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Pyrus transcription factor PyERF3 as well as recombinant expression vector and application thereof

A technology for expressing vectors and transcription factors, which is applied in the field of plant genetic engineering to reduce agricultural costs and achieve environmental friendliness.

Active Publication Date: 2018-02-13
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few reports on AP2 / ERF transcription factors involved in anthocyanin biosynthesis

Method used

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  • Pyrus transcription factor PyERF3 as well as recombinant expression vector and application thereof
  • Pyrus transcription factor PyERF3 as well as recombinant expression vector and application thereof
  • Pyrus transcription factor PyERF3 as well as recombinant expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Expression analysis of the PyERF3 gene of the present invention in red-skinned pear varieties with different coloring patterns

[0035]It has been shown in previous reports that anthocyanin biosynthesis is regulated by the transcriptional regulatory complex. We selected the transcription factors screened in the red / green pericarp RNA-seq data (Yang et al., 2015) of the previous study and performed correlation analysis with the candidate gene PyMYB114. Interestingly, five APETALA2 / ERFs (AP2 / ERFs) transcription factors were found to be up-regulated in the pericarp of red-skinned pear 'Red Eggplant', and showed a consistent expression pattern with PyMYB114. The naming method of the AP2 / ERFs gene is based on the gene with the highest similarity sequence in the comparison results of the NCBI database as the reference. In this study, we selected 6 red-skinned pear varieties at different development stages to verify whether the expression pattern of the candidate ge...

Embodiment 2

[0039] Cloning and recombinant vector construction of embodiment 2 PyERF3 gene of the present invention

[0040] RNA was extracted from the peel of 'Red Eggplant' pear, and the first-strand cDNA obtained by reverse transcription was used to amplify the full-length PyERF3 gene. RNA was extracted using Plant Total RNA Isolation Kit Plus (Foregene, RE-05022), and operated according to the operating instructions provided by the kit. First-strand cDNA was synthesized using First Script Strand cDNASynthesis SuperMix (Transgene, AE301-02) reverse transcription kit (operated according to the instructions provided by the kit). Amplified gene primer pair is PyERF3-F1: 5'-ACTAGTGGATCCAAA gaattc ATGTTTTTGGGGTACAGTCGGG-3' (SEQ ID No. 3); PyERF3-R1:

[0041] 5’-CAGGACTCTAGAAGTACT ctcgag TCAACTGGATGAGGATGGATTGTTGC-3' (SEQ ID No. 4). Ultra-Fidelity DNA Polymerase Super-Fidelity DNA Polymerase (P505-d1) was purchased from Novozyme Biotechnology Company. The reaction system for amplif...

Embodiment 3

[0044] Example 3 Co-transformation of the PyERF3 gene of the present invention with other co-acting factors PyMYB114 and PybHLH3 leads to the synthesis of tobacco anthocyanins

[0045]The constructed recombinant vector was used to transiently transform tobacco leaves mediated by Agrobacterium. The results showed that the accumulation of anthocyanin content could not be observed by transforming one transcription factor alone, but the accumulation of a small amount of anthocyanin in tobacco leaves could be observed by co-transformation of two transcription factors PyMYB114 and PybHLH3. However, co-transformation of PyERF3 with three transcription factors, PyMYB114 and PybHLH33, can accelerate anthocyanin biosynthesis, showing deeper color and stronger response ( figure 2 A). In addition, co-transformation of PyMYB114 and PyMYB10 also resulted in weak pigmentation. Tobacco leaves with anthocyanin accumulation were tested for pigment content by colorimeter and spectrophotometer...

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Abstract

The invention discloses a pyrus transcription factor PyERF3 as well as a recombinant expression vector and application thereof. A transcription factor PyERF3 gene which is separated from 'Starkrimson'pyrus and has an effect of promoting pyrus peel anthocyanin biosynthesis has a nucleotide sequence shown as SEQ ID No.1, and a coded amino acid sequence is shown as SEQ ID No.2 in a sequence table. The transcription factor is subjected to instantaneous conversion in tobacco, strawberry and pear fruits by virtue of an agrobacterium mediated genetic transformation method. Biological function verification proves that the PyERF3 gene cloned in the invention and other transcription factors PyMYB114 and PybHLH3 form transcriptional control complex so as to promote the biosynthesis of the pyrus peelanthocyanin. Discovery of the gene PyERF3 provides novel gene resources for promoting molecular breeding of pyrus peel anthocyanin synthesis and provides novel genetic resources for implementing green agriculture. Development and utilization of the genetic resources contribute to reduction of the agricultural cost and realization of environment friendliness.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and relates to pear transcription factor PyERF3 and its recombinant expression vector and application, in particular to an AP2 / ERF family member involved in the biosynthesis of anthocyanins in pear peels, which is isolated and cloned from 'Red Eggplant' pears PyERF3 gene and its application. Background technique [0002] Anthocyanin is a plant secondary metabolite biosynthesized in the flavonoid metabolic pathway, which is a water-soluble pigment. Widely present in the plant kingdom, it makes stems, leaves, flowers and fruits appear brightly colored. Anthocyanins in flowers mainly attract pollinators, while anthocyanins in seeds and fruits are synthesized to attract birds and animals for feeding, which is more conducive to seed dispersal (Holton et al., 1995). Anthocyanins also play an important role in plant disease resistance, such as in UV protection, antioxidant activity (Bieza et a...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/08A01H6/74
CPCC07K14/415C12N15/8261
Inventor 吴俊姚改芳张绍铃明美玲谷超汪润泽杨广艳刘海楠
Owner NANJING AGRICULTURAL UNIVERSITY
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