Eggplant flavanone 3-hydroxylase SmF3H as well as gene and application thereof

A technology of flavanone and hydroxylase, which is applied in the fields of application, genetic engineering, oxidoreductase, etc., can solve the problems of unexamined and lagging eggplant anthocyanin research, and achieve the effect of great application value

Inactive Publication Date: 2015-11-25
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of genome-related information, research on eggplant anthocya

Method used

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  • Eggplant flavanone 3-hydroxylase SmF3H as well as gene and application thereof
  • Eggplant flavanone 3-hydroxylase SmF3H as well as gene and application thereof
  • Eggplant flavanone 3-hydroxylase SmF3H as well as gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, the cloning of eggplant SmF3H gene

[0041] 1. Acquisition of plant material

[0042] The plant material used in this experiment is the excellent eggplant germplasm resource A. spp. The phenotype of Lanshanhexian eggplant was purple stem, purple flower and purple fruit. The experimental materials were cultivated in artificial plastic greenhouses in the Aerospace Breeding Base of Pujiang Town, Minhang District, Shanghai. Seedlings are raised, grown and fruited under natural conditions. The peel of eggplant was collected for RNA and DNA extraction.

[0043] 2. Extraction of RNA and DNA

[0044]Total RNA was extracted by the TRIzol method (TRIzol was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.). The integrity of RNA was identified by formaldehyde denaturing gel electrophoresis, and then the purity and concentration of RNA were determined on a spectrophotometer (ThermoScientificNANODROP1000Spectrophotometer).

[0045] Total DNA was extrac...

Embodiment 2

[0055] Example 2, Sequence Information and Homology Analysis of Eggplant F3H Gene

[0056] The full-length CDS open reading frame sequence of the new eggplant F3H gene of the present invention is 1101bp, and the detailed sequence is shown in SEQ ID NO.1. According to the CDS open reading frame sequence, the amino acid sequence of eggplant F3H is deduced, with a total of 366 amino acid residues, a molecular weight of 41077.9 Daltons, and a theoretical isoelectric point (pI) of 5.38. The detailed sequence is shown in SEQ ID NO.3. The genomic DNA of SmF3H includes 3 exons and 2 introns, and the detailed sequence is shown in SEQ ID NO.2.

[0057] The CDS open reading frame sequence of eggplant F3H and the amino acid sequence of its encoded protein were identified by BLAST program in the Non-redundantGenBank+EMBL+DDBJ+PDB and Non-redundantGenBankCDStranslations+PDB+SwissProt+Superdate+PIR databases. Source retrieval, it was found that it has 88.57% identity with the potato F3H g...

Embodiment 3

[0058] Embodiment 3, promoter cloning and sequence analysis of eggplant F3H gene

[0059] Using the GenomeWalkingKit kit (Bao Biological Engineering (Dalian) Co., Ltd.), design primers

[0060] SP1 (SEQ ID NO.9): 5'-CCCAATCTTCACATGCCTTAACAATAT-3'

[0061] SP2 (SEQ ID NO.10): 5'-CCATTCAACGATATGACTGGAATCTC-3'

[0062] SP3 (SEQ ID NO.11): 5'-AGCCACTTTTGGACGCTCTTCTTCG-3'

[0063] Using DNA as a template, three rounds of PCR amplification were performed, and the amplified product was recovered and connected to the pMD-19T (Bao Biological Engineering (Dalian) Co., Ltd.) vector, transformed into E. coli DH5α, and screened positive by α complementation and colony PCR Clones were sent to Yingwei Jieji (Shanghai) Trading Co., Ltd. for sequencing. Sequencing results found that 395bp of eggplant F3H gene promoter was obtained. The sequence was submitted to two cis-element online prediction software, PLACE and PlantCARE, for analysis, and it was found that it contained the light-resp...

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Abstract

The invention discloses eggplant flavanone 3-hydroxylase SmF3H as well as a gene and application thereof; nucleotide sequences of cDNA and gDNA of the gene are respectively as shown in SEQ ID NO.1 and SEQ ID NO.2; and the amino sequence coded by the gene is as shown in SEQ ID NO.3. The nucleotide sequence of the promoter sequence of the gene is as shown in SEQ ID NO.4. The gene is expressed in root, stem, leaf, flower, peel and pulp, and the expression amount in the stem is significantly higher than that in other tissues. Under low-temperature stress, the expression amount achieves a maximum value at 12thh, which is 2.34 times as much as that unprocessed. An over-expression vector of the eggplant SmF3H is constructed and is converted into arabidopsis thaliana, discovering that anthocyanin contents in stem and fruit pod are significantly improved. The invention provides theoretical basis for improving plant quality by virtue of gene engineering technology in the future so as to obtain highly anti-oxidative medicines or food; therefore, the eggplant flavanone 3-hydroxylase SmF3H is quite high in application value.

Description

technical field [0001] The invention relates to key enzymes and coding genes in the eggplant anthocyanin synthesis pathway, in particular to an eggplant flavanone 3-hydroxylase gene SmF3H, which belongs to the field of biotechnology. Background technique [0002] The human body will inevitably produce a large number of free radicals due to metabolic activities. As a strong reducing substance, anthocyanins can remove free radicals in the human body and delay the aging of the body. The synthetic pathway of anthocyanins is a part of the synthetic pathway of flavonoids, and it is also a very extensive and in-depth plant secondary metabolic pathway studied at present, which has been elaborated in the main model plants such as Arabidopsis and petunia. The biosynthesis of anthocyanins is mainly divided into three stages: the first stage is from phenylalanine to coumaryl-CoA, which is a common step in many secondary metabolisms; the second stage is from coumaryl-CoA to dihydroflavon...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/113C12N15/11C12N15/84C12P17/06A01H5/00
CPCY02P20/52C12N9/0071C12N15/8205C12N15/825C12P17/06C12Y114/11009
Inventor 陈火英蒋明敏刘杨任丽李静刘卫徐龙
Owner SHANGHAI JIAO TONG UNIV
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