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Application of rice OsBBTI4 protein gene to improving resistance of rice to rice blast

A technology of resistance to rice blast and rice, applied in the field of genetic engineering, can solve the problems that no research on the biological function of pathogenic fungi has been reported, and there are few studies on trypsin inhibitors, so as to improve plant defense ability and plant disease resistance , Improve the effect of resistance

Active Publication Date: 2018-09-11
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, there are few studies on trypsin inhibitors in rice, and there are no reports on the biological functions of trypsin inhibitors in rice to pathogenic fungi.

Method used

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  • Application of rice OsBBTI4 protein gene to improving resistance of rice to rice blast
  • Application of rice OsBBTI4 protein gene to improving resistance of rice to rice blast
  • Application of rice OsBBTI4 protein gene to improving resistance of rice to rice blast

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Experimental program
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Effect test

Embodiment 1

[0025] 1) Extraction of total RNA

[0026] The japonica rice variety Nipponbare was selected, and when the rice seedlings grew to about two weeks, the leaves were immediately frozen in liquid nitrogen and stored in a -80°C refrigerator. Take part of the leaves, grind them with a mortar, transfer them into a 1.5ml EP tube filled with Trizol lysate, shake fully, extract the total RNA, and identify the quality of the total RNA by electrophoresis.

[0027] 2) Cloning of rice trypsin inhibitor protein gene OsBBTI4 and construction of plant expression vector

[0028] Design primers at both ends:

[0029] P1: 5-ATGAGCAACACCACCATGGC-3 (SEQ ID No. 3),

[0030] P2: 5-CTAGTTCTCCGCTCGGGGTT-3 (SEQ ID No. 4)

[0031] The total RNA obtained in step 1) was reverse-transcribed to synthesize the first strand of cDNA, which was used as a template for PCR amplification with high-fidelity Pfu enzyme. The PCR program was as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C fo...

Embodiment 2

[0035] Identification of disease resistance of OsBBTI4-OX transgenic lines:

[0036] Punch inoculation: When the transgenic rice grows to 5-8 weeks of leaf age, select the second leaf to inoculate with blast fungus, then put the rice in the plant growth box for 24 hours in the dark, then transfer to 12 hours of light, 12 hours Grow under dark conditions for 10-14 days; observe the expansion of the lesion, investigate the disease state, and count the area and biomass of the lesion. Compared with the control, the transgenic rice plant with obvious resistance is the obtained blast-resistant transgenic plant.

[0037] The results showed that OsBBTI4 overexpression transgenic plants had enhanced disease resistance to Magnaporthe oryzae physiological race RB22 compared with wild type (WT) ( image 3 ), at the same time, the relative lesion area (Table 1) and relative fungal biomass (Table 2) of transgenic lines and wild-type diseased leaves were counted. About 50% of ( Figure 4 )...

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Abstract

The invention discloses application of a rice trypsin inhibitor protein gene OsBBTI4 to improving the resistance of rice to rice blast. The cDNA nucleotide sequence of the rice trypsin inhibitor protein gene OsBBTI4 is shown as SEQ ID No. 1 or the degenerate sequence of SEQ ID No. 1. A transgenosis experiment proves that the overexpression of the gene improves the resistance of rice to rice blast.Therefore, the gene can serve as a target gene to be transferred into a plant, thus the plant disease resistance can be improved, and plant species improvement can be conducted. The transferred trypsin inhibitor can also protect other proteins from being degraded by inhibiting hydrolysis of protease, and accordingly the plant defense capability is effectively improved.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the application of a rice OsBBTI4 protein gene in improving rice blast resistance. Background technique [0002] Trypsin inhibitor (Trypsin inhibitor, TI) is ubiquitous in animals, plants and microorganisms in nature. At present, the trypsin inhibitors that have been studied more mainly come from the main grains of human beings, such as legumes and choline, and have also been reported in crucifers. In the animal kingdom, trypsin inhibitors also exist in the blood, pancreas, and microorganisms such as yeast and Streptomyces. [0003] Protease inhibitors can be divided into four categories: serine, cysteine, aspartic acid, and metalloproteinase inhibitors. Trypsin inhibitors belong to the serine proteinase inhibitor (Serpin) family. Among them, the most extensive and in-depth studies are Kunitz type inhibitors and Bowman-Birk type inhibitors, namely Kunitz type Try...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/46C07K14/81C12N15/29
CPCC07K14/8117C12N15/8282
Inventor 宁约瑟张重阳
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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