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Coding sequences and applications of 4-coumarate coenzyme A ligase protein family of isatis indigotica fortune

A technology of coumaroyl coenzyme and protein family, which is applied in the biological field and can solve the problems such as the undiscovered Isatis indigotica 4-coumaroyl coenzyme A ligase protein and the like

Inactive Publication Date: 2015-07-29
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Have not yet found relevant reports related to Isatis indigo 4-coumaroyl-CoA ligase protein and application mentioned in the present invention

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  • Coding sequences and applications of 4-coumarate coenzyme A ligase protein family of isatis indigotica fortune
  • Coding sequences and applications of 4-coumarate coenzyme A ligase protein family of isatis indigotica fortune
  • Coding sequences and applications of 4-coumarate coenzyme A ligase protein family of isatis indigotica fortune

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Embodiment 1

[0020] Cloning of Isatis indigo 4-coumaroyl-CoA ligase family gene

[0021] 1. Tissue Isolation

[0022] The leaves of Isatis indigo (I. indigotica) were collected from the Botanical Garden of the School of Pharmacy, Second Military Medical University. After collection, the leaves were immediately frozen in liquid nitrogen.

[0023] 2. Isolation of mRNA

[0024] Take part of the tissue, grind it with a mortar, add it to a 50ml tube filled with lysate, shake it fully, and then transfer it into a glass homogenizer. After homogenization, transfer to a new 50ml tube, and use TRIzol (TRIzol Reagents, Gibco, NY, USA) reagent to extract total RNA. The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis.

[0025] 3. Cloning of the gene

[0026] 3.1. Synthesis of first-strand cDNA

[0027] 3.2. PCR amplification of the coding regions of Isatis indigo 4-coumaroyl-CoA ligase family genes (Ii4CL1, Ii4CL2, Ii4CL3)

[0028] According to the coding seque...

Embodiment 2

[0042] Sequence information and homology analysis of Isatis indigo Ii4CL gene family

[0043] Ii4CL1 full-length cDNA is 1967bp (GenBank accession number: GU937875 ), containing a 1629bp open reading frame (ORF), encoding 543 amino acids, an isoelectric point (pI) of 8.59, and a molecular weight of about 59.5kDa;

[0044] The full-length cDNA of Ii4CL2 is 1975bp (Genbank accession number: KC430622 ), containing a 1692bp ORF, encoding 564 amino acids, with an isoelectric point (pI) of 5.09 and a molecular weight of about 62.2kDa;

[0045] The full-length cDNA of Ii4CL3 is 1932bp (Genbank accession number: KC430623), contains a 1629bp ORF, encodes 543 amino acids, has an isoelectric point (pI) of 5.52, and a molecular weight of about 59.1kDa.

[0046] BLAST results showed that Ii4CL1, Ii4CL2, Ii4CL3 had high similarity with other plant 4CLs proteins. figure 1 is the alignment of the amino acid sequence of Ii4CL protein with the amino acid sequence of 4CL protein in Arabido...

Embodiment 3

[0048] Preparation and enzyme activity assay of Isatis indigo Ii4CL family protein

[0049] According to the protein coding sequences of Isatis indigo Ii4CL1, Ii4CL2 and Ii4CL3, primers for amplifying the complete coding reading frame (corresponding to about 20 or more nucleotides at the 5' and 3' ends of the coding sequence respectively) were designed respectively, and the forward and reverse primers Restriction endonuclease sites (this depends on the selected Pet32a vector) were introduced on the above, so as to construct the expression vector. Ii4CL1, Ii4CL2 and Ii4CL3 were cloned into the Pet32a vector under the premise of ensuring the correct reading frame. The identified expression vectors were transformed into Escherichia coli DH5α using the CaCl2 method, and the engineering bacteria DH5α-pet32a-Ii4CL1, DH5α-pet32a-Ii4CL2, DH5α-pet32a respectively containing the expression vectors of pet32a-Ii4CL1, pet32a-Ii4CL2, and pet32a-Ii4CL3 were screened and identified -Ii4CL3....

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Abstract

The present invention provides three sections of coding sequences of 4-coumarate coenzyme A ligase protein family of isatis indigotica fortune, wherein the coding sequences are respectively represented by SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3. The present invention further provides amino acid sequences of the 4-coumarate coenzyme A ligase protein family of the isatis indigotica fortune, wherein the amino acid sequences are characterized in that the amino acid sequences are represented by SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6. The present invention further provides applications for changing biosynthesis of lariciresinol by regulating the expression of the 4-coumarate coenzyme A family protein of the isatis indigotica fortune. According to the present invention, the important application values are provided in the fields of breeding and biological energy sources.

Description

technical field [0001] The present invention relates to the nucleotide sequence of Isatis indigo 4-coumaroyl-CoA ligase protein family and its application. The present invention also relates to the amino acid sequence of Isatis indigo 4-coumaroyl-CoA ligase protein family, belonging to the field of biotechnology . Background technique [0002] Isatis indigo is a cruciferous plant, and its root is used as medicine. It is the commonly used traditional Chinese medicine Radix Isatidis. , Japanese encephalitis, mumps, acute and chronic hepatitis, herpes zoster, etc. The study of chemical constituents and pharmacological activity showed that agaricin is an important active substance basis for the antiviral effect of Isatis indigo. Therefore, improving the content of agaricin is the key to obtaining high-quality Isatis indica strains with high antiviral activity, and has important application value; meanwhile, alaricin belongs to the lignans, and reduces the content of alaricin i...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N9/00A01H5/00
Inventor 肖莹周洵杨颖博陈万生张磊
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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