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Method for improving insect resistance of plant by using RNAi technique

一种抗虫性、植物的技术，应用在生物技术和植物学领域，能够解决死灰复燃、转基因抗虫植物抗性下降等问题

Inactive Publication Date: 2008-06-11
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there have been research reports that as time goes by, the resistance of various transgenic insect-resistant plants is declining, and the phenomenon of large-scale and rare insect pests in the past has begun to resurface.

Method used

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  • Method for improving insect resistance of plant by using RNAi technique
  • Method for improving insect resistance of plant by using RNAi technique
  • Method for improving insect resistance of plant by using RNAi technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] The expression characteristics of embodiment 1 GIP gene

[0113] 1. Expression of GIP gene in cotton bollworm

[0114] In order to detect the expression of GIP gene in cotton bollworm, the inventors used RT-PCR method to detect the expression of GIP in different tissues. The method is as follows: the mRNA is extracted from the midgut, fat body, Malpighian duct, genitalia, and brain tissue of the cotton bollworm respectively, the GIP gene is amplified by the conventional RT-PCR method, and the obtained amplification product is detected by agarose electrophoresis. Among them, the primers used in RT-PCR are shown in Table 1.

[0115] Table 1

[0116] GIP+

5'-GTGCTTTGATGAGACTCTTCG-3'

SEQ ID NO: 5

GIP-

5'-TACATTTGCTATTTATAATTTGC-3'

SEQ ID NO: 6

[0117] The results of electrophoresis of RT-PCR products are shown in image 3 a. In the figure, 1-5 successively represent the expression of GIP gene in the midgut, fat body, Malpighian d...

Embodiment 2

[0124] Embodiment 2 Isolation of GIP gene

[0125] 1 μl Asian cotton bollworm (Helicovepa armigera) (about 10 6 pfu) After gradient dilution of the cDNA library (ZAPexpress), use the following specific primers for PCR to determine the minimum working concentration for PCR:

[0126] GIP2+: 5'-GAAGATTTTCTCGATAAGGAAG-3' (SEQ ID NO: 7);

[0127] GIP2-: 5'-ATATAAAGCACTGTGCCACTAAG-3' (SEQ ID NO: 8).

[0128] Soak the 96-well plate in 70% ethanol for several hours, shake it dry, and irradiate it with ultraviolet light for 15-30 minutes, then add 200-300μl LB (containing 10mM MgSO 4 / 0.2% maltose). Take an appropriate dilution of the library (1000-fold dilution) solution and mix it with 400 μl XL1-Blue bacteria solution, shake and incubate at 37°C for about 30 minutes, and add 4 μl of the mixture to each well. Plates were incubated overnight at 37°C. After amplification, 5 μl of bacterial solution was taken from each column of 8 wells and mixed evenly, and a total of 12 columns w...

Embodiment 3

[0130] Example 3 For the construction of dsGIP, the isolation of dsGST1 gene

[0131] Using the Asian cotton bollworm (Helicovepa armigera) cDNA library (ZAP express) as a template, use gene-specific primer pairs:

[0132] Primer pairs used to obtain GIP gene fragments:

[0133] GIPF: 5'-GAAGATTTTCTCGATAAGGAAG-3' (SEQ ID NO: 7), and

[0134] GIPR: 5'-ATATAAAGCACTGTGCCACTAAG-3' (SEQ ID NO: 8); and

[0135] Primer pairs used to obtain fragments of the GST1 gene (GenBank accession number EF033109):

[0136] GSTF: 5'-GACCTTGGCAGACCTCAG-3' (SEQ ID NO: 9), and

[0137] GSTR: 5'-CCAGCTCGAACCACTTTT-3' (SEQ ID NO: 10);

[0138] Perform PCR amplification to obtain GIP and GST1 fragments for constructing dsGIP and dsGST1 expression vectors, respectively.

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Abstract

The invention discloses a method of enhancing the plant insect resistance, which ensures that insect gene expression dsRNA in a plant body is processed to form siRNA in the plant body through the transferring gene method, the RNA is interfered to the target gene through the phytophagy insect fetching food and coming into the plant body, and therefore the expression of the target gene is restrained. The invention discloses a new method which can utilize the plant as the medium to restrain the insect growth through the RNA interference mechanism and enhance the plant insect resistance for the first time.

Description

technical field [0001] The present invention belongs to the fields of biotechnology and botany. In particular, the present invention relates to a novel method for improving the resistance of plants to insects. Background technique [0002] In agriculture, insect pests have always been an important factor affecting crop yields. People have to invest a lot of manpower and material resources every year to suppress pests and increase crop yields. [0003] With the increasing resistance of agricultural pests to pesticides, and for the consideration of environmental protection and sustainable development, there is an urgent need for new pest-resistant methods. The emergence of transgenic insect-resistant plants has alleviated this contradiction and made a significant contribution to the development of agriculture, such as transgenic insect-resistant soybeans, Bt insect-resistant cotton, etc. However, studies have reported that with the passage of time, the resistance of various...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/12C12N15/82
CPCC12N15/8218C12N15/8286Y02A40/146
Inventor 陈晓亚毛颖波林芝萍王凌健
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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