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RcTAT gene, RNAi expression vector and application thereof

An expression vector and gene expression technology, applied in the field of genetic engineering to achieve the effect of increasing the content

Active Publication Date: 2021-02-19
TIBET AGRI & ANIMAL HUSBANDRY COLLEGE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, whether there is a tyrosine transamination pathway in the biosynthetic pathway of salidroside, that is, tyrosine generates 4-HPAA through a series of reactions under the action of TAT and 4-hydroxy-phenylpyruvate decarboxylase is currently unknown. See the report

Method used

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  • RcTAT gene, RNAi expression vector and application thereof
  • RcTAT gene, RNAi expression vector and application thereof
  • RcTAT gene, RNAi expression vector and application thereof

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Experimental program
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Effect test

Embodiment 1

[0028] Embodiment 1, cloning of tyrosine aminotransferase (Tyrosine Aminotransferase, TAT) gene

[0029] Total RNA was isolated from different tissues of the plant material Rhodiola rosea using an RNA extraction kit (Tiangen Biotech, Beijing, China) according to the manufacturer's protocol. The integrity of the RNA was detected by agarose gel electrophoresis, and the RNA concentration was measured by NanoDrop 2000C (Thermo Fisher Scientific Inc, MA, USA) and the purity of the RNA was determined by the OD260 / OD280 value. cDNA was synthesized according to the instructions of the first-strand synthesis kit. The synthesized cDNA was stored in a -20°C refrigerator for subsequent gene cloning.

[0030] According to the database on the NCBI website, download the nucleotide sequences of TAT from other species that have been reported. At the same time, the comparison was carried out through the BLAST plate, and then the primers for amplifying the full length of Rhodiola daflora were ...

Embodiment 2

[0036] Example 2, analysis of tissue expression pattern and induction pattern of RcTAT

[0037] According to the RcTAT sequence, real-time fluorescent quantitative PCR (qPCR) technology was used to detect the relative expression level of RcTAT in the roots and leaves of Rhodiola grandiflora, and the internal reference was phosphoglycerate kinase gene (PGK). For qPCR analysis, kits purchased from Bio-Rad were used; the qPCR system was Bio-Rad IQ5. For each sample and the PGK internal reference gene, 3 replicates and 1 negative control were set up, and the relative quantification of gene expression was carried out with the internal reference gene PGK as the standard, using 2 -ΔΔC(t) Calculation of gene expression in each tissue. At the same time, the melting curve analysis was carried out after the amplification, and the procedure was as follows: gradually increase from 60°C to 95°C, take a reading every 0.5°C, keep warm for 5 seconds each time, and continuously record the chan...

Embodiment 3

[0039] Example 3, prokaryotic expression verification of the function of the RcTAT gene

[0040] 1. Prokaryotic expression and protein purification of RcTAT

[0041] The coding region of RcTAT was cloned using primers containing KpnI and HindIII restriction sites, and the coding region was inserted into the protein expression vector pQE30 by restriction ligation to obtain the RcTAT prokaryotic expression vector pQE30-RcTAT. The primer sequences are as follows:

[0042] RcTAT-KpnI-F: 5′- ggggtacccc atggagcaagaagcagcagc-3' (SEQ ID NO. 13);

[0043] RcTAT-HindIII-R: 5′- cccaagcttggg tttcgcgaccctgtcataca-3' (SEQ ID NO. 14).

[0044] The recombinant vector was introduced into Rosseta Escherichia coli for recombinant protein expression. Shake culture at 37°C in LB medium with chloramphenicol and ampicillin, bacterial solution OD 600 When it reaches 0.6, add 0.5mM IPTG, induce at 25°C for 6 hours, collect the bacteria by centrifugation at 8000rpm for 5min, and resuspend 50m...

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Abstract

The invention discloses an RcTAT gene, an RNAi expression vector and an application thereof. The RcTAT gene sequence is shown as SEQ ID No. 12. After the rhodiola crenulata TAT gene is subjected to interference expression, the contents of bioactive substances tyrosol and salidroside in the hairy root of rhodiola crenulata are obviously increased, which indicates that the expression quantity of RcTAT and the synthetic quantity of salidroside are in negative correlation, namely, the RNAi interference TAT expression can effectively increase the content of salidroside, and RcTAT does not participate in the synthesis of salidroside but is a potential competitive branch of salidroside biosynthesis. Therefore, a new high-yield salidroside variety can be obtained by silencing the RcTAT gene, and the RcTAT gene has excellent market prospect and economic value.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to Rhodiola daflora TAT (RcTAT) gene, an RNAi carrier constructed by using the gene and application thereof. Background technique [0002] Rhodiola grandis belongs to the Rosaceae Sedum family plant, and it mainly grows in alpine grasslands and rock crevices at an altitude of 3500-5000 meters in Tibet. Although it grows in a very harsh environment, it has become a very rare Tibetan medicine plant because of its special adaptability. It also enjoys the reputation of "snowy ginseng" and "plant gold" among the people. At present, many companies and enterprises have put Rhodiola rosea as a raw material to produce oral liquids, beverages, medicines, food, etc., which have been put on the market in large quantities. The dual-purpose effect of Rhodiola daflora for medicine and food also makes its demand continue to increase. [0003] The active ingredients in Rhodiola rosea are...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/113C12N15/82A01H5/12A01H6/74
CPCC12N9/1096C12N15/113C12N15/8218C12N15/8243C12Y206/01005C12N2310/14
Inventor 兰小中廖志华陈敏权红曾俊岚
Owner TIBET AGRI & ANIMAL HUSBANDRY COLLEGE
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