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Strobilanthes cusia BcTSA gene, protein encoded by same and application of Strobilanthes cusia BcTSA gene

A horse blue and gene technology, applied in the field of horse blue BcTSA gene and its encoded protein, can solve the problems that there are no protein reports yet

Active Publication Date: 2019-07-05
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] After searching the literature of the prior art, it is found that there is no report about the horse blue BcTSA gene and its encoded protein

Method used

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  • Strobilanthes cusia BcTSA gene, protein encoded by same and application of Strobilanthes cusia BcTSA gene
  • Strobilanthes cusia BcTSA gene, protein encoded by same and application of Strobilanthes cusia BcTSA gene
  • Strobilanthes cusia BcTSA gene, protein encoded by same and application of Strobilanthes cusia BcTSA gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Horse Blue BcTSA Gene Screening

[0057] 1. Transcriptome data acquisition of Malan root, stem and leaf materials

[0058] TRNzolA+ method was used to extract the total RNA of 9 samples of roots, stems, and leaves of different organs of the horseshoe crab, and Agilent 2100 Bioanalyzer was used to detect the quality of RNA and determine its concentration. According to the instructions of Oligotexm RNA Midi Kit (Qiagen), mRNA was isolated and enriched from qualified total RNA, and the first strand of cDNA was synthesized according to the instructions of SMART PCR cDNA synthesis kit (Clonetech), and then according to the instructions of Advantage 2PCR kit (Clonetech) cDNA second strand synthesis. According to the instructions of the Pure PCR purification kit (Invitrogen), the >300bp dsDNA was recovered, and PolyA was removed by enzyme digestion. According to the instructions of the QIAquick PCR Purification Kit (Qiagen), the above digested products were purifi...

Embodiment 2

[0066] Embodiment 2: Cloning of horse blue BcTSA gene

[0067] 1. Extraction of total RNA and DNA from the horse blue genome

[0068] Take an appropriate amount of fresh horse blue leaves and quickly grind them into powder in liquid nitrogen, then take about 100mg of powder and add it to a 1.5ml EP tube pre-filled with plant tissue lysate, shake and mix well, and then follow the TIANGEN RNAprep Pure plant total RNA respectively and TIANGEN Plant Genomic DNA (gDNA) Extraction Kit instructions to extract horse blue total RNA and gDNA. The quality of RNA and gDNA was identified by agarose gel electrophoresis, and then the RNA and gDNA concentrations were determined on a NanoDrop 2000C.

[0069] 2. Cloning of horse blue BcTSA gene

[0070] Using the extracted total RNA as a template, horse blue cDNA was synthesized using the Whole Gold TransScript First-Strand cDNASynthesis Supermix kit.

[0071] Design gene-specific primers based on the sequence of the BcTSA gene:

[0072] Fo...

Embodiment 3

[0077] Embodiment 3: Bioinformatics analysis of horse blue BcTSA gene

[0078] Using SMART (http: / / smart.embl-heidelberg.de / ) software, it is predicted that the BcTSA protein contains a typical Trp_syntA conserved domain and the Trp_syntA domain covers the amino acid length from position 57 to position 314 on the entire protein sequence (such as figure 2 Shown in A) is the signature conserved domain of TSA proteins, and most of the proteins containing this domain in plants are proteins involved in the synthesis of tryptophan.

[0079] Through SWISS-MODEL, in which TSA uses Crystal structure of Tryptophan synthasealpha chain homolog BX1 (PDB id: 1rd5.1) as the reference template, the sequence identity between TSA and the reference template reached 59.46%, and the three-dimensional structure of BcTSA protein was constructed (such as figure 2 Shown in B), from the structural point of view, BcTSA contains all the functional domains of 1rd5.1. BX1 is a representative enzyme that...

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Abstract

The invention relates to a Strobilanthes cusia BcTSA gene, protein encoded by the same and application of the Strobilanthes cusia BcTSA gene. The nucleotide sequence of the Strobilanthes cusia BcTSA gene is as shown in SEQ ID No. 1, and the nucleotide sequence of the protein encoded by the Strobilanthes cusia BcTSA gene is as shown in SEQ ID No. 2. The BcTSA gene is separated from Strobilanthes cusia, cloning and biological information analysis of the Strobilanthes cusia BcTSA gene is completed, and the understanding of indole alkaloid biological synthesis is perfected. The Strobilanthes cusiaBcTSA gene can control indole alkaloid synthesis, the protein encoded by the Strobilanthes cusia BcTSA gene is applicable to the indole alkaloid synthesis and used for increasing the yield of indolealkaloid, and a high-yield and stable plant material is provided for the large-scale production of the indole alkaloid.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a horse blue BcTSA gene and its encoded protein and application. Background technique [0002] Baphicacanthus cusia, widely distributed in Southwest my country, South China and East China, is an important medicinal plant of Acanthaceae. Indigo Daisy, which is processed from its stems and leaves, is the best quality product in Fujian. It is known as Jianqingdai and is an authentic medicinal material in Fujian. The main functional ingredients of horse blue and Qingdai are indigo and indirubin. In particular, indirubin has become the index component of Qingdai and its original plant Malan. Indirubin is a bisindole alkaloid, which is considered to have anti-tumor effects. It is clinically used to treat chronic myeloid leukemia and is a Chinese patent medicine " The main active ingredients of "Huangdai Tablets" and "Danggui Aloe Vera Pills". At present, the researches on S. indigo and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/82A01H5/00A01H6/20A01H6/46
CPCC12N9/88C12Y402/0102C12N15/8243
Inventor 张磊刁勇郭志英谭何新黄豆豆陈越
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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