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Holotrichia parallela embryonic cell line and application thereof

A technology of dark gill beetles and cell lines, applied in the field of insect cell biology, can solve the problems of long life cycle, labor-intensive and time-consuming, and less research on Coleoptera beetles

Active Publication Date: 2020-06-05
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Before the 1980s, the research and application of Bt mainly focused on Lepidoptera and Diptera pests, while there were relatively few studies on Coleoptera Scarabidae
[0005] At present, live insects are mainly used in the screening of insecticidal active substances, bioassays and insecticidal mechanism studies at home and abroad. Due to the long cycle of live bioassays, it is laborious and time-consuming, and the sensitivity of insects is relatively affected by factors such as seasons, temperatures, and insect strains. Big
Especially grub pests have a long life cycle, and the black beetle generally needs to complete one generation in one year, and it is difficult to raise, which has become a bottleneck that limits the screening of insecticidal active substances, bioassays and in-depth research on insecticidal mechanisms.

Method used

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  • Holotrichia parallela embryonic cell line and application thereof
  • Holotrichia parallela embryonic cell line and application thereof
  • Holotrichia parallela embryonic cell line and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Establishment of the dark beetle cell line

[0022] (1) Primary culture of beetle beetle embryo cells

[0023] The adult black beetles were raised indoors, and the eggs were collected every day after the adults mated and laid eggs for cell culture. The method of primary culture of cells refers to the method of Zheng Guiling et al. (2010) (Zheng Guiling, Li Changyou, Zhou Hongxu, Li Shuwen, Li Guoxun, Xue Ming. 2010. Establishment of two new cell lines of cotton bollworm embryos and their response to baculovirus infection . Entomological Acta, 53 (2): 167-174.), the embryonic cells of separation are inoculated in the TNM-FH substratum containing 15% fetal bovine serum (FBS), cultured statically in 28 ℃ of incubators, according to According to the growth state of the cells, fresh medium of different proportions was replaced every 15-20 days.

[0024] The embryonic cells of the dark beetles cultured for 24 hours began to adhere to the wall, gradually elongated...

Embodiment 2

[0028] Example 2: Cloning and screening of beetle cell lines

[0029] (1) Cloning of the cell line of the dark beetle

[0030] The Hp-9 cell line is derived from the embryonic tissue of the black beetle, which may contain a variety of cell components, and the biological characteristics are not completely consistent. Cloning and screening of the Hp-9 cell line can be continued to obtain cell lines with different biological properties. Therefore, the present invention clones the Hp-9 cell line, and tests the sensitivity of different clones to the Bt Cry8 insecticidal crystal protein, so as to obtain excellent sensitive cell lines for subsequent research.

[0031] Take the Hp-9 cells that have been subcultured for 15 times, dilute the cells to a concentration of 16 cells per milliliter with 10% FBS TNM-FH medium, and clone the cells using a 384 (24×16) well cell cloning plate, each Add 2 mL of cell suspension to each large well, and culture at 28°C for 2 hours, observe and reco...

Embodiment 3

[0039] Example 3: Molecular identification of the dark beetle cell line Hp-9-2

[0040] (1) Extraction of cellular genomic DNA

[0041] Take the Hp-9-2 cells in the logarithmic growth phase and the eggs of the black beetle, refer to MagExtractor TM -Genome-genome DNA extraction kit (Toyobo Biotechnology Co., Ltd.) instruction manual, extract the genomic DNA of cells and eggs, and use 0.8% agarose gel to perform electrophoresis to detect the extraction effect of genomic DNA.

[0042] (2) Amplification and analysis of mitochondrial cytochrome oxidase I subunit (COI) gene fragment

[0043] Using the genomic DNA of Hp-9-2 cells and the eggs of the black beetle as templates, the mitochondrial cytochrome oxidase I subunit (COI) gene universal primer HCO 2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3', SEQ ID NO:3) was used as a template and LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′, SEQ ID NO:4) for PCR amplification of its COI gene fragment, the PCR reaction system is: 10×PCR buffer 5 μL, ...

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Abstract

The invention provides a cell line sensitive to Bt Cry8 insecticidal proteins for killing holotrichia parallela larvae. The cell line is a holotrichia parallela cell line QAU-Hp-E-9-2, and the preservation number of the cell line is CCTCC No:C201867. The cell line can be applied to activity screening of Bt insecticidal crystal proteins for killing the holotrichia parallela larvae and in-vitro biological assay. Holotrichia parallela embryonic tissue is utilized as a material, the cell line of the holotrichia parallela is constructed and subjected to cloning and screening, one cell line sensitive to the Bt Cry8 insecticidal proteins is obtained, biological characteristics related to the cell line are researched, and the material is provided for screening, in-vitro biological assay and insecticidal mechanism research of active insecticidal proteins of the holotrichia parallela.

Description

technical field [0001] The invention belongs to the technical field of insect cell biology, and specifically relates to a black beetle cell line and application thereof, which can be used for the toxicity determination and insecticidal mechanism research of Bacillus thuringiensis Cry8 insecticidal crystal protein for killing grub pests. Background technique [0002] The dark gill beetle (Holotrichia parallela) is a worldwide agricultural pest, distributed in more than 20 provinces in China, and is the main underground pest of peanuts, beans, grain crops and lawns in many areas. Both adults and larvae (commonly known as grubs) can cause damage. The adults mainly feed on the leaves, flowers and other ground organs of the host plant; while the larvae live and feed in the soil all year round, biting off the roots of the host seedlings, biting the tender stems of plants and other The underground organization has led to a decline in the quality of agricultural products, lack of se...

Claims

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Application Information

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IPC IPC(8): C12N5/07C12N15/11C12Q1/6888C12Q1/02C12R1/91
CPCC12N5/0601C12Q1/6888G01N33/5044C12N2503/02G01N2500/10
Inventor 李长友郑桂玲李苗苗李洁张保华于乾龙
Owner QINGDAO AGRI UNIV
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