Low-temperature inducible promoter PCPI and application thereof

A low-temperature induction and promoter technology, applied in the field of genetic engineering, can solve the problem of no low-temperature induction promoter of edible fungi, and achieve the effect of wide application prospects

Active Publication Date: 2015-05-13
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there has been no low-temperature-inducible promoter identified from Pleurotus ostreatus, and there are no reports on low-temperature-induced promoters of other edible fungi in China, which is of great significance for stress-resistant breeding of edible fungi

Method used

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  • Low-temperature inducible promoter PCPI and application thereof
  • Low-temperature inducible promoter PCPI and application thereof
  • Low-temperature inducible promoter PCPI and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Through the analysis of the upstream promoter sequence of the lectin gene lec of the Pleurotus ostreatus variety "Shen Ping 35" (provided by the Jilin Agricultural University, and publicly promoted and applied), it was found that the promoter region has multiple dehydration, low temperature response and ABA stress-responsive cis-acting element.

[0024] Select the Pleurotus ostreatus variety "Shen Ping 35", inoculate it on a PDA medium plate and grow it for one week, culture it at 4°C for 24 hours, take the mycelium, grind it with liquid nitrogen, add it to a 1.5ml EP tube containing the lysate, shake and mix well Finally, use the kit RNAiso Plus to extract total RNA, use formaldehyde denaturing gel electrophoresis to identify the quality of RNA, and then use a spectrophotometer to measure the concentration of RNA; perform reverse transcription according to the instructions of the reverse transcription kit from the US company FERMENTAS, and synthesize cDNA first The pri...

Embodiment 2

[0026] According to the cDNA sequence of the low-temperature-induced promoter of Pleurotus ostreatus, design primers to amplify the complete coding reading frame, add an XbaI restriction site in the upstream, and add a NcoI restriction site in the downstream, upstream primer: CAG CATCTG CGACCACCCTAGCACG, downstream primer: CAAGTG AGTGCCGATGGAT, using the cDNA obtained in Example 1 as a template for PCR amplification, the cDNA of the low-temperature promoter sequence was cloned into the pMD-18T vector, and further cloned into the binary expression vector pCAMBIA1302 to construct pCAMBIA1302 -PCP1-GFP Carrier, enzyme digestion electrophoresis results see Figure 4 ,PCR detection picture see Figure 5 . The constructed plant expression vector was transformed into tobacco by the method of Agrobacterium infection, and after the tobacco seedlings in the MS screening medium were subjected to low temperature stress at 4°C for 12 hours, the control was the transgenic tobacco culti...

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Abstract

The invention discloses a sequence of a low-temperature inducible promoter PCPI which is an induced promoter discovered in oyster mushroom and related to low-temperature stress. Experiments show that a constructed plant expression vector carrying the low-temperature inducible promoter of oyster mushroom and GFP gene is transferred into tobacco; the GFP gene can be expressed under low-temperature stress but cannot be expressed in case of no low-temperature stress. The gene used in the invention is from edible mushroom and has a preferred codon suitable for edible mushrooms and a genetic engineering receptor of the gene is mainly suitable for pleurotus citrinopileatus, pleurotus eryngii pleurotus nebrodensis and other edible mushrooms. The low temperature stress selective marker has a wide application prospect in culture of transgenic plants and edible mushrooms.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a low temperature inducible promoter PCP1 and its application. technical background [0002] Inducible promoter (Inducible Promoter) means that under the stimulation of certain specific physical or chemical signals, this type of promoter can greatly increase the transcription level of genes. During the long-term evolution process, plants can adapt to changes in light, temperature, water and other environments within a certain range by activating the expression of different genes. The promoters of these genes usually contain relatively conservative cis-acting elements, which can be used to infer the possible functions of new genes, and the promoters of these environmental response genes can also be fused with stress-resistant genes, so that transgenic plants can better adapt adversity. Inducible promoters include light, temperature, hormone responsive promoters, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/82A01H5/00A01H15/00C12Q1/68
Inventor 付永平盛立柱李玉宋冰李丹戴月婷王欣欣郭昱秀张春兰
Owner JILIN AGRICULTURAL UNIV
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