Protease inhibitor of black nightshade and application thereof

A technology of protease inhibitors and genes, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of insect death, insect inability to decompose protein, and affect insect growth and development, so as to improve insect resistance and plant insect resistance sexual effect

Inactive Publication Date: 2008-10-08
SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Serine-type protease inhibitors in plant protease inhibitors can inhibit the protease activity in the digestive tract of feeding insects, so that the

Method used

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  • Protease inhibitor of black nightshade and application thereof
  • Protease inhibitor of black nightshade and application thereof
  • Protease inhibitor of black nightshade and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0033] Example 1 The acquisition of NPIB and the construction of its expression vector

[0034] 1. Acquisition of NPIB and construction of plant expression vector pAH2b

[0035] According to the partial sequence of SaPIN2b, the present invention obtains NPIB cDNA through RT-PCR with solanum nigrum mRNA, and then uses primers ZF259 and ZF260 to introduce Bam HI and Kpn I restriction sites. Then connect it with pGEM-T easy (PROMEGA), transform Escherichia coli DH5α, and screen the cloned transformants with blue and white spots. Extract the plasmid of the positive transformant, carry out enzyme digestion test with Kpn I and Bam HI endonuclease, and recover the restriction fragment. Digest the pHANNIBAL vector with KpnI and Bam HI (the pHANNIBAL vector has a CaMV 35S promoter and NOS terminator that provide gene expression, and has Amp resistance), and the pHANNIBAL fragment after digestion is gel-recovered, ligated with the NPIB cDNA fragment, and ligated The product was transf...

Example Embodiment

[0046] Tobacco Transformation and Kanamycin Resistance Screening of Example 2 Agrobacterium-Mediated

[0047] Inoculate the corresponding Agrobacterium strains from the plate in YEB liquid medium containing corresponding antibiotics (kanamycin 50mg / ml, rifampin 50mg / L), and culture at 180rpm at 28°C for two days; then dilute the Agrobacterium with liquid TRM medium liquid to OD 600 About 0.2 to 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, and blot the liquid on sterile filter paper. The explants were transferred to TRM solid plates containing corresponding antibiotics (100 μg / ml kanamycin, 250 μg / m...

Example Embodiment

[0048] Embodiment 3 Kanamycin-resistant transgenic plants are detected by PCR

[0049] Primers used in this example:

[0050] 5' primer sequence: ZF56 5'-TCCCACTATCCTTCGCAAGACCC-3';

[0051] 3' end primer sequence: ZF171 5'-CCGCTCGAGGCATTTCATTGATTAG-3';

[0052] 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. Select the material identified as positive by PCR, the result is as follows figure 2 As shown, the corresponding bands were amplified in the transgenic plants and the positive control.

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Abstract

The invention discloses gene of black nightshade protease inhibitor and an application thereof. The gene sequence is shown as SEQ ID No:1, and the coded amino acid sequence is shown as SEQ ID No:2. The NPIB gene provided by the invention is the gene which is separated from the solanaceae plant black nightshade, through the coded protease inhibitor has remarkable inhibitory effect to the activation of trypsin and chymotrypsin, and the plant resistance against insect can be enhanced. The expression vector of a plant is constructed by adopting NPIB gene as target gene in the invention, the excess expression is performed under the driving of the cauliflower mosaic virus (CAMV)35S promoter, the insect resistant ability of the transgenic plant is enhanced, the gene can be applied in the research of insect-resistant plant genetic engineering and the biological control of the phosphorus wing item of insect such as cotton bollworm etc, and invention has important application value in the plant insect-resistant genetic engineering field, the medicine and the molecular biological field.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, and relates to a solanum protease inhibitor gene and application thereof. Background technique [0002] Insect pests have always been 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% to 30%, and the annual loss is about hundreds of billions of dollars. It has become the direction of modern agricultural development to introduce insect-resistant genes into crop cells by means of genetic engineering and make them stably expressed and inherited, and to breed new varieties of insect-resistant crops, so as to obtain high and stable yields of crops. [0003] At present, insect-resistant genes widely used in plant genetic engineering can be divided into three categories according to their sources: one is the insecticidal crystal protein gene derived from the microorgan...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/63C12N15/82C12N15/70C12N15/74C12N5/10C12N1/15C12N1/19C12N1/21C07K14/415A01H1/00
CPCY02A40/146
Inventor 徐增富罗鸣王兆玉吴锋
Owner SUN YAT SEN UNIV
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