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Gene descended from antifreeze peptide insect and preparation method and application thereof

An insect and gene technology, applied in the field of insect antifreeze peptide gene and its preparation

Active Publication Date: 2013-09-11
上海金粮农业科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the current research results, its antifreeze activity is not as high as that of insects [Fei Yunbiao et al., Acta Entomology, 2000, 43(1): 98-102]

Method used

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  • Gene descended from antifreeze peptide insect and preparation method and application thereof
  • Gene descended from antifreeze peptide insect and preparation method and application thereof
  • Gene descended from antifreeze peptide insect and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Synthesis of Antifreeze Peptide CFAFP3I Gene from Insects by Gene Synthesis

[0045] (1) Test method:

[0046] Resynthesis of the insect antifreeze peptide CFAFP3I gene following the plant-preferred code.

[0047] The synthetic primers of CFAFP3I gene are as follows:

[0048] CFAFP3I-1Z:

[0049] GAA, GGA, TCC, ATG, AAA, TGC, CTG, ATG, CTC, ATC, ATG, GCT, CTT, GCT, ATC, ATT, AAC, ACC, GTC, AGC

[0050] CFAFP3I-1F:

[0051] CAG, ACA, GTT, GGC, TGT, TAG, TGT, TAG, TGC, AAG, TAC, CGT, CAC, TGC, TGA, CGG, TGT, TAA, TGA, TAG

[0052] CFAFP3I-2Z:

[0053] CAC, TAA, CAG, CCA, ACT, GTC, TGC, CAA, CAG, TAA, GTG, TGA, CAA, GAG, TAC, TCT, GAC, TAA, CTG, TTA

[0054] CFAFP3I-2F:

[0055] ACC, AGT, GCA, GGT, GGT, GCC, ATA, GAC, TTC, AGA, CTT, GTC, AAC, ATA, ACA, GTT, AGT, CAG, AGT, ACT

[0056] CFAFP3I-3Z:

[0057] ATG, GCA, CCA, CCT, GCA, CTG, GTT, CTC, GTT, TCG, ATG, GTG, TCA, CCA, TCA, CCA, GCA, GCA, CCA, GCA

[0058] CFAFP3I-3F:

[0059] CTG, CTG, ATC, TTA, CAG, CCT, ...

Embodiment 2

[0080] Arabidopsis transformation

[0081] (1) Test method:

[0082] 1. Transformation of Agrobacterium by electric shock method

[0083]1) Preparation of Agrobacterium GV3101 competent, the method refers to MicroPulserTM Electroporation Apparatus Operating Instructions and Application Guide (BIO-RAD Company) [Raineri et al., Bio.Tech., 1990, 8: 33-38].

[0084] 2) Take 50 μL of GV3101 competent cells, add 1 μL of DNA, and transfer to a 0.2 cm electric shock cup for transformation (400Ω, 2.5KV, 25 μf). Add 1 mL of LB medium containing 1 wt% mannitol to recover the culture for 2 hours (28° C., 250 rpm). Take 10 μL and 100 μL respectively and apply on LB plates (rifampicin 50 μg / mL, gentamicin 50 μg / mL, chloramphenicol 100 μg / mL).

[0085] 2. Plant material

[0086] Wild-type Arabidopsis (Arabidopsis thaliana) Ecotype Arabidopsis Columbia.

[0087] 3. Culture of Arabidopsis

[0088] 1) The Arabidopsis seeds were subjected to vernalization treatment at 4°C for 2-3 days, in ...

Embodiment 3

[0106] Antifreeze Analysis of Insect Antifreeze Peptide CFAFP3I Gene Transformed Plants of the Present Invention

[0107] (1) Test method:

[0108] The transformed plants were selfed and homozygous for three generations to obtain homozygous transformants, and the seeds were harvested. After sowing, grow for two weeks at 22°C. Wild-type and transgenic Petri dish seedlings were then used for low temperature resistance experiments. The wild-type and transgenic seedlings were cultured in a refrigerator at 4°C for 48 hours, then frozen at -20°C for 30 minutes, then returned to a refrigerator at 4°C overnight for recovery, and then returned to the culture room to resume cultivation the next day. The results showed that there were great differences in the survival rate of wild-type and transgenic Arabidopsis seedlings.

[0109] (2) Test results:

[0110] The phenotypic differences between transgenic and wild-type Arabidopsis treated with antifreeze are as follows: Figure 4 show...

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Abstract

The invention relates to a gene descended from insect antifreeze peptide and a preparation method and application thereof. The nucleotide sequence of the gene is shown in SEQ ID NO 1, the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO 2; and the gene is the CFAFP3I gene which is prepared by modifying the CFAFP3I gene of insect antifreeze peptide according to the preferred codons of plant through the gene synthesis method. When the gene is used to transform Arabidopsis thaliana, the anti-freezing capability of the transgenic Arabidopsis thaliana can be increased. The gene of the invention plays an extremely important role in the cultivation of the anti-freezing plant variety and has important theoretical significance and practical significance.

Description

technical field [0001] The invention belongs to the field of crop genetics and breeding, and specifically relates to an antifreeze peptide gene derived from insects, a preparation method and application thereof. Background technique [0002] Plants have sufficient adaptability and resistance to environmental changes and adverse environments. This stress resistance is not only controlled by the genetic type of phylogenetic evolution, but also restricted by physiological and ecological factors in phylogeny. Adversity will induce plants to express a large number of specific proteins, and the proteins induced by different stresses are not exactly the same, with both crossover and differences, and the same stress induces different proteins in different parts of the same plant. The synthesis of these proteins varies even more among different plant species [Kasuga et al., Nature Biotechnol., 1999, 17:287-291]. Therefore, many proteins are involved in the plant's response to abioti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/435C12N15/10C12N15/84A01H5/00
Inventor 朱波姚泉洪彭日荷熊爱生薛永高峰付晓燕田永生赵伟金晓芬
Owner 上海金粮农业科技发展有限公司
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