Pear transcription factor PyHY5, and recombinant expression vector and application thereof

A technology for expressing vectors and transcription factors, which is applied in the field of plant genetic engineering, can solve problems such as the regulation of anthocyanins by transcription factors that have not been seen, and achieve environmental friendliness and reduce agricultural costs.

Active Publication Date: 2019-05-28
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the study of Hongpi pear, there has been no report of transcription factors regulating anthocyanin transport-related gene GST so far.

Method used

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  • Pear transcription factor PyHY5, and recombinant expression vector and application thereof
  • Pear transcription factor PyHY5, and recombinant expression vector and application thereof
  • Pear transcription factor PyHY5, and recombinant expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Changes in the content of anthocyanins in different tissues and different development stages of 'Hongzaosu' and its green buds of the present invention

[0029] The total anthocyanin content of 'Hongzaosu' in different tissues was significantly different. At 20 days after flowering, the content of 'Hongzaosu' was 3.26 mg. g-1FW in young leaves, the young fruit was slightly lower than the leaves, and the petals and young stems were the lowest. During fruit development, the total anthocyanin content of 'Hongzaosu' fruit decreased to a relatively low level at the fruit swelling stage (60DAFB) until the fruit ripening stage (120DAFB). In 20DFAB, the highest value is 2.49mg.g-1FW, the lowest value is 0.86mg.g-1FW. The content of anthocyanins in the green mutants of ‘Hongzaosu’ was always low, with only slight changes in different tissues and developmental stages. The total anthocyanin content of the red tissue of 'Hongzaosu' was significantly higher than that of ...

Embodiment 2

[0030] Example 2 Analysis of the expression pattern of the PyHY5 gene in different tissues of 'Hongzaosu' and its green buds of the present invention

[0031] Through the analysis of RNA-seq data, it was found that the expression level of PyHY5 gene in all red tissues of 'Hongzaosu' was significantly higher than that of its green buds. In addition, there is an anthocyanin metabolism-related structural gene PyGSTf12, The expression levels of PyMYB114 (Yao et al., 2017), PyMYB10 (Zhai et al., 2015) were consistently higher in red tissues than in green tissues ( figure 1 ). The forward primer used for RT-qPCR analysis of PyGSTf12 is 5'-GGTGATGGTTTGCCTTTTGG-3' (SEQ ID NO. 15), the reverse primer is 5'-AACTCGGGTCGCTTGTGCT-3' (SEQ ID NO.16); the forward primer used by PyHY5 It is 5'-GGGCCATCATAAGGTACTACGC-3' (SEQ ID NO.9), and the reverse primer is 5'-CACCAAATGCCCCAGTTTAGC-3' (SEQ ID NO.10); the forward primer of PyMYB114 gene is 5'-GCAGACAGAGGTGGTTGAACT-3' ( SEQ ID NO.11), the re...

Embodiment 3

[0032] Example 3 The cloning of PyHY5 gene and the construction of recombinant vector in 'Red Zaosu' of the present invention

[0033] RNA was extracted from the peel of 'Hongzaosu' pear, and the first-strand cDNA obtained by reverse transcription was used to amplify the full-length PyHY5 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). The amplification gene primer pair is PyHY5-F1: 5'-CGCTCTAGAACTAGTggatccATGCAAGAGCAGGCGACG-3' (SEQ ID No.3); PyHY5-R1: 5'-GTCGACGGTATCGATaagcttTCAATCCGCATTTGCACTACC-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 amplification i...

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Abstract

The invention discloses a pear transcription factor PyHY5 and application of a recombinant expression vector of the pear transcription factor PyHY5. The nucleotide sequence of a transcription factor PyHY5 gene which is separated from 'Red Zaosu' pears and has a function of promoting anthocyanin biosynthesis of pear peels is as shown in SEQ ID No. 1, and the encoded amino acid sequence of the transcription factor PyHY5 gene is as shown in SEQ ID No. 2. Transcription factors PyHY5 and PyMYB114 are co-transformed into strawberry and pear fruits by an agrobacterium-mediated transformation method;according to biological function verification, the cloned PyHY5 gene and a cofactor PyMYB114 thereof interact to promote the transport function of anthocyanin in the pear peels; through discovery of new functions of the PyHY5, new genetic resources are provided for molecular breeding that promotes accumulation of the anthocyanin in the pear peels, and new genetic resources are provided for the implementation of green agriculture; the development and utilization of the genetic resources is beneficial to agricultural cost reduction and environmental friendliness.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and relates to pear transcription factor PyHY5 and its recombinant expression vector and application, in particular to a bZIP family member PyHY5 that is involved in the biosynthesis of anthocyanins in pear peels by separating and cloning from 'Hongzaosu' pears Genes and their applications. Background technique [0002] The stems, leaves, flowers, and fruits of plant tissues exhibit a range of bright colors ranging from red to purple, and this bright color is associated with the accumulation of anthocyanins. Anthocyanins are water-soluble pigments that play an important role in plant disease resistance and protection against ultraviolet radiation (Bieza et al., 2001), and are generally considered to have antioxidant capacity (Veeriah et al., 2006), preventing human It plays an important role in neurological and cardiovascular diseases, cancer and diabetes (Konczak et al., 2004). [0003...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/08A01H6/74
Inventor 吴俊姚改芳张绍铃杨广艳张明月汪润泽谢智华
Owner NANJING AGRICULTURAL UNIVERSITY
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