Construction of Plant Transient Expression Analysis Vector Matching With Yeast Single Hybrid System
College of life science and engineering, Lanzhou University of technology, Gansu 730050
Field Crop, 2020, Vol. 3, No. 5
Received: 04 Jun., 2020 Accepted: 06 Jun., 2020 Published: 06 Jun., 2020
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This article was first published in Molecular Plant Breeding ( ISSN1672-416X，CN46-1068/S) in Chinese, and here was authorized to translate and publish the paper in English under the terms of Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
In order to verify the plant transcription factors and their key domains resolved by yeast single hybrid system in plant cells/protoplasts, a set of plant transient expression analysis system matched with yeast single hybrid system was constructed, and transient expression analysis was carried out in Arabidopsis leaf protoplasts. The results showed that the reporter gene GUS could be activated in Arabidopsis protoplasts by the effector vector constructed from the transcriptional activation region (CRII) of AtDPBF4. This result is consistent with the result of CRII of AtDPBF4 in yeast cells. In the report vector, six heterozygous promoters containing UASGal1 could induce the GUS transcription of the downstream report gene with an enzyme activity of 0.96 nmol 4-MU·mg-1·min-1; three heterozygous promoters containing UASGal1 could induce GUS gene with an activity of 0.73 nmol 4-MU·mg-1·min-1. The heterozygous promoter with 6 UASGal1 was stronger than that with 3 UASGal1, but its activity was not twice as high as that of 3 UASGal1, only 31.5% higher than that of 3 UASGal1. This indicates that increasing the repeat sequence of the GAL4 binding site (UASGal1) in the reporter gene can increase the expression of the downstream reporter gene, but this increase in activity does not have a linear relationship with the number of repeats of UASGal1.
Yeast single hybrid system; Plant transient expression analysis system; Vector construction
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