Application of OsHin1 gene in prevention and control of gramineous meloidogyne

A root-knot nematode and genetic technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of short life cycle, difficult prevention and control, etc., and achieve the effect of broad application prospects

Active Publication Date: 2021-11-02
RICE RES INST GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the nematode has a wide range of hosts, a short life cycle and is very resistant to water, it is difficult to control it with conventional methods.

Method used

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  • Application of OsHin1 gene in prevention and control of gramineous meloidogyne
  • Application of OsHin1 gene in prevention and control of gramineous meloidogyne
  • Application of OsHin1 gene in prevention and control of gramineous meloidogyne

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Recognition of rice OsHin1 gene to nematode infestation

[0028] 1. Rice Culture and Nematode Inoculation

[0029] Wild-type Nipponbare rice grown for 2 weeks was taken, and each plant was inoculated with 200 root-knot nematodes of Gramineae, and rice roots not inoculated with nematodes were used as a negative control. The rice culture conditions are: temperature 28°C, light / dark cycle 16h / 8h.

[0030] 2. Rice total RNA extraction and cDNA synthesis

[0031] Nipponbare rice roots of each group were taken at different periods after inoculation, and total RNA was extracted.

[0032] The total RNA of rice was obtained using the RNAprep pure Plant Kit method, and the specific steps were as follows:

[0033] (1) Take 100 mg Nipponbare rice root and quickly grind it into powder in a mortar filled with liquid nitrogen, then transfer it to 450 μL RL solution (1% β-mercaptoethanol), and shake vigorously.

[0034] (2) Transfer the lysate to a CS filter column and ce...

Embodiment 2

[0052] Cloning and vector construction of embodiment 2 rice OsHin1 gene

[0053] 1. Cloning of OsHin1 gene

[0054] The reference for obtaining the rice cDNA is the same as in Example 1.

[0055] The full-length cDNA sequence of the OsHin1 gene contains a 675bp open reading frame (SEQ ID NO: 1), encoding 224AA (SEQ ID NO: 2).

[0056] Using rice cDNA as a template, the OsHin1 gene was amplified by the following specific primers:

[0057] OsHin1-BamH1-F: ATGAGCAAGGAGAAGCAC;

[0058] OsHin1-Pst1-R: CACCTCGACGCTGCACCC.

[0059] 2. Vector construction of OsHin1 gene

[0060] The 35s promoter in pCAMBIA1305 was replaced with the maize ubiquitin promoter Ubi by double enzyme digestion to obtain the pCAMBIA1305:Ubi rice expression vector. The OsHin1 fragment containing the BamH1 / Pst1 restriction site amplified in step 1 is connected into the pCAMBIA1305:Ubi expression vector to obtain the overexpression vector pCAMBIA1305.1:Ubi:OsHin1( image 3 ).

[0061] Mutation targets CAA...

Embodiment 3

[0062] Example 3 Rice OsHin1 Gene Overexpression and Transformation of Mutant Plants

[0063] The wild-type Nipponbare rice callus was transformed by carrying the overexpression vector of Example 2 and the gene knockout vector in Agrobacterium, respectively.

[0064] After antibiotics and PCR screening, 12 rice positive transformed plants with OsHin1 gene mutation and 25 rice positive transformed plants with OsHin1 gene overexpressed were obtained.

[0065] Sequencing analysis was performed on the OsHin1 gene knockout lines, and three homozygous lines were obtained for later experiments ( Figure 6 ). At the same time, qRT-PCR checks the transcription of the OsHin1 gene in the mutant rice, and the results are shown in the figure Figure 7 As shown in A.

[0066] Finally, through qRT-PCR analysis, three overexpressed plants with higher OsHin1 gene expression were selected for later experiments ( Figure 8 A).

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Abstract

The invention discloses application of an OsHin1 gene in prevention and control of gramineous meloidogyne, and belongs to the technical field of plant disease prevention and control. The nucleotide sequence of the OsHin1 gene is as shown in SEQ ID NO. 1, and the coded amino acid sequence is as shown in SEQ ID NO. 2. Overexpression of the OsHin1 gene can reduce infection of the gramineae meloidogyne, and knockout of the OsHin1 gene can improve infection of the gramineae meloidogyne, so that the OsHin1 gene can be applied to preparation of products for preventing and controlling the gramineae meloidogyne and cultivation of rice capable of resisting the gramineae meloidogyne.

Description

technical field [0001] The invention relates to the technical field of plant disease prevention and control, and more specifically relates to the application of the OsHin1 gene in the prevention and control of root-knot nematode Pseudomonas graminearum. Background technique [0002] Rice is the staple food for more than half of the world's population. Meloidogyne.graminicola is one of the most serious plant parasitic nematodes in rice, which can cause up to 87% loss of rice yield. M. graminearum is widely distributed, especially in the main rice producing areas of the world, such as India, Nepal, Myanmar, Bangladesh, Laos, Georgia, Libya, Thailand, Philippines, Indonesia, Pakistan, Singapore, Vietnam, etc. Both countries and regions have been reported. In recent years, root-knot nematode graminaceae has been found successively in Guangdong, Guangxi, Fujian, Yunnan, Hunan, Sichuan and Jiangxi in my country, and has caused serious damage in some areas, threatening the safety...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8285
Inventor 陈建松李治文
Owner RICE RES INST GUANGDONG ACADEMY OF AGRI SCI
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