Gene sequence related to flavone composite in radix scutellariae and application thereof

A technology of flavonoids and sequences, applied in the direction of plant gene improvement, application, genetic engineering, etc., can solve problems that have not been reported

Active Publication Date: 2018-12-21
SHANGHAI CHENSHAN BOTANICAL GARDEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since baicalein and wogonin do not contain the 4'-hydroxyl on the B ring, and no enzymes capable of removing the 4'-hydroxyl of naringenin have been reported in plants.

Method used

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  • Gene sequence related to flavone composite in radix scutellariae and application thereof
  • Gene sequence related to flavone composite in radix scutellariae and application thereof
  • Gene sequence related to flavone composite in radix scutellariae and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] This example is to clone the scutellaria baicalensis flavone synthase (SbFNSII-2) gene, flavone 6-hydroxylase (SbF6H) gene, flavone 8-hydroxylase (SbF8H) gene and construct corresponding plant overexpression vectors and yeast expression vectors.

[0050] (1) Design and synthesis of SbFNSII-2, SbF6H, SbF8H gene primers;

[0051] Firstly, the transcriptome of Scutellaria baicalensis was deeply sequenced, and then, according to the comparison of the existing SbFNSII-2 and SbF6H gene sequences in Genbank, some of the encoded proteins were selected based on the related sequences of related species reported in Labiatae plants For sequences with a similarity >60%, after drawing the phylogenetic tree, get the relevant gene sequences in Scutellaria baicalensis, use primerpremier5.0 to design the full-length primers, and add the Gateway recombination site to the primers of SbFNSII-2, SbF6H, and SbF8H point (Gateway is underlined).

[0052] SbFNSII-2-F:

[0053] GGGGACAAGTTTGT...

Embodiment 2

[0079] This example is to verify the protein functions of the Scutellaria baicalensis SbSbFNSII-2, SbF6H and SbF8H genes in the yeast system.

[0080] Express the proteins of Scutellaria baicalensis SbSbFNSII-2, SbF6H and SbF8H genes in yeast.

[0081] The yeast expression vectors pYES-dest52-SbFNSII-2, pYES-dest52-F6H, pYES-dest52-F8H were transformed into Saccharomyces cerevisiae WAT11 strain by chemical transformation method to obtain the yeast strain containing the target gene, and simultaneously transform the yeast strain containing pYES-dest52 As a negative control, the empty plasmid was grown in SD solid medium containing glucose and lacking Uracil (Ura) at 28°C for 24-48h.

[0082] Then a single clone was picked and grown in 10 mL of SD liquid medium containing glucose and lacking His at 28° C. and 200 rpm for 24 h until the OD600 reached 2-3. Collect the thalline, and wash off the glucose in the thalline with sterile water. The bacteria were resuspended in 20 mL of ...

Embodiment 3

[0087] This example is to verify the protein functions of the Scutellaria baicalensis SbFNSII-2, SbF6H, and SbF8H genes in the exogenous plant Arabidopsis thaliana.

[0088] The plant expression vectors pK7WG2R-SbFNSII-2, pH7WG2-SbF6H and pH7WG2-SbF8H were transformed into the Agrobacterium tumefaciens strain GV3101pMP90 by electric shock transformation, and the empty plasmid containing the CaMV 35S promoter was transformed into the strain as a negative control.

[0089]Wild-type Arabidopsis col-0 was used as transgenic material. Arabidopsis thaliana seeds were sown into the soil, stored in the dark at 4°C for two days, and then transferred to a culture room with a light time of 16 hours, a temperature of 23°C and a humidity of 50% for cultivation. Arabidopsis was transformed by inflorescence dipping. After harvesting the seeds, the T1 generation seeds were sown on MS medium containing kanamycin (Kan) resistance for selection, and then transferred to soil. The positive transg...

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Abstract

The invention discloses a gene related to flavone composite in radix scutellariae. The gene includes one or several in flavone synthetase gene with a sequence shown in SEQ ID No.1, flavone 6-site hydroxylase gene with a sequence shown in SEQ ID No.3 and flavone 8-site hydroxylase gene with a sequence shown in SEQ ID No.5. The invention further provides primer composite for amplifying the gene, protein encoded by the gene, a recombinant vector, host cell or transgenic cell line containing the gene and application thereof. The protein encoded by the gene disclosed by the invention can participate in synthesizing baicalein and wogonin and catalyzing synthesis and hydroxylation reaction of flavone matters of the similar structures, can provide very good basis for producing effective active matters, provides theoretical basis for producing baicalein, wogonin and aglycone of the baicalein and the wogonin in a large scale and establishes solid basis for industrially producing other related flavonoid compound.

Description

technical field [0001] The invention relates to the field of genetic engineering of medicinal plants, in particular to gene sequences involved in the synthesis of flavonoids in Scutellaria baicalensis and applications thereof. More specifically, it involves cloning a gene of flavone synthase and two genes of flavone hydroxylase, and transforming the above genes can effectively increase the content of flavone secondary metabolites in Scutellaria baicalensis. Background technique [0002] Scutellaria baicalensis, a perennial herb of the Lamiaceae Scutellaria genus, has been used as a traditional medicinal plant for two thousand years; it is mainly used to treat upper respiratory tract infections, lung-heat cough, damp-heat jaundice, pneumonia, dysentery and other diseases. Modern medical research shows that the main active ingredient in Scutellaria baicalensis is the flavonoids that exist in large quantities in its root: baicalein, wogonin and its glycosylated product baicalin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/11C12N9/02C12N15/82C12N15/81C12N1/19C12R1/865
CPCC12N9/0071C12N9/0073C12N15/81C12N15/8243C12Y114/11022
Inventor 凯西·马丁赵清崔孟颖柳洁
Owner SHANGHAI CHENSHAN BOTANICAL GARDEN
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