Bioassary method for identifying insect-resistance performance of transgenic insect-resistance plant

A technology for insect-resistant plants and bioassays, which is applied to the measurement/inspection of microorganisms, biochemical equipment and methods, and measuring devices, and can solve problems such as failure, no insect resistance, and poor insect resistance of transformed plants

Inactive Publication Date: 2007-05-23
HUAZHONG NORMAL UNIV
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AI Technical Summary

Problems solved by technology

At present, the bioassay of transgenic insect-resistant poplar is mainly done through the indoor insect resistance test. The indoor insecticidal test is easy to control, has good repeatability, and the results are relatively accurate. The transformed plants have poor or no insect resistance, which means the failure of the whole experiment

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  • Bioassary method for identifying insect-resistance performance of transgenic insect-resistance plant
  • Bioassary method for identifying insect-resistance performance of transgenic insect-resistance plant
  • Bioassary method for identifying insect-resistance performance of transgenic insect-resistance plant

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Embodiment Construction

[0031] The bioassay procedure for insect resistance of transgenic insect-resistant plants is shown in Figure 4. The operation steps are as follows:

[0032] 1. Extraction of total plant protein:

[0033] ① Take 1.0 g of fresh leaves of positive plants detected by PCR (polymerase chain reaction), place them in a mortar, add liquid nitrogen and PVP (polyvinylpyrrolidone), and quickly grind them to powder;

[0034] ② Add 3.5 ml of plant protein extract to the mortar and continue grinding for 30 seconds;

[0035] ③ Place the above mixture at 4°C overnight;

[0036] ④ Centrifuge at 5000 rpm for 10 minutes at 4°C;

[0037] ⑤ Take the supernatant and centrifuge at 10,000 rpm for 20 minutes at 4°C;

[0038] ⑥The supernatant was taken to measure the protein concentration by Bradford method.

[0039] 2. Determination of protein toxicity:

[0040] ① Inoculate Hi5 cells (2-3×10 5 cells / ml), add 100 microliters of cell culture medium to each well, and culture overnight at 28°C;

[00...

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Abstract

It is an identifying transgenic insect-resistant plants insect-resistance biometric detection method. Using vitro cultured trichoplusia Ni cells as material, through the insect cell toxicity of the transgenic insect-resistant plants protein, determine the insect-resistance of the transgenic insect-resistant plants. As the succinate dehydrogenase in the live cell mitochondria and MTT react to produce blue-violet crystal product A Za, and the color depth of A Za is proportional to the number of live cells, and in the 570nm wavelength, measuring its OD value, to calculate the number of the live cells, and the succinate dehydrogenase of died cells disappears, which can not restore MTT. Using insect cells measuring the transgenic insect-resistant plants insect-resistance, it is easy to observe and analyze transgenic insect-resistant plants killing mechanism from the molecules level. Compared vitro cultured insect cells with the keeping or field live insects, it has good homogeneity, and not vulnerable to the external environment and live insect body wall, intestinal protease activity, fluid pH, and other factors. The method is simple, convenient and saving manpower and resources.

Description

technical field [0001] The invention relates to a bioassay technology for identifying insect resistance of transgenic insect-resistant plants. It belongs to bioengineering and biogenetic engineering technology. technical background [0002] Pests are an important factor restricting the high and stable yield of crops. How to effectively control pests and reduce losses has received widespread attention from governments and scientists in various countries. The disadvantages of traditional chemical control have become more and more prominent. The non-specific mode of action of chemical pesticides not only kills pests, but also kills beneficial insects and natural enemies of pests; Drug resistance, the use of more and more drugs has formed a vicious circle; excessive use of chemical pesticides not only seriously damages the natural ecological balance, but also directly threatens the safety of humans and animals. However, biological control can overcome the above shortcomings. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/00C12Q1/18
Inventor 郑进洪华珠康薇
Owner HUAZHONG NORMAL UNIV
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