Application of isatis indigotica fort IiAP2/ERF049 gene to regulating and controlling synthesis of lignan compound

A technology of woad and gene, applied in the field of genetic engineering, to achieve the effect of reducing pollution, great application prospects, and increasing content

Active Publication Date: 2016-11-23
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report about the IiAP2 / ERF049 gene of Isatis indigo and its application in regulating the synthesis of lignans.

Method used

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  • Application of isatis indigotica fort IiAP2/ERF049 gene to regulating and controlling synthesis of lignan compound
  • Application of isatis indigotica fort IiAP2/ERF049 gene to regulating and controlling synthesis of lignan compound
  • Application of isatis indigotica fort IiAP2/ERF049 gene to regulating and controlling synthesis of lignan compound

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: Screening of Isatis indigo AP2 / ERF transcription factor

[0048] Arabidopsis thaliana AP2 / ERF transcription factors were downloaded from the Arabidopsis database (DATF, http: / / datf.cbi.pku.edu.cn / ) and the Chinese cabbage database (http: / / brassicadb.org / brad / ) respectively 159 (Arabidopsisthaliana AP2 / ERFs, AtAP2 / ERFs) and 321 Chinese cabbage AP2 / ERF transcription factors (Chinese cabbageAP2 / ERFs, BraAP2 / ERFs), according to their amino acid sequences to establish the Brassicaceae AP2 / ERF database. Use the TBLASTN algorithm to compare this database with the Isatis indigo transcriptome database (the e-value value is set to 10 -5 ), identify and extract genes homologous to Brassicaceae AP2 / ERF in the Isatis indigo transcriptome database as Isatis indigo AP2 / ERF candidate genes.

[0049] Bioinformatics software Vector NTI 11.5 and MEGA 5.05 were used to analyze the nucleotide sequence of the Isatis indigo AP2 / ERF candidate gene obtained above, extract the ope...

Embodiment 2

[0052] Embodiment 2: the cloning of woad IiAP2 / ERF049 gene

[0053] 1. Isatis indica DNA and total RNA extraction

[0054] Take about 100 mg of Isatis indigo sterile seedlings, grind them into powder quickly in liquid nitrogen, and use the CTAB method to extract Isatis indigo DNA; take another about 100 mg of Isatis indigo sterile seedlings, quickly grind them into powder in liquid nitrogen, add the pre-installed plant tissue cracking solution in a 1.5ml centrifuge tube, shake and mix well, and then follow the TIANGEN TRNzol-A + Total RNA was extracted from Isatis indica according to the instructions of the total RNA extraction kit. Use 1% agarose gel electrophoresis to identify the quality of DNA and total RNA, and use a Nano drop 2000C spectrophotometer (Thermo Scientific, San Jose, California, USA) to detect the purity and concentration of DNA and total RNA;

[0055] 2. Isatis indigo IiAP2 / ERF049 gene cloning

[0056] Using 1 μg of the extracted total RNA as a template, ...

Embodiment 3

[0064] Example 3: Analysis of the subcellular localization of the woad IiAP2 / ERF049 gene

[0065] According to the content of the bioinformatics analysis in Example 2, the IiAP2 / ERF049 gene is an AP2 / ERF transcription factor with an AP2 domain. In order to further verify the nature of IiAP2 / ERF049 gene as a transcription factor, we constructed the subcellular localization vector of IiAP2 / ERF049 gene, and confirmed that IiAP2 / ERF049 is localized in the nucleus by transforming rice protoplasts, which conforms to the characteristics of transcription factors.

[0066] 1. Construction of subcellular localization vector

[0067] In this embodiment, according to the pCAMBIA1301-GFP vector sequence and the IiAP2 / ERF049 gene ORF region sequence, design primers with restriction sites, wherein the forward primer contains the Nco I restriction site (5'-AA CCATGG GAATGGTGAGCTTAAGAAGG-3', SEQ ID NO: 6), the reverse primer contains a Spe I restriction site (5'-GG ACTAGT GGTAGAAAGTGTACTGA...

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Abstract

The invention relates to the field of gene engineering, in particular to an AP2/ERF family IiAP2/ERF049 transcription factor related to the content of a lignan compound in isatis indigotica fort, a nucleotide sequence for coding the transcription factor and an application of the transcription factor to increasing the content of lariciresinol in the isatis indigotica fort. An overexpression carrier of the IiAP2/ERF049 is built, the isatis indigotica fort is subjected to genetic transformation through the pivotal transcription factor IiAP2/ERF049, a bottleneck of lariciresinol synthesis is broken, and a high-yield isatis-indigotica-fort hairy-root strain is obtained; efficient regulating and controlling target points are provided for increasing the yield of the lariciresinol, and the application has enormous application prospects in the aspects that the content of the lariciresinol is increased, supplying shortage of the lariciresinol is relieved, the cost in traditional Chinese medicine extracting is reduced, pollution is reduced, and an excellent isatis-indigotica-fort strain is cultivated.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the application of the IiAP2 / ERF049 gene of Isatis indigo in regulating the synthesis of lignan compounds. Background technique [0002] Woad (Isatis indigotica Fort.), an annual or biennial herb of the Cruciferae family, was first recorded in "Shen Nong's Materia Medica", and its root is commonly used as a traditional Chinese medicine Radix Isatis, which is a commonly used antiviral Chinese herbal medicine in my country. It is used in modern clinics for influenza, Japanese encephalitis, mumps, acute and chronic hepatitis, herpes zoster, etc., and plays an important role in the prevention and treatment of atypical pneumonia (SARS) and influenza A H1N1 virus effect. Through in vitro antiviral experiments, our research group has confirmed that the lignan compounds represented by larch resin are the material basis for the antiviral activity of Isatis indigo. In recent ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82C12N1/21A01H5/00
CPCC07K14/415C12N15/8243
Inventor 马瑞芳张磊陈万生肖莹
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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