Tulipa fosteriana flavonol synthase TfFLS protein and coding gene thereof

A flavonol synthase, synthase technology, applied in genetic engineering, plant gene improvement, enzymes and other directions, can solve the problem of lack of coding gene sequence and so on

Inactive Publication Date: 2014-04-30
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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  • Tulipa fosteriana flavonol synthase TfFLS protein and coding gene thereof
  • Tulipa fosteriana flavonol synthase TfFLS protein and coding gene thereof
  • Tulipa fosteriana flavonol synthase TfFLS protein and coding gene thereof

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Experimental program
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Effect test

Embodiment 1

[0044] The cloning of embodiment 1, tulip TfFLS gene

[0045] 1. Acquisition of plant material

[0046] Healthy, uniform-sized tulip bulbs (Tulipa fosteriana 'Shangnongzaoxia', approved by the Shanghai Crop Variety Approval Committee. Number: Shanghai Nongpin Huahua 2011 No. 004) were planted as usual and managed in the field until the flowers were fully open. Petal tissue was collected when the petals were fully colored for RNA extraction.

[0047] 2. Extraction of RNA

[0048] Total RNA was extracted using the "RNA prep pure plant total RNA extraction kit" (RNA prep pure Plant Kit: Tiangen Biochemical Technology (Beijing) 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 (Thermo Scientific NANODROP1000 Spectrophotometer).

[0049] 3. Full-length cloning of genes

[0050] According to the conserved amino acid sequences of FLS genes in other s...

Embodiment 2

[0059] Example 2, Sequence Information and Homology Analysis of Tulip TfFLS Gene

[0060]The full-length CDS open reading frame sequence of tulip TfFLS is 999bp, and the detailed sequence is shown in SEQ ID NO.1; the amino acid sequence of tulip TfFLS is deduced according to the CDS open reading frame sequence, with a total of 332 amino acid residues and a molecular weight of 37672 Dalton, the isoelectric point (pI) is 5.39, and the detailed sequence is shown in SEQ ID NO.2.

[0061] The CDS open reading frame sequence of tulip TfFLS and the amino acid sequence of its encoded protein were nucleated in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR databases using BLAST program Nucleotide and protein homology searches. Table 2 is the homology comparison (GAP) result of the nucleotide sequence of tulip TfFLS gene of the present invention and onion (Alliumcepa) FLS gene mRNA; As can be seen from Table 2, tulip TfFLS g...

Embodiment 3

[0068] Example 3, the expression difference of TfFLS gene of tulip in different developmental stages of flowers and in different tissues of tulip

[0069] 1. Acquisition of materials

[0070] During the four different developmental stages of tulip flowers (buds, petals are not colored; buds, petals are beginning to color; flowers are partially open, petals are not fully colored; flowers are fully open, petals are fully colored), the bulbs, aboveground stems, leaves and For the petals (mixed samples of petals at each coloring stage), the samples were wrapped in aluminum platinum paper and immediately put into liquid nitrogen, and then transferred to a -80°C ultra-low temperature refrigerator for storage until use.

[0071] 2. Extraction of RNA

[0072] RNA prep pure plant total RNA extraction kit (RNA prep pure Plant Kit: Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract the petals of tulip flowers at different developmental stages and the RNA in differ...

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Abstract

The invention discloses a Tulipa fosteriana flavonol synthase TfFLS protein and a coding gene thereof. The protein is a protein composed by an amino acid sequence shown in SEQ ID No. 2 or a protein composed of an amino acid sequence obtained through replacement, deletion or addition of one or more amino acids of the amino acid sequence shown in the SEQ ID No. 2 and having the activity of Tulipa fosteriana flavonol synthase. The invention further provides a nucleic acid sequence used for coding the protein, and the nucleic acid sequence is shown in SEQ ID No. 1. The Tulipa fosteriana flavonol synthase TfFLS gene provided by the invention expresses a recombinant flavonol synthase protein in Escherichia coli cells and enables dihydrokaempferol to produce kaempferol, which proves that the gene has the functions of flavonol synthase. With the TfFLS gene, substances, receptors, inhibitors, antagonists or the like interacting with TfFLS can be screened by using a variety of conventional screening methods.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and relates to a key enzyme in the synthetic pathway of tulip flavonoids and its coding gene, in particular to a tulip flavonol synthase TfFLS protein and its coding gene. Background technique [0002] Flavonoids are a class of polyphenolic compounds that widely exist in nature. According to different modifications to the central C ring, they can be divided into major categories such as flavonoids, isoflavones, flavanones, flavonols, flavanols, and anthocyanins. Among them, flavonoid alcohols are the most common, accounting for about 1 / 3 of the total flavonoids. Flavonols are widely distributed in a variety of plants. They are inhibitors of several kinases related to intracellular signal transduction and cell transformation, and have a variety of biological activities, because they can selectively block intracellular signal transduction pathways , so flavonoids are considered as potential ...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/53C12N15/70
CPCC12N9/0071C12Y114/11023
Inventor 袁媛史益敏唐东芹马晓红陶秀花
Owner SHANGHAI JIAO TONG UNIV
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