector pHun4c12, and transformed into Agrobacterium. After Agrobacterium-mediated transformation, T 0 Generation plants, mutants were discovered by sequencing. This results in the insertion of an "A" at 83 distances from the start codon, thereby forming a frameshift and causing a mutation. After breeding, the F1 generation was obtained and used for experiments.

[0071] The wild-type "Xindao No. 1" and the gene-silenced plant CYP71A1-KO were subjected to a feed selection experiment (the steps are the same as in Example 1). Depend on Figure...

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Abstract

The invention discloses application of a rice gene CYP71A1 and 5-hydroxytryptamine in regulating and controlling pest resistance of a rice plant. The application has the advantages that a pest resistance experiment is performed by a mutant induced by gamma-rays and a wild type, so that the rice plant with gene CYP71A1 mutation has higher pest resistance in comparison with the wild type, and the application of expression inhibition of gene CYP71A1 in improving the pest resistance of the rice plant is demonstrated; meanwhile, by regulating the change of the content of the 5-hydroxytryptamine in the plant, the pest resistance of the plant is improved by decreasing the content of the 5-hydroxytryptamine, and the application of inhibition of the 5-hydroxytryptamine in improving the pest resistance of the rice plant is demonstrated.

Description

technical field [0001] The invention relates to the fields of plant genetic engineering and rice molecular breeding, in particular to the application of rice genes CYP71A1 and 5-hydroxytryptamine in regulating rice plant insect resistance. Background technique [0002] Rice is an important food crop, especially for Asian countries, it is very important to ensure its stable production. Nilaparvata lugens is a piercing-sucking insect, which penetrates the leaf sheath to absorb the nutrients of rice through the piercing-sucking mouthparts. area of ​​death. In addition, rice planthoppers can also transmit a variety of rice virus diseases. [0003] At present, the control of rice planthoppers in this field is mainly chemical control, which leads to the increase of drug costs, the increase of pest resistance, the residue of harmful substances, and environmental pollution. [0004] In the study of N. lugens resistance, researchers have excavated more than 20 resistance sites, bu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/82A01H5/00A01H6/46
CPCC12N9/001C12N15/8213C12N15/8286C12Y103/03009
Inventor 芦海平舒庆尧富昊伟娄永根叶恭银张渭章
Owner ZHEJIANG UNIV
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