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Cell apoptosis gene segment for reducing egg laying amount of small brown rice planthopper and application thereof

A gene fragment, the technology of the small planthopper, applied in the field of agricultural science, achieves the effect of facilitating large-scale popularization and application, reducing the amount of eggs laid, and the amount of eggs laid

Active Publication Date: 2020-03-31
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is the problem that chemical pesticides must be used in the existing methods for preventing and treating SBPH

Method used

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  • Cell apoptosis gene segment for reducing egg laying amount of small brown rice planthopper and application thereof
  • Cell apoptosis gene segment for reducing egg laying amount of small brown rice planthopper and application thereof
  • Cell apoptosis gene segment for reducing egg laying amount of small brown rice planthopper and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1, the method for cloning the Lsbcl-2 fragment of the L. striatellus cell apoptosis gene.

[0033] Specific steps are as follows:

[0034] Step 1, take 40-50 ovaries of female adult SBPH, and extract total RNA by TRizol method;

[0035] Step 2, using total RNA as a template, reverse transcription to synthesize the first strand of cDNA;

[0036] Step 3: Obtain gene fragment sequences from the SBPH genome, and perform homology comparisons in the NCBI database (http: / / www.ncbi.nlm.nih.gov / ) to predict cell apoptosis in SBPH For the gene Lsbcl-2, the upstream primer P1 (SEQ ID NO.3) and the downstream primer P2 ( SEQ ID NO.4), carry out PCR amplification;

[0037] Upstream primer P1 (SEQ ID NO.3): 5'-CGTCTGCTGAAGTGGTGGAT-3'

[0038] Downstream primer P2 (SEQ ID NO.4): 5'-TCAGGAAACACCTCTCGCAC-3'

[0039] The PCR amplification system is as follows: 4 μL 5× reaction buffer buffer, 2 μL dNTP, 1 μL cDNA template in step (1), 2 μL upstream primer P1 whose sequence is...

Embodiment 2

[0041] Example 2, the dsRNA synthesis and recovery of the apoptosis gene Lsbcl-2 of SBPH.

[0042] Specific steps are as follows:

[0043] Step 1. Based on the verified gene sequence, use Primer-Blast in the NCBI database (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) to design the upstream primer P3 (SEQ ID NO.6) and downstream primer P4 (SEQ ID NO.7)

[0044] Upstream primer P3 (SEQ ID NO.6):

[0045] 5′-taatacgactcactatagggGTGAGCGATTTGCCTTTGGG-3′

[0046] Downstream primer P4 (SEQ ID NO.7):

[0047] 5′-taatacgactcactatagggGTAGACCGCCATCCGATTGT-3′

[0048] Control GFP gene design upstream primer P5 (SEQ ID NO.8) and downstream primer P6 (SEQ ID NO.9)

[0049] Upstream primer P5 (SEQ ID NO.8):

[0050] 5′-taatacgactcactatagggAGAATGAGTAAAGGAGAAGAACTTTTC-3′

[0051] Downstream primer P6 (SEQ ID NO.9):

[0052] 5′-taatacgactcactatagggAGATTTGTATAGTTCATCCATGCCATGT-3′

[0053] The PCR amplification system is as follows: 10 μL 2× reaction buffer buffer, 2 μL upstream prime...

Embodiment 3

[0070] Example 3, experiment of feeding dsRNA on SBPH.

[0071] Specific steps are as follows:

[0072] Step 1, uniformly mixing the SBPH feed with the dsRNA (concentration of 4000 ng / μL) of the apoptosis gene;

[0073] Step 2, prepare a plurality of double-pass glass tubes, seal one end of the double-pass glass tubes with gauze and rubber bands, and use an electric insect sucker to suck 2-3 instar SBPH nymphs into the double-pass glass tubes;

[0074] Step 3: Seal the other end of the double-way glass tube with Parafilm, and use a pipette to draw 100 μL of dsRNA mixture of SBPH feed and apoptosis gene (treatment group) or 100 μL of SBPH feed and control GFP gene The dsGFP mixture (control group) was dropped in the center of the parafilm, and another parafilm was used to seal the mixture of SBPH feed and apoptosis dsRNA between the two layers of parafilm;

[0075] Step 4, place the double-pass glass tube filled with the mixture of SBPH feed and apoptosis dsRNA at a temperatu...

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Abstract

The invention discloses a cell apoptosis gene segment for reducing the egg laying amount of the small brown rice planthopper and application thereof. The gene sequence is shown as SEQ ID NO.1 and theamino acid sequence is shown as SEQ ID NO.2. According to the invention, dsRNA of a small brown rice planthopper cell apoptosis gene segment is synthesized through a molecular biological technique; and the egg laying amount is reduced obviously by using an egg laying amount and a gene capable of serving as an effective target for biological control of the small brown rice planthopper is found. ThedsRNA sequence for synthesizing the small brown rice planthopper cell apoptosis gene segment is shown as SEQ ID NO.5, so that the gene is effectively silenced and the degradation of RNA enzyme can bewell resisted. Meanwhile, the synthesis cost is low and thus the cell apoptosis gene segment is suitable for large-scale application and popularization; the dsRNA of the cell apoptosis gene of the small brown rice planthopper has a significant reduction effect on the egg laying amount of the small brown rice planthopper by the RNAi technology; and thus a novel path is provided for controlling pests through the RNAi technology and the use of chemical pesticides is reduced.

Description

technical field [0001] The invention relates to the field of agricultural science and technology, in particular to a cell apoptosis gene fragment for reducing the egg production of the striatellus striatellus and its application. Background technique [0002] As the main pest in my country's grain areas, SBPH, in addition to directly eating and harming food crops, can also spread a variety of crop viruses and indirectly harm crops, causing 30-50% or even no harvest of food crops every year. In my country, chemical pesticides are an important and main means of SBPH control. However, the continuous and single use of chemical pesticides for a long time has resulted in the resistance of SBPH to various pesticides in varying degrees. In order to achieve a satisfactory control effect, farmers continue to increase the use of pesticides, but the control effect is not satisfactory and the environmental pollution is aggravated, forming a vicious circle. Therefore, in agricultural pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/10C07K14/435C12N15/113A01N57/16A01P7/04
CPCC07K14/43563C12N15/1096C12N15/113A01N57/16C12N2310/14C12N2330/30C12Q2531/113
Inventor 郭燕
Owner SUN YAT SEN UNIV
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