Specific promoter and method for culturing disease-resistant transgenic plant

A promoter, coding gene technology, applied in the fields of botanical equipment and methods, angiosperms/flowering plants, plant products, etc., can solve the problems of plant dwarfing, yield decline, growth retardation, etc.

Inactive Publication Date: 2011-08-17
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Abstract
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Problems solved by technology

However, AP2 / ERF transcription factor overexpression can also produce adverse effects such as growth retardation, plant dwarfing, and yield decline while improving the stress tolerance of transgenic plants (Yang Wenlong, Liu Jingmei, Liu Qiang, Gong Yandao, Zhao Nanming.Isolation and characterization of a DREB-like transcription factor gene from tall fescue. Journal of Nuclear Agriculture, 2006, 20(3): 187-192)

Method used

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  • Specific promoter and method for culturing disease-resistant transgenic plant
  • Specific promoter and method for culturing disease-resistant transgenic plant
  • Specific promoter and method for culturing disease-resistant transgenic plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, the cloning of tissue-specific promoter RSS1P

[0042] A pair of specific primers (RSS1P-F1 and RSS1P-R1) were designed according to the 5' end sequence of the existing rice sucrose synthase gene, and the target sequence was the upstream sequence (1715bp) of the 5' end non-coding region.

[0043] RSS1P-F1: 5'-CTCCTTTCATTTTCAGTGCAAATGTG-3';

[0044] RSS1P-R1: 5'-CCAATGGTGGTCAGAGACG AG-3'.

[0045] Genomic DNA was extracted from the young leaves of rice variety "Nipponbare" according to the improved CTAB method.

[0046] Using genomic DNA as a template, specific primers (RSS1P-F1 / RSS1P-P1) were used for PCR amplification to obtain PCR amplification products. Taq plus DNA polymerase (Takara) was used for PCR amplification; the reaction parameters were: pre-denaturation at 94°C for 5 min; 30 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 3 min; and extension at 72°C for 10 min. The PCR product was recovered and purified by low-melting point agarose ...

Embodiment 2

[0048] Example 2, Construction of recombinant expression vector (pA20-EnRSS1P::TiERF1) and acquisition of enhanced RSS1P (EnRSS1P)

[0049] 1. Construction of recombinant expression vector pA20-EnRSS1P::TiERF1

[0050] Such as figure 1 The recombinant expression vector pA20-EnRSS1P::TiERF1 was constructed as shown.

[0051] 1. According to the improved CTAB method (Murray M G, Thompson W F. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 1980, 8(19): 4321-4325), the genomic DNA of the young leaves of the rice variety "Nipponbare" was extracted .

[0052] 2. Using the genomic DNA extracted in step 1 as a template, perform PCR amplification with a primer pair composed of RSS1P-F2 / RSS1P-R2 to obtain a PCR amplification product.

[0053] RSS1P-F2: 5′-AGA CTCCTTTCATTTTCAGTGCAAATG-3' (Sph I recognition sequence in the box);

[0054] RSS1P-R2: 5′-ATA CCAATGGTGGTCAGAGAC-3' (BamH I recognition sequence in box).

[0055] 3. The PCR amplified product ...

Embodiment 3

[0071] Activity comparison of embodiment 3, RSS1P, EnRSS1P and Ubi promoter

[0072] 1. Preparation of recombinant plasmids

[0073] The pAHC20 plasmid has a Ubi promoter-driven GUS gene expression cassette.

[0074] RSS1P shown in sequence 1 of the sequence listing was synthesized. RSS1P was inserted between the HindIII and BamH I sites of the pAHC20 vector plasmid to obtain recombinant plasmid A (pRss1p:GUS). In recombinant plasmid A, RSS1P promotes the expression of GUS gene.

[0075] Digest the pAHC20 vector with restriction endonuclease Bgl II and BamH I to recover a small fragment (UBI-INTRON fragment); digest the recombinant plasmid A with restriction enzyme BamH I to recover a large fragment (vector backbone); The large fragment and the small fragment were ligated to obtain recombinant plasmid B (pEnRss1p::GUS). In recombinant plasmid B, EnRSS1P promotes the expression of GUS gene.

[0076] 2. Transient expression experiment

[0077] Using Xu Huijun et al. (Xu H-...

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Abstract

The invention discloses a specific promoter and a method for culturing disease-resistant transgenic plant. The promoter provided by the invention is DNA (deoxyribonucleic acid) shown as a sequence 2 in a sequence table. The method provided by the invention is as follows: the promoter and the coding gene of TiERF1 protein are introduced to a starting plant; the promoter is used to start the expression of the coding gene of the TiERF1 protein to obtain a transgenic plant the disease resistance of which is stronger than that of the starting plant. The amino acid sequence of the TiERF1 protein isshown as the sequence 4 in a sequence table. The transgenic wheat obtained through the method disclosed by the invention has enhanced resistance on banded sclerotial blight and uninfluenced agronomictrait, thus having important practical value.

Description

technical field [0001] The invention relates to a special promoter and a method for cultivating disease-resistant transgenic plants. Background technique [0002] Wheat sheath blight, also known as wheat sharp eyespot, is an important disease harmful to wheat production in my country, mainly caused by Rhizoctonia cerealis CAG-1 (Chen Yanxi, Tang Wenhua, Zhang Dunhua, Jian Xiaoying.A preliminary study on etiology of sharp eye-spot of wheat in China. Acta Plant Protection 1986, 13(1): 39-44). Sheath blight destroys the phloem tissue of the stalks of wheat, which not only causes the plants to be prone to lodging, but also causes poor nutrient transportation, makes the seeds dry, and even causes withered and white ears, resulting in reduced yield. Sheath blight can generally reduce wheat production by 10%-30%, and severe plots can reduce production by more than 50%, or even no grains. Coupled with the continuous improvement of wheat production level in recent years, the change ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N1/21C12N1/15C12N5/10C12N1/19C12N15/63A01H5/00
Inventor 张增艳李钊庄洪涛杜丽璞徐惠君
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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