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Tulip flavanone-3-hydroxylase TfF3H protein and encoding gene thereof

A technology of flavanones and tulips, applied in genetic engineering, oxidoreductase, plant genetic improvement, etc., can solve problems such as unexamined

Inactive Publication Date: 2014-03-05
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no literature report related to tulip F3H protein and its coding gene sequence

Method used

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  • Tulip flavanone-3-hydroxylase TfF3H protein and encoding gene thereof
  • Tulip flavanone-3-hydroxylase TfF3H protein and encoding gene thereof
  • Tulip flavanone-3-hydroxylase TfF3H protein and encoding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 , the cloning of tulip TfF3H gene

[0048] 1. Acquisition of plant material

[0049] Healthy, uniform-sized tulip bulbs (Tulipa fosteriana 'Shangnongzaoxia', approved by the Shanghai Crop Variety Approval Committee. No.: Shanghai Nongpin Huahua 2011 No. 004) were planted and managed in the field according to the routine, and the flowers were fully opened. When the petals are fully colored, the petal tissue is collected for RNA extraction;

[0050] 2. Extraction of RNA

[0051] Total RNA was extracted with "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 NANODROP1000Spectrophotometer);

[0052] 3. Full-length cloning of genes

[0053] According to the amino acid conserved sequence of F3H gene in ...

Embodiment 2

[0065] Example 2 , Sequence Information and Homology Analysis of TfF3H Gene of Tulip

[0066] The full-length CDS open reading frame sequence of tulip TfF3H of the present invention is 1101bp, and the detailed sequence is as shown in SEQ ID NO.3. According to the CDS open reading frame sequence, the amino acid sequence of tulip TfF3H is deduced, with a total of 366 amino acid residues , the molecular weight is 40669.2 Daltons, the isoelectric point (pI) is 5.10, and the detailed sequence is shown in SEQ ID NO.4;

[0067] The CDS open reading frame sequence of Tulip TfF3H and the amino acid sequence of its encoded protein were nuclearized in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR databases using the BLAST program Nucleotide and protein homology search, it was found that it has 83% identity with the lily F3H gene (GenBank accession number AB699160.1) at the nucleotide level, such as figure 1 and figure ...

Embodiment 3

[0068] Example 3 , Expression differences of tulip TfF3H gene in different developmental stages of flowers and in different tissues of tulip

[0069] 1. Obtaining materials: During the four different development stages of tulip flowers (buds, petals are not colored; buds, petals are beginning to be colored; flowers are partially open, petals are not fully colored; flowers are fully open, petals are fully colored), and other tulips are collected in the field. For the petals, the leaves, aboveground stems, stamens, pistils, and petals (the mixed samples of petals at each coloring stage) were collected at the same time, and the samples were wrapped in aluminum platinum paper and immediately put into liquid nitrogen, and then transferred to -80°C ultra-low temperature Store in the refrigerator for later use;

[0070] 2. Extraction of RNA: RNA prep pure Plant Total RNA Extraction Kit (RNA prep pure Plant Kit: Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract...

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Abstract

The invention relates to a tulip flavanone-3-hydroxylase TfF3H protein and an encoding gene thereof. The protein is composed of amino acid sequences represented by the SEQ ID No.4, or the protein is composed of amino acid sequences which have been processed by replacing, removing, or adding one or several amino acids, and has a tulip flavanone-3-hydroxylase activity. The invention also provides a nucleic acid sequence which encodes the protein mentioned above. The flavanone-3-hydroxylase is an important branch point catalyzing enzyme in the flavonoid synthesis route, and is a pivot of a whole flavonoid compound metabolic route. After the tulip flavanone-3-hydroxylase gene is transferred to orange red morning glories through establishing an antisense expression vector, flower color of more than 50% of genetically modified morning glories becomes lighter, and the total anthocyanin content of corresponding flowers is reduced by 30% to 50%. By utilizing the tulip-flavanone-3-hydroxylase gene TfF3H, people can change the flower color by utilizing a gene engineering technology, and obtain an effective technical means for creating novel flower colors.

Description

technical field [0001] The invention relates to a key enzyme in the synthetic pathway of tulip anthocyanins and its coding gene, in particular to a tulip flavanone-3-hydroxylase TfF3H protein and its coding gene, and belongs to the field of biotechnology. Background technique [0002] Flavonoids (Flavonoids) include anthocyanins and other flavonoid pigments, which are a large class of secondary metabolites in plants, which make the flowers, leaves, fruits, and stems of plants produce rich colors, and play a role in insect pollination, auxin transportation, and protection. It plays an important role in protecting leaves from ultraviolet rays, inhibiting pests and diseases, and inducing root nodules. In the synthetic pathway of flavonoids, flavanone-3-hydroxylase (F3H) is the third enzyme following flavanone-3-hydroxylase (CHS) and chalcone isomerase (CHI). Studies have shown that F3H is an important branch point catalytic enzyme, which may be the center of the whole metaboli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53
CPCC12N9/0071C12Y114/11009
Inventor 袁媛史益敏唐东芹马晓红
Owner SHANGHAI JIAOTONG UNIV
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