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Application of endogenous small RNA of Chilo suppressalis in improving rice insect resistance

A rice and gene technology, applied in the direction of DNA / RNA fragments, applications, plant genetic improvement, etc., to achieve the effect of easy prediction, low risk, and small fragments

Inactive Publication Date: 2017-07-28
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, this technology is widely used in functional genomics research, variety improvement and medical fields, but the application of amiRNA technology in transgenic insect-resistant breeding has not been reported.

Method used

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  • Application of endogenous small RNA of Chilo suppressalis in improving rice insect resistance
  • Application of endogenous small RNA of Chilo suppressalis in improving rice insect resistance
  • Application of endogenous small RNA of Chilo suppressalis in improving rice insect resistance

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: the extraction of the small RNA sample of Chilo suppressalis

[0050] The life cycle of Chilo suppressalis includes four stages of growth and development: egg, larva, pupa and adult. The larval stage is usually divided into 5 instars. The present invention collects samples of six growth and development stages of the stem borer, including eggs, 1-2 instar larvae, 3-4 instar larvae, 5th instar larvae, pupae and adults. After collecting the samples of Chilo borer in various stages, put them into the liquid nitrogen tank and store them first. After all the samples are collected, use Trizol reagent for extraction. The method is as follows:

[0051] 1) Weigh 0.1 g of Chilo stem borer sample and grind it into powder in liquid nitrogen.

[0052] 2) Add 5ml Trizol reagent to quickly mix the ground sample powder, and distribute it in five 1.5ml RNAase-free centrifuge tubes.

[0053] 3) Leave it at room temperature for a few minutes until the mixture completely me...

Embodiment 2

[0065] Embodiment 2: the recovery and Solexa sequencing of the small RNA sample of Chilo suppressalis

[0066] The small RNA fragments below 80 nt were recovered by gel electrophoresis from the RNA samples of the 6 stages of Chilo suppressalis, and then the recovered small RNA fragments were connected with specific 3' end adapters and 5' end adapters. The 3' end primers were used for reverse transcription, and the obtained products were amplified by PCR with 5' and 3' end primers for high-throughput small RNA sequencing.

[0067] Through Solexa sequencing, each sample obtains a small fragment data volume of about 1×108. After obtaining these data, the analysis process is as follows figure 1 Place. First, the 5' and 3' end adapters were removed from the obtained fragment sequences, and the low-quality sequences were removed to select fragments with a length of 18-30nt. The processed fragments were compared with the Genbank database, and the conserved rRNA, scRNA, snoRNA, snRN...

Embodiment 3

[0071] Embodiment 3: verification of small RNA cus-15 expression

[0072] The new miRNA csu-15 of C. borer predicted by high-throughput sequencing and bioinformatics needs to be verified by experiments such as stem-loop RT-PCR (Chen et al.2005) and re-sequencing of RT-PCT products its truthfulness and accuracy. Its method is as follows.

[0073] According to the predicted candidate miRNA results, specific reverse transcription primers and PCR primers were designed, and the primers involved are shown in Table 1.

[0074] Table 1 Primer sequences required for stem-loop RT-PCR

[0075]

[0076] Firstly, DNase I was used to remove the genomic DNA in the samples, and then the 6 samples of C. borer were reverse-transcribed with the specific reverse transcription primer 15-specific-R. The system was as follows:

[0077] RNA sample: 1.0 μg

[0078] DNase I: 0.5 μl

[0079] 10×DNase I buffer 1.0μl

[0080] Supplement DEPC-treated ddH 2 0 to 10 μl

[0081] React at 37°C for 15 ...

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Abstract

The invention belongs to the technical field of gene engineering, in particular relates to application of Chilo suppressalis (Walker) endogenesis small RNA in rice inset resistance improvement, and aims to verify the function and study the application of an obtained Chilo suppressalis endogenesis small RNA sequence. By means of high-flux small RNA sequencing and bioinformatics analysis, a DNA sequence of Chilo suppressalis endogenesis small RNA csu-15, and the nucleotide sequence of the DNA sequence is as shown in SEQ ID NO:1. The sequence of csu-15 is adopted to establish an artificial microRNA (amiRNA) expression carrier and convert rice. The in-vitro stalk insect inoculation experiment shows that Chilo suppressalis growth can be remarkably inhibited in transgenic rice.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. Specifically, it relates to the application of endogenous small RNA of Chilo suppressalis in the improvement of rice insect resistance. An endogenous small RNA sequence of Chilo suppressalis (Walker) was cloned, and the functional verification of the sequence and its application in rice insect resistance improvement were studied. Background technique [0002] Rice is one of the main food crops in the world, and it is the staple food of about half of the world's population. In agricultural production, pests and diseases will lead to large-scale production reduction of rice production, resulting in huge economic losses. Chilo borer is one of the main pests of rice, widely distributed in Asia, Oceania, North Africa, southern Europe and other countries; in my country except Qinghai and Tibet, all provinces have the occurrence of Chilo borer (Sheng Chengfa et al. 2003). In recent ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84C12N15/113A01H5/00
Inventor 陈浩江山张志伟林拥军
Owner HUAZHONG AGRI UNIV
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