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Rice source insect-resistant related gene OsHR1 as well as encoded product and application thereof

A technology of gene encoding and transgenic plants is applied to the rice-derived insect resistance-related gene OsHR1 and its encoded products and application fields, which can solve the problems of not effectively curbing pest damage, environmental pollution, and accelerating the evolution of pest resistance.

Active Publication Date: 2015-03-25
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, facts have proved that the extensive use of pesticides has not only failed to effectively curb pest damage, but has caused more serious environmental pollution and accelerated the evolution of pest resistance

Method used

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  • Rice source insect-resistant related gene OsHR1 as well as encoded product and application thereof
  • Rice source insect-resistant related gene OsHR1 as well as encoded product and application thereof
  • Rice source insect-resistant related gene OsHR1 as well as encoded product and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1, OsHR1 Gene cloning and sequence analysis

[0024] (1) Extraction of total RNA from rice stems and synthesis of cDNA

[0025] Weigh 100 mg of ground rice stalks (damaged by SSB), use SV Total RNA Isolation System (Promega) to extract total RNA and detect its concentration and purity; 1 μg of total RNA is used for cDNA synthesis (PrimeScript? RT-PCR Kit, TaKaRa), please refer to the product manual for specific operation.

[0026] (2) OsHR1 gene cloning

[0027] Using the above cDNA as a template, amplify the cDNA containing the 5' and 3' non-coding regions OsHR1 Fragments, reaction systems and procedures refer to PrimeSTAR ? HS DNA Polymerase (TaKaRa) instructions. The amplified product was detected by electrophoresis ( figure 1 ), recovered and purified using AxyPrep? DNA Gel Extraction Kit (Axygen); the purified fragments were ligated into pMD19-T vector (TaKaRa) and transformed into E. coli TG1 cells; positive clones were selected for preservati...

Embodiment 2

[0032] Embodiment 2, after the rice stem borer and brown planthopper damage OsHR1 Expression feature analysis

[0033] (1) Treatment of Chilo suppressalis: Insert one 3rd instar SSB larvae into the base of the rice stem, and start timing after the SSB bores. h Cut off 2-3 cm stalks from the damaged part, immediately immerse them in liquid nitrogen, and store them at -80°C for later use; healthy rice stalks not connected to SSB were used as a control.

[0034] (2) Treatment of brown planthopper: A cylindrical glass cover (4 cm in diameter, 8 cm in height, 48 air-ventilating holes with a diameter of 0.8 mm evenly distributed on the wall of the tube) was fixed at the base of the rice stem, and 15 The head of BPH pregnant female adults, the top is sealed with sponge; 0, 0.5, 1, 2, 4, 8, 12, 24, 48 hours after treatment, the outer leaf sheath of the damaged part is cut off, immediately immersed in liquid nitrogen, and stored at -80 °C Standby; take the healthy rice leaf sheath put ...

Embodiment 3

[0043] Embodiment 3, OsHR1 Obtaining rice lines with gene overexpression

[0044] (1) Vector construction

[0045] Design primers HR1-F3 and HR1-R3 for amplification OsHR1 The ORF fragment, the product was cla I and Bam H Ⅰ After double enzyme digestion, insert the CaMV35S promoter of the expression vector pCAMBIA1301 ( image 3 ), and transferred to Agrobacterium strains for preservation and sequencing. The primer information is as follows:

[0046] HR1-F3 (SEQ ID No. 11): 5'-CC ATCGAT ATGACTATAGAACTTCTT-3'

[0047] HR1-R3 (SEQ ID No.12): 5'-CG GGATCC TCACGGCGAGGCCGCTG-3'

[0048] (2) Plant transformation

[0049] After the rice seeds were dehulled and sterilized, they were placed in NBD medium at 28°C and cultured in the dark; 10 days later, the generated callus was peeled off and co-cultivated with the above-mentioned Agrobacterium on NBDC; 3 days later, the callus was washed away For excess Agrobacterium, the co-infected callus was screened on NBDS medium co...

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Abstract

The invention discloses a rice source insect-resistant related gene OsHR1 as well as an encoded product and application thereof. The OsHR1 has a DNA sequence shown in SEQ ID No.1, and the length is 1019bp; the complete encoding frame of the gene consists of 81st-posiion nucleotide to 875th-posiiont nucleotide in SEQ ID No.1; small molecular weight protein of 264 amino acid residues can be encoded. The research results show that the OsHR1 is closely related to the insect resistance of rice, and the resistance of rice to chilo suppressalis and brown planthopper is improved due to an expression product of the gene. The rice source insect-resistant related gene OsHR1 can be widely applied to crop breeding, particularly rice insect-resistant breeding.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a rice-derived insect resistance-related gene OsHR1 And its coding products and applications. Background technique [0002] Rice ( Oryza sativa ) is an important food crop in our country and even in the world, and its production surplus is directly related to our country's food security. Since the beginning of this century, rice pests, especially rice planthoppers, have become increasingly rampant, causing great damage to rice production in my country. According to statistics, in 2005, the brown planthopper in Zhejiang Province ( Nilaparvata lugens , brown plant hopper, BPH) accounted for 86% of the total rice planting area with an occurrence of more than 300,000 heads per mu, and 42% of the total rice planting area with more than 1 million heads per mu. The highest field occurrence reached more than 10 million heads per mu , resulting in a direct output loss of 1.2 million ton...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415A01H5/00
Inventor 吕静周书行鞠红平娄永根
Owner ZHEJIANG UNIV