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A kind of highland barley feruloyl tyrosyltransferase gene and its use

A technology of feruloyl tyramide and transgenic plants, which is applied in the field of genetic engineering and can solve problems such as the absence of feruloyl tyramide in barley

Active Publication Date: 2021-09-10
AGRI RES INST TIBET ACADEMY OF AGRI & ANIMAL HUSBANDRY SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There is no report of feruloyl tyramine in highland barley

Method used

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  • A kind of highland barley feruloyl tyrosyltransferase gene and its use
  • A kind of highland barley feruloyl tyrosyltransferase gene and its use
  • A kind of highland barley feruloyl tyrosyltransferase gene and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Isolation and prokaryotic expression of HVUL4H14252 gene

[0034] This example is mainly about the method of HVUL4H14252 gene acquisition, vector construction and prokaryotic expression.

[0035] (1) Construction of gene fragments and vectors

[0036] Weigh 1 gram of fresh barley leaves, extract barley RNA, synthesize cDNA with M-MLV Reverse Transcriptase from Thermo Fisher Company, and design primers as follows:

[0037] F: CGCGGATCCATGGAGATGACGAGCAGCTCG

[0038] R:ATAAGAATGCGGCCGCTCAATCCAACGAGTAGCAGATCTGC

[0039] Perform PCR amplification to obtain fragments of the target band size. The PCR product was purified with a gel extraction kit (GelExtraction Kit D2500-02, OMEGA).

[0040] The nucleotide sequence (SEQ ID NO.1) of the amplified target fragment HVUL4H14252 gene is:

[0041]ATGGAGATGACGAGCAGCTCGATGGTGAAACCGGCGTACGCCGTGCCGCACCCGCTCGTCGGCGACAAGGTCCCGCTGACCGTCTTCGACCGCGCCGCCCTGGACATCTTCGTCCCCACCGTGCTCGCCTACCCCGCGCCGGCGCCGTCCAACGAGGCTCTCAGGGAAGGGCTCC...

Embodiment 2

[0057] The construction of embodiment 2 transgenic tobacco

[0058] ①Transform the transient expression vector containing the target gene (provided by Professor George Lomonossoff, John InnesCentre) into Agrobacterium (EHA105);

[0059] ②Pick positive Agrobacterium clones in 500ul LB containing corresponding antibiotics (kn), and culture them for 20-24 hours;

[0060] ③Transfer 200ul into 5ml LB containing the corresponding antibiotic (kn), shake at 28°C at 220rpm to OD=2.0.

[0061] ④ Centrifuge at 10000rpm at room temperature for 2 minutes to collect the bacteria, resuspend the bacteria with the transformation buffer prepared in advance, and shake on the shaker for 3 hours; the composition and concentration of the buffer working solution are as follows: 10mM MES (pH5.7), 10mM MgCl2, 100μM acetosyringone (acetosyringone, AS).

[0062] ⑤Take the prepared 1ml syringe, remove the needle, select a syringe with a smooth outlet to inhale the bacterial solution, take the tobacco (...

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Abstract

The invention provides a feruloyl tyramide acyltransferase gene HVUL4H14252, the invention also provides a method for preparing a recombinant vector, a recombinant bacteria and a transgenic plant comprising the gene, the invention also provides a feruloyl tyramide acyltransferase and its preparation method. The feruloyl tyramide acyltransferase of the invention can synthesize traditional Chinese medicine monomer feruloyl tyramide, and has excellent application prospect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the application of a highland barley feruloyl tyrosyl transferase gene. Background technique [0002] The active ingredient of traditional Chinese medicine, feruloyl tyramide, has the functions of scavenging free radicals and antioxidation, and also has antibacterial, melanin synthesis, and insulin secretion stimulating activities. At present, feruloyl tyramide is mainly extracted from traditional Chinese medicines such as Shimanzi and Digupi. Due to the limited natural resources of traditional Chinese medicine, only relying on direct extraction cannot meet people's long-term needs. [0003] Highland barley, English name of highland barley: hulless barley is a kind of cereal crop of Poaceae Barley. Because of the separation of inner and outer glumes and bare kernels, it is also called naked barley, yuan wheat, and rice barley. It is mainly produced in China's Tibet, Qinghai, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N15/70C12N1/21C12N9/10A01H5/00A01H6/46A01H6/82C12R1/19
CPCC12N9/1029C12N15/8243C12Y203/0111
Inventor 曾兴权徐齐君王玉林原红军扎桑韦泽秀顿珠加布杨春葆于明寨尼玛扎西
Owner AGRI RES INST TIBET ACADEMY OF AGRI & ANIMAL HUSBANDRY SCI