Chinese cabbage activating protein 2/ethylene responsive factor (AP2/ERF) transcription factor gene and application thereof

A technology of transcription factor and gene, applied in cabbage AP2/ERF transcription factor gene and its application field, can solve the problem of limited research on disease resistance process and regulation mechanism, achieve the effect of improving bacterial wilt resistance ability and important application value

Inactive Publication Date: 2012-10-24
FUJIAN AGRI & FORESTRY UNIV
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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 Chinese cabbage, ERF transcription factors participate in the process of plant disease resistance and its regulatory mechanism. research is still quite limited

Method used

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  • Chinese cabbage activating protein 2/ethylene responsive factor (AP2/ERF) transcription factor gene and application thereof
  • Chinese cabbage activating protein 2/ethylene responsive factor (AP2/ERF) transcription factor gene and application thereof
  • Chinese cabbage activating protein 2/ethylene responsive factor (AP2/ERF) transcription factor gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, BrERF11 Isolation of full-length cDNA

[0032] Using the amino acid of Arabidopsis AtERF11 (AB055882) as a probe, the EST sequence of Chinese cabbage was obtained from GenBank (www.ncbi.nlm.gov / ), and the contig analysis was carried out by DNAMAN, and the sequences containing the conserved domain of ERF protein were respectively obtained. For the consensus sequence, specific primers were designed according to the consensus sequence using PRIMER5 software. The primers for detecting the target band in the high-temperature-treated Chinese cabbage cDNA library (the cDNA library uses Chinese cabbage leaves treated at 42°C for 2 h as the initial material) are as follows:

[0033] forward, 5′-CGTTACGCCGCCGAGAT-3′

[0034] reverse, 5'-TCAGGCTTGGGAGGGAG-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°C for 4...

Embodiment 2

[0041] Embodiment 2, BrERF11 Expression pattern analysis

[0042] The experimental materials were 2-week-old Chinese cabbage seedlings, and the leaves were sprayed with 10% ethanol solution containing 5 mM salicylic acid (SA) and 0.1 mM methyl-jasmonic acid (MeJA) respectively, (the corresponding control Spray the leaves with 10% ethanol solution for Chinese cabbage seedlings of the same seedling age). Respectively with 0.1 mM abscisic acid (Abscisic acid, ABA), 0.1 mM H 2 o 2 and 10 mM ethephon (Ethephon) aqueous solution sprayed the leaves, and the corresponding control was water. The leaves of the treated plants and the corresponding leaves of the control plants were collected at different times (0-48h), and immediately frozen in liquid nitrogen and placed at -70°C for subsequent use BrERF11 Gene expression pattern analysis.

[0043] After extracting RNA from leaves of treated and control plants, select high-quality equivalent RNA with reference to PrimerScript from ...

Embodiment 3

[0045] Example 3, Construction of Plant Expression Vector pMDC32-BrERF11

[0046] The present invention uses gateway technology (Walhout et al., 2000) to construct an overexpression vector for transgenic dicotyledonous plants.

[0047] Dicotyledonous plant overexpression vector uses pDONR207 as the entry vector, and the destination vector is the pMDC32 vector with 2×CaMV35S promoter, amplified BrERF11 The primers used in the ORF region are:

[0048] forward, 5′- AAAAAGCAGGCTTCATGGCGCCGACAGCTAAAACGAC-3′;

[0049] reverse, 5′-AGAAAGCTGGGTCCTAATTCTCAGGCTTGGGAGGGAG-3′

[0050] The outer adapter primers are:

[0051] attB1: 5'- GGGGACAAGTTTGTACAAAAAAAGCAGGCT-3'

[0052] attB2: 5'- GGGGACCACTTTGTACAAGAAAGCTGGGT-3'

[0053] Using the positive clone plasmid obtained by screening in Example 1 as a template, using forward / reverse as a primer (10 μM), through Primer STAR TM HS DNA polymerase (Takara) was used for two rounds of amplification, and attB linkers were added on both si...

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Abstract

The invention relates to a Chinese cabbage AP2 / ERF transcription factor gene and applications thereof and belongs to the technical field of plant genetic engineering. A complementary deoxyribonucleic acid (cDNA) sequence of the gene is represented as SEQIDNo.1. The Chinese cabbage AP2 / ERF transcription factor gene is applied in nightshade tobacco bacterial wilt-resisting genetic engineering. When transgenic tobacco is compared with wild tobacco, the overexpression of a Chinese cabbage brassinosteroid ethylene responsive factor 11 (BrERF11) gene in the tobacco can significantly improve the bacterial wilt-resisting ability of the transgenic tobacco, so that the Chinese cabbage AP2 / ERF transcription factor gene has an important application value in the plant bacterial wilt-resisting genetic engineering and can promote the development of the tobacco bacterial wilt-resisting genetic engineering greatly.

Description

technical field [0001] The invention relates to a cabbage AP2 / ERF transcription factor gene and application thereof, belonging to the technical field of plant genetic engineering. technical background [0002] Chinese cabbage( Brassica rapa L. ssp. pekinensis ) is one of the most important cruciferous vegetables in Asia. Its growth process is easily affected by various diseases and abiotic adversity, and the hidden dangers in the environment and health caused by the over-reliance on pesticides and chemical fertilizers have prompted people to adopt more sustainable and practical countermeasures from genetic improvement, which is A more in-depth analysis and understanding of the stress-resistant molecular mechanism of cabbage is needed from the molecular level. [0003] Under the stimulation of external environmental stress, plants maintain their normal growth and development through a series of defense response mechanisms such as transcriptional activation of defense res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/00
Inventor 何水林赖燕黄木坤肖于华戚爱华官德义林金辉陈成聪
Owner FUJIAN AGRI & FORESTRY UNIV
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