Black nightshade pest-resistant gene and uses thereof

An insect-resistant gene, Solanum nigrum technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problem of protease inactivation, etc., and achieve the effect of improving insect resistance and plant insect resistance

Inactive Publication Date: 2008-01-30
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Almost all protease inhibitors will bind to their target protease to form a stable, non-e

Method used

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  • Black nightshade pest-resistant gene and uses thereof
  • Black nightshade pest-resistant gene and uses thereof
  • Black nightshade pest-resistant gene and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0034] Embodiment 1: the acquisition of solanum protease inhibitor gene A and the construction of plant expression vector

[0035]According to the sequence of solanum protease inhibitor gene IIa (SaPIN2a), gene-specific primers were designed, and the 5' untranslated region (UTR) was obtained by rapid amplification of cDNA ends. Use GeneRacer 5'primer and GeneRacer 5'nested primer and reverse gene-specific primer 2A-KDELSAC1 to amplify cDNA as a template, and clone the RT-PCR product into the pMD-18T vector to obtain the vector p5'RACE- TV. A pair of primers 2a-full-1 and 2a-full-2 were synthesized to amplify the solanum protease inhibitor IIa gene cDNA from the transcription start point to the UAA stop codon from p5'RACE-TV-1. The product was also cloned into pMD-18T to obtain pF-TV. The pF-TV was digested with BamH I+Sac I to obtain the NPIA gene, which is 496 bp long (see SEQ ID NO: 1).

[0036] The obtained NPIA gene replaced the GUS gene on pBI121, and the plasmid was n...

Example Embodiment

[0049] Example 2: Agrobacterium-mediated tobacco transformation and kanamycin resistance selection

[0050] Inoculate the corresponding Agrobacterium strain from the plate in 5ml YEB liquid medium (kanamycin 50mg / ml, rifampicin 50mg / L), and culture at 28°C 180 rpm for two days; then dilute the Agrobacterium solution to OD with liquid TRM medium 600 About 0.2-0.3. The sterile leaves were cut into leaf discs and placed in diluted Agrobacterium solution for 5 minutes. Get a small amount of cut leaves and put them into the liquid TRM medium without Agrobacterium, and move them to plates without or with Kan (50mg / L) respectively as positive and negative controls for transformation. Incubate in the dark at 28°C for two days; rinse the explants at least 5 times with liquid TRM medium. Blot the liquid on sterile filter paper. The explants were transferred to TRM solid plates (containing 100 μg / ml kanamycin and 250 μg / ml carbenicillin) and cultured at 28°C. Change the medium every ...

Example Embodiment

[0053] Example 3: PCR detection of kanamycin-resistant transgenic plants

[0054] Primers used to identify pF-121 (F series)

[0055] 5' primer sequence: ZF56 5'-TCCCACTATCCTTCGCAAGACCC-3'

[0056] 3' end primer sequence:

[0057] ZF103 5'GCGGATCCTTAGAAATAAGCAGTGGTCT-3'

[0058] Primers used to identify pBI-121 (B series)

[0059] 5' primer sequence: ZF56 5'-TCCCACTATCCTTCGCAAGACCC-3'

[0060] 3' end primer sequence: ZF47 5'-TCACCGAAGTTCATGCCAGT-3'

[0061] The PCR reaction conditions were: 95°C, 5 minutes for 1 cycle, followed by 95°C for 50 seconds, 58°C for 40 seconds, 72°C for 40 seconds, 35 cycles, and then 72°C for 10 minutes. Materials identified as positive by PCR were selected. The results are shown in Table 2 and Figure 2.

[0062] Table 2

[0063] positive

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Abstract

The invention discloses an anti-insect gene of solanum nigrum and applications thereof. The gene sequence is shown as in SEQ ID NO: 1. The protease inhibitor coded by the anti-insect gene of solanum nigrum of the invention has remarkable inhibiting effect on the activity of trypsin and chymotrypsin. The over-expression of the gene ensures transgenic plants to obtain obvious resistance to cotton bollworm and spodoptera litura. The gene has important application value in the anti-insect genetic engineering field and medicine and molecular biology field.

Description

technical field [0001] The invention relates to the technical field of plant insect-resistant genes, in particular to a Solanum solanum insect-resistant gene and its application. Background technique [0002] Insect pests are one of the important reasons for agricultural production reduction. According to statistics, the average economic loss of various crops due to insect pests reaches 20%-30%, and the annual loss is about hundreds of billions of dollars. The birth of plant insect-resistant genetic engineering provides a brand-new way to control pests. Since this method has many advantages such as safety, effectiveness, reduced investment and environmental pollution, since the first report of insect-resistant transgenic plants in 1987, the research on plant insect-resistant genetic engineering has achieved rapid development. [0003] At present, the insect-resistant genes widely used in plant insect-resistant genetic engineering can be divided into three categories accord...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82
CPCY02A40/146
Inventor 徐增富罗鸣王兆玉李华鹏蔡颖鹏
Owner SUN YAT SEN UNIV
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