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Hot pepper adipocyte protein 2 (AP2)/ethylene responsive factor (ERF) transcription factor gene and application thereof

A transcription factor and gene technology, applied to pepper AP2/ERF transcription factor gene and its application field, can solve problems such as limited research, achieve important application value and improve the effect of bacterial wilt resistance

Inactive Publication Date: 2012-11-07
FUJIAN AGRI & FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, research on the function and mechanism of ERF transcription factors has mainly focused on a few model plants such as rice, Arabidopsis, and tobacco. For the non-model plant pepper, ERF transcription factors participate in the plant disease resistance process and regulatory mechanism. The research is still quite limited, only a small number of genes have been reported

Method used

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  • Hot pepper adipocyte protein 2 (AP2)/ethylene responsive factor (ERF) transcription factor gene and application thereof
  • Hot pepper adipocyte protein 2 (AP2)/ethylene responsive factor (ERF) transcription factor gene and application thereof
  • Hot pepper adipocyte protein 2 (AP2)/ethylene responsive factor (ERF) transcription factor gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, CaERF105 Isolation of full-length cDNA

[0032] Using the amino acid of Arabidopsis ERF104 (At5g61600) as a probe, the pepper EST sequence was obtained from GenBank (www.ncbi.nlm.gov / ), and the contig analysis was performed by DNAMAN to obtain the conserved domain of ERF protein. For the consensus sequence, specific primers were designed according to the consensus sequence using PRIMER5 software. Detected in pepper homogenized cDNA library treated with 5 mM SA (library construction is based on equal amount of total RNA of pepper plants treated with SA for 0 h, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h as the initial material) Primers to the band of interest are as follows:

[0033] forward -F: 5'-CAACGAACGCAAGCCATCT -3',

[0034] reverse -R:5'-GCCTTAGCCGCATCCAC-3'.

[0035] The number of amplification cycles was 35, and the PCR reaction conditions were pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72...

Embodiment 2

[0041] Embodiment 2, CaERF105 Expression pattern analysis

[0042] The experimental materials were pepper plants with the same growth period for 25 days. The leaves were sprayed with 5 mM salicylic acid (SA) and 0.1 mM methyl-jasmonic acid (MeJA) dissolved in 10% ethanol, respectively, ( The corresponding control is 10% ethanol solution). The leaves were sprayed with 10 mM Ethephon dissolved in water, and the corresponding control was water. Take eight-week-old pepper plants and divide them by 10 8 cfu / mL R. solanacearum for root irrigation. Leaves of treated plants and corresponding control plants were collected at different times (0-48 / 96 h), and immediately snap-frozen in liquid nitrogen and placed at -70°C for subsequent analysis.

[0043] Select high-quality equivalent RNA and refer to PrimerScript from TaKaRa Company TM RT reagent Kit for reverse transcription. 1 μL of the reverse-transcribed single-stranded cDNA diluted 10-fold was used as a template. capsicum ...

Embodiment 3

[0045] Example 3, Transient expression analysis of CaERF105, GCC-box and CRT / DRE cis-acting elements

[0046] The present invention adopts BiolisticPDS-1000 / He (BioRad) gene gun to carry out gene gun bombardment method to carry out instantaneous expression analysis. The onion epidermis (1 cm × 1 cm) was torn off and cultured on solid MS (2% agar, 3% sucrose, pH 5.8), and cultured at 25°C in the dark for 1 day. 10 μg of the PK7WG2 plasmid containing the target gene and 10 μg of the pBT10-GUS-2GCC plasmid bombarded the onion epidermis at the same time, spread it on MS and cultured it in the dark for 18 hours, then observed it under the X-Gluc staining microscope and took pictures. Such as image 3 As shown, after X-Gluc staining, the expression of GUS gene can be detected in the onion epidermis co-transformed with CaERF105 and pBT10-GUS-2GCC, but the expression of GUS gene cannot be detected in the onion epidermis co-transformed with pBT10-GUS, indicating that CaERF105 Can be ...

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Abstract

The invention relates to a hot pepper adipocyte protein 2 (AP2) / ethylene responsive factor (ERF) transcription factor gene and an application thereof and belongs to the technical field of plant genetic engineering. The complementary deoxyribonucleic acid (cDNA) sequence of the gene is shown as sequence identifier (SEQID) No.1, and the hot pepper AP2 / ERF transcription factor gene is applied to the tobacco bacterial wilt resistant genetic engineering. Compared with wild type tobaccos, the overexpression of the hot pepper CaERF105 gene in the tobacco can remarkably improve the bacterial wilt resistant capabilities of a transgentic tobacco, an extremely important application value can be achieved in the bacterial wilt resistant genetic engineering of solanaceae plants, and the development of the bacterial wilt resistant genetic engineering of solanaceae plants including the tobaccos can be vigorously facilitated.

Description

technical field [0001] The invention relates to a capsicum AP2 / ERF transcription factor gene and application thereof, belonging to the technical field of plant genetic engineering. technical background [0002] During the long-term evolution process, plants have formed a set of complex physiological and biochemical mechanisms to adapt to the impact of external environmental stimuli on their growth and development. When infected by pathogenic bacteria, plants can generate a rapid defense response through the intrinsic immune system. The innate immune system of plants is mainly composed of immune responses triggered by microorganisms or pathogen-associated molecular patterns (MAMP- or PAMP-triggered immunity, PTI) and immune responses triggered by effector proteins (effector-triggered immunity, ETI). PTI is a response mechanism that generates a rapid defense response at the infection site mediated by pattern recognition receptors (PRRs) on the surface of plant cells, and is a...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415A01H5/00
Inventor 何水林赖燕林明周芦粮党峰峰官德义林菁虞露
Owner FUJIAN AGRI & FORESTRY UNIV
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