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BdREF2 gene for regulating asafetide anabolism and application of BdREF2 gene

A technology of anabolic and ferulic acid, applied in the field of genetic engineering, can solve problems such as affecting the degradability of plant biomass and no research reports

Active Publication Date: 2018-12-21
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, Arabidopsis thaliana produced by chemical mutagenesis with ethyl methanesulfonate (EMS) REF1 In the cell wall of the mutant, the content of ferulate was reduced by about 50% compared with the wild-type control, so REF1 is an important gene involved in the regulation of ferulic acid biosynthesis, but REF1 Whether changes in gene expression can affect the degradability of plant biomass has not been reported so far

Method used

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  • BdREF2 gene for regulating asafetide anabolism and application of BdREF2 gene
  • BdREF2 gene for regulating asafetide anabolism and application of BdREF2 gene
  • BdREF2 gene for regulating asafetide anabolism and application of BdREF2 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: BdREF2 gene segregation

[0028] (1) PCR amplification of gene fragments: using the In-Fusion SMARTer Directional cDNA LibraryConstruction Kit (TaKaRa product), the mRNA of Brachypodium distachydium (provided by the School of Life Sciences, Shandong Agricultural University, Tai’an, Shandong) was extracted by conventional methods, and the construction of Brachypodium distachydium Brachypodium cDNA library. Using the library cDNA as a template, the following primer pairs were used for PCR amplification:

[0029] Forward primer sequence REF2-F: 5′-CCATCTTCTTTCAGCCAAGC-3′ (SEQ.ID.NO.3)

[0030] Reverse primer sequence REF2-R: 5′-TACGCAAGGAGCACTCAG-3′ (SEQ.ID.NO.4)

[0031]RT-PCR amplification uses KOD-Plus-Neo high-fidelity DNA polymerase system (TOYOBO product), the reaction system is: 10×PCR Buffer 5μL, 2mM dNTPs 5μL, 25mM MgSO 4 3 μL, 1.5 μL each of 10 μM forward and reverse primers, 1 μL of cDNA template, 1 U of KOD-Plus-Neo enzyme, with sterile dH 2 O ...

Embodiment 2

[0034] Example 2: BdREF2 Gene expression patterns in Brachypodium distachyon

[0035] (1) Preparation of total RNA from different tissues and organs of Brachypodium distachyon: young leaves, mature leaves, young stems, mature stems, young ears and roots of Brachypodium distachyon were collected from six different tissues and organs, respectively, about 20 days after heading. 0.1 g of each tissue material was used to prepare total RNA samples using MiniBEST Plant RNA Extraction Kit (TaKaRa product), and then the integrity and concentration of RNA samples were detected by 1% agarose gel electrophoresis and UV spectrophotometer respectively.

[0036] (2) mRNA reverse transcription reaction: 1 μg total RNA was used as the starting material for the reverse transcription reaction, and the reaction volume was 20 μL according to the steps introduced in the instruction manual of PrimeScript™ RTreagent Kit (TaKaRa product). The synthesized cDNA was used as a template for real-time fluo...

Embodiment 3

[0044] Example 3: BdREF2 prokaryotic expression of genes

[0045] (1) BdREF1 Construction of gene prokaryotic expression vector: based on the cloned BdREF2 Gene sequence, using PrimerPremier 6.0 to design specific PCR primers for amplification BdREF2 ORF region sequence without terminator.

[0046] The primer sequences are as follows:

[0047] REF2-OF: 5′-AG CATATG GGAAGCAACGAGGGAGG-3' (SEQ ID NO.9), the underlined part is Nde I digestion recognition site.

[0048] REF2-OR: 5′-AA GCGGCCGC ATACCAAGGCGAGTCAGGGA-3' (SEQ ID NO.10), the underlined part is not I digestion recognition site.

[0049] With the pTA2- obtained in step (2) of Example 1 BdREF2 Plasmid DNA (cloning the PCR product into the pTA2 vector) was used as a template, and the KOD-Plus-Neo high-fidelity DNA polymerase system (TOYOBO product) was used to amplify the ORF region of the gene. The PCR product was first purified with the SanPrep Column PCR Product Purification Kit (Shanghai Sangong Product)...

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Abstract

The invention relates to a BdREF2 gene for regulating asafetide anabolism and application of the BdREF2 gene, and belongs to the field of gene engineering. According to the BdREF2 gene disclosed by the invention, a coniferous aldehyde dehydrogenase gene named BdREF2 is separated from cDNA library of brachypodium distachyon; the BdREF2 gene in a brachypodium distachyon genome is knocked out by adopting a CRISPER / Cas9 gene editing system; it is found that the mutation of the BdREF2 can decrease the content of ferulic acid ester in plant cell walls while the degradation efficiency of biomass is obviously improved; the result indicates that the BdREF2 gene can be applied to biomass biodegradable genetic improvement of gramineous plants. It is firstly and directly proved that an REF homologousgene involved in regulating the biosynthesis of ferulic acid can be applied to improving the genetic improvement on biomass degradation and transformation efficiency of the gramineous plants, and hasimportant value for resource efficient utilization of biomass such as gramineae energy grass, forages and crop straws.

Description

technical field [0001] The invention relates to a method for regulating the synthesis and metabolism of plant ferulic acid BdREF2 A gene and its application belong to the field of genetic engineering. Background technique [0002] Lignocellulose (lignocellulose) is the main form of biomass resources. It has the characteristics of abundant reserves, sustainable regeneration, and environmental friendliness. Its efficient conversion and utilization has great potential in solving energy and environmental problems. Among them, judging from the natural biological attributes of raw materials, ecological and economic attributes, and energy attributes that are easy to convert and utilize, miscanthus, hybrid pennisetum, switchgrass and other gramineous lignocellulosic herbaceous plants have obvious comprehensive advantages and are ideal energy plants , its biomass can be used for cellulosic ethanol or biogas fermentation. At the same time, they are also important forage resources, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/70C12N15/82A01H5/00A01H6/46
CPCC12N9/0008C12N15/70C12N15/8243
Inventor 蒋建雄齐旭莉孙建中
Owner JIANGSU UNIV
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