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Promoter and application thereof

A promoter and reaction technology, which is applied in the field of genetic engineering, can solve the problems of limited oil production effect of engineered microalgae strains and hinder the development of microalgae genetic engineering, and achieve the effects of increasing yield, saving production cost, and increasing biomass

Active Publication Date: 2017-07-04
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the oil production effect of engineered microalgae strains obtained by this method is limited
Currently, in the study of oil production in Phaeodactylum tricornutum, there is only one widely used promoter, which greatly hinders the development of microalgae genetic engineering

Method used

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  • Promoter and application thereof
  • Promoter and application thereof
  • Promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1: Cloning of promoter and GUS gene

[0079] First, the genomic DNA of Phaeodactylum tricornutum was extracted using the plant DNA extraction kit from Magene. The primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd., purified by PAGE method, and diluted to 100 μM when used. The promoter PPt48882 was cloned in the DNA of Phaeodactylum tricornutum, and the GUS gene was cloned in the universal vector pCAMBIA1301 (purchased from Shanghai Jielan Biotechnology Co., Ltd.). Wherein the cloning conditions are all adopted PCR method, using KOD-plus-Neo enzyme (TOYOBO, JAPAN) PCR to obtain, the PCR primers of promoter PPt48882 and GUS gene are shown in Table 1, and the PCR reaction system is shown in Table 2 and Table 3 respectively , the promoter of Phaeodactylum tricornutum gene Pt48882 has not been confirmed. The present invention predicts the core element of the PPt48882 promoter through multiple prediction software, and takes 1103bp of it as the promo...

Embodiment 2

[0089] Embodiment 2: Construction of recombinant vector

[0090] The above-cloned promoter PPt48882, GUS gene and fcpA terminator (277bp, synthesized by Shanghai Sangon Biotech Co., Ltd.) were homologously recombined using the ClonExpress MultiS One Step Cloning kit (VazymeBiotech Co., Ltd, nanjing) Connected to the carrier containing the bleomycin resistance expression cassette, the vector containing the bleomycin resistance expression cassette is made of the pPt-ApCAT vector (the construction steps of the pPt-ApCAT vector refer to Niu et al., 2011) to convert the chloramphenicol resistance The sex gene is obtained by replacing the bleomycin gene with conventional genetic engineering means known to those skilled in the art, and a recombinant expression vector containing the promoter PPt48882 and the GUS gene (such as figure 1 shown). The kit used is the one step cloning KIT from Novizyme Biotechnology Co., Ltd.

Embodiment 3

[0091] Embodiment 3: Transformation of Phaeodactylum tricornutum and screening of transformed algae

[0092] Referring to the electroporation method of Niu et al. (2012), the recombinant vector was integrated into the genome of Phaeodactylum tricornutum. Transformed algae with resistance were obtained through 4-5 times of bleomycin selection.

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Abstract

The invention relates to the field of gene engineering, and especially relates to a promoter and an application thereof. The promoter of gene Pt48882 is cloned from Phaeodactylum tricornutum, and the nucleotide sequence of the promoter is represented by SEQ ID NO.1. The promoter PPt48882 can promote high-level expression of downstream gene in the growth period of microalga cells, so the promoter can be applied to the gene engineering, and has good application prospect in the gene engineering.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a promoter and its application. Background technique [0002] At present, the world's energy shortage and environmental pollution have become a huge challenge faced by human society. Therefore, it has become the consensus of all countries in the world to find a new type of renewable and clean energy that can replace fossil energy. Biodiesel, whose performance is basically equivalent to petrochemical diesel, and has good environmental protection and biodegradability, has become the most widely used and fastest growing ideal renewable energy in the world. At present, biodiesel refers to the formation of mono-alkyl fatty acid esters through transesterification of oils (mainly triglycerides) from living organisms and alcohols (methanol or ethanol). [0003] The principle of microalgae to produce biodiesel is to use the high-efficiency expression of microalgae oil synthesis-related...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/79C12N1/13C12P7/64C12R1/89
CPCC07K14/405C12N15/79C12N2830/34C12P7/6463C12P7/649Y02E50/10
Inventor 李宏业邹立功陈嘉雯杨维东刘洁生
Owner JINAN UNIVERSITY
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