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Citrus fruit epidermis high-efficiency expression promoter and application thereof

A high-efficiency expression and promoter technology, applied in the field of plant biology, can solve the problems of disrupting the metabolic balance of plants, wasting plant materials and energy, and affecting the normal growth and development of plants.

Active Publication Date: 2019-08-13
INST OF FRUIT & TEA HUBEI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the constitutive promoter drives the target gene to be continuously overexpressed in the whole plant, which not only wastes the material and energy in the plant, but also disrupts the metabolic balance of the plant and affects the normal growth and development of the plant.
The expression of foreign genes in plant pollen and edible parts also brings controversy over the safety of genetically modified organisms

Method used

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  • Citrus fruit epidermis high-efficiency expression promoter and application thereof
  • Citrus fruit epidermis high-efficiency expression promoter and application thereof
  • Citrus fruit epidermis high-efficiency expression promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1 pCAMBIA2300-P1080-GUS construction

[0018] The applicant used four kinds of citrus fruit outer skin transcriptome databases to screen a super-highly expressed gene, which was found in the sweet orange genome database ( http: / / citrus.hzau.edu.cn / orange / ) in the gene number is Cs2g31080. Through quantitative PCR analysis of different tissues, the cDNA of citrus roots, leaves, fruit outer skin and pulp were used as templates, and the primers qF: 5'-GCAAGGAGGTGAAAGGAGTT-3' and qR: 5'-TTTGAAGGCTGATCCGACTAAG-3' were used for quantitative PCR amplification . The citrus housekeeping gene β-actin was used as an internal reference, and the primers were: aF 5'-GCTGCCTGATGGTCAAGTC-3' and aR: 5'-AGTTGTAGGTGGTCTCGTGAA -3'. The reaction was carried out on an ABI 7500 real-time quantitative PCR instrument (Applied Biosystems, USA), 10 μL of reaction system, including 5 μL of SYBER Green PCR Mix, 0.5 μL of each primer (10 μmol L-1), about 200 ng of cDNA template, and e...

Embodiment 2

[0023] Example 2 Agrobacterium-mediated Transformation of Nicotiana benthamiana Leaves and Micro Tom Tomato Green Mature Fruits

[0024]Take about 1 μg of the pCAMBIA2300-P1080-GUS vector plasmid and add it to 200 μL of Agrobacterium EHA105 competent cells. After standing on ice for 30 minutes, treat with liquid nitrogen for 5 minutes. Medium, 28°C, 180rpm shaker culture for 4-5h. 4°C, centrifuge at 3000rpm for 5 minutes to collect the bacterial liquid, resuspend with 200 μL LB liquid medium, take 100 μL and spread on LB solid medium (containing 100 mg / L kanamycin and 25 mg / L rifampicin). The petri dish was sealed with Parafilim sealing film and cultured at 28°C for 2-3 days, and the monoclonal bacteria were picked and multiplied, using the specific primer F: 5'- CTGCAG CTGTGCGGCAATAATTTCTTT-3' and R:5'- GGATCC TCGGACGTCCATTATATACAGA-3' was used to screen positive clones by PCR. The verified Agrobacterium EHA105 plus 30% glycerol was stored at -80°C for later use.

[00...

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Abstract

The invention discloses a citrus fruit epidermis high-efficiency expression promoter and application thereof. The citrus fruit epidermis high-efficiency expression promoter is named as P1080, and itsnucleotide sequence is shown in SEQ ID NO.1. It is found through quantitative PCR analysis of different tissues of citrus that the P1080 promoter regulates the strong expression activity of a downstream Cs2g31080 gene in citrus fruit epidermis and low expression in leaves, roots and pulp; the P1080 promoter is fused with a marker gene GUS, native tobacco leaves and Micro Tom tomato fruits are transiently transformed by agrobacterium EHA105 mediation, and it is proved that the P1080 promoter has similar expression activity with a positive control (strong promoter CaMV35s). The P1080 promoter can be used for constructing transgenic plants with fruit epidermis specific expression.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, and in particular relates to a high-efficiency expression promoter for citrus fruit epidermis and its application. Background technique [0002] Citrus (Citrus) is the world's largest fruit. my country is one of the origins of citrus, and its output and area account for about 1 / 4 of the world. Citrus fruit epidermis is also called yellow cortex, which is the main carrier of fruit appearance and color, and can also be used as the main edible part of some citrus varieties (such as kumquats). At the same time, the yellow cortex is rich in flavonoids, phenols and other chemically active components such as citrus essential oils, which can be used to prevent and control pests and bacteria. [0003] The expression regulation of plant genes includes pre-transcriptional regulation (such as DNA modification), transcriptional regulation, post-transcriptional regulation, translation and post-translationa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N1/21A01H5/08A01H6/82
CPCC12N15/8235
Inventor 邱文明孙中海徐育海仝铸何秀娟肖翠
Owner INST OF FRUIT & TEA HUBEI ACAD OF AGRI SCI