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New application of banana antioxidant-related protein mabbi1 and its gene

A related protein and antioxidant technology, applied in applications, genetic engineering, plant genetic improvement, etc., can solve problems such as affecting oxidative damage tolerance

Active Publication Date: 2020-08-18
GUANGZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no literature reporting whether the banana antioxidant-related protein MaBBI1 gene can affect the tolerance of oxidative damage in bananas

Method used

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  • New application of banana antioxidant-related protein mabbi1 and its gene
  • New application of banana antioxidant-related protein mabbi1 and its gene
  • New application of banana antioxidant-related protein mabbi1 and its gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: The expression of MaBBI1 gene is induced by methyl viologen (MV), heavy metal cadmium, low temperature, mannitol (Manntiol) and hormones jasmonic acid (JA), salicylic acid (SA), gibberellin (GA), Regulation of abscisic acid (ABA).

[0043] In this study, two-week-old rooted seedlings of Musa acuminata were used as experimental materials, cultured in 1 / 2 MS liquid medium for 3 days in a 23°C culture room (16h light / 8h dark), and methyl violet Ethanol (100μM), CdCl 2 (0.5mM), 4°C, mannitol (200μM), jasmonic acid (100μM), salicylic acid (100μM), gibberellin (100μM), ABA (100μM), treatment. Collect 0.5 g each of banana leaves and roots after stress treatment for 0h, 1h, 6h, and 24h for extraction of total RNA. RNA was extracted according to the instructions of HiPure Plant RNA Kits (R4151) from Magen Company. cDNA was reverse transcribed using a two-step method using total RNA as a template. The synthesis of cDNA strands was carried out according to the instr...

Embodiment 2

[0048] Example 2: Cloning of MaBBI1 gene and construction of yeast recombinant expression vector MaBBI1-pYES260

[0049] The leaves of the Brazilian banana seedlings were taken, and the RNA of the young leaves was extracted and reverse-transcribed into cDNA. Using the cDNA as a template, primers MaBBI1YEF: 5'-TACCGAGCTCGGATCCATGAGGTACAACATGGTGG-3' and MaBBI1YER: 5'-TAGATGCATGCTCGAGCTACTGGGCGAGGAGCG-3' (SEQ ID NO.7 and SEQ ID NO.8), using high-fidelity Taq enzyme PCR to amplify the full length of the cDNA reading frame of the MaBBI1 gene. For the PCR system used, refer to the instruction manual of PrimeSTAR HS DNA Polymerase with GC Buffer from TaKaRa Company. The amplified DNA fragments were used according to the instructions of HiPure Gel Pure DNAKits from Magen Company. The recovered fragment was used for insertion into the yeast expression vector pYES60. The Saccharomyces cerevisiae expression vector pYES260 was digested with BamHI and XhoI to recover the linearized plasm...

Embodiment 3

[0050] Example 3: Expression of MaBBI1 gene in Saccharomyces cerevisiae improves tolerance of yeast to oxidative stress

[0051] Saccharomyces cerevisiae strains Δskn7 and Δyap1 (purchased from Euroscarf, European Yeast Research Center, http: / / web.uni-frankfurt.de / fb15 / mikro / euroscarf / , strain numbers Y02900 and Y00569), the above-mentioned yeasts were transformed with pYES260 plasmid and recombinant MaBBI1-pYES260. Since the MaBBI1 gene is placed under the regulation of the yeast galactose-induced promoter PGAL1 (see figure 1 shown), MaBBI1-pYES260 was transformed into Saccharomyces cerevisiae and grown on the growth selection synthetic medium (Selective Growth Synthetic Medium, SD medium) supplemented with galactose, and the heterologous overexpression of MaBBI1 in yeast could be induced.

[0052] The method used for yeast transformation is the lithium acetate conversion method, and the specific steps are as follows:

[0053] 1) Inoculate a single colony of the yeast ...

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Abstract

The invention discloses the application of the banana antioxidant-related protein MaBBI1 and its encoding gene in improving plant tolerance to oxidative stress and resistance to pathogenic bacteria. The expression of this gene in the engineering strain Saccharomyces cerevisiae can improve the tolerance of the yeast to H2O2 stress, indicating that the MaBBI1 protein has the ability to resist oxidation, and at the same time, the induced isolated and purified MaBBI1 protein can also inhibit the growth of pathogenic bacteria in vitro, indicating that the MaBBI1 protein has the effect of inhibiting the growth of pathogenic bacteria in vitro. It has the function of antimicrobial peptide. The gene has the potential of being applied to plant disease-resistant genetic engineering breeding, and the tolerance of the transgenic plant to pathogenic bacteria can be changed by regulating the expression of the gene in the plant.

Description

technical field [0001] The invention belongs to the field of biological genetic engineering, in particular to banana antioxidant-related protein MaBBI1 (Musaacuminata Bowman-Birk inhibitor 1 ) and its coding gene and its application in the regulation of Saccharomyces cerevisiae tolerance to oxidative stress and / or resistance to pathogenic bacteria. Background technique [0002] Previous studies have shown that most adversity stresses in nature can cause oxidative damage to cells in varying degrees, H 2 o 2 As a kind of reactive oxygen species, its large accumulation in cells can disrupt the balance of oxidative metabolism and cause oxidative damage to cells. One of the ways of anti-oxidation is to prevent the release of superoxide anion groups so as to achieve the purpose of anti-oxidation. BBI (Bowman-Birk inhibitor) is a plant protease inhibitor rich in cysteine, which can inhibit serine proteases such as trypsin, chymotrypsin and elastase. BBI can inhibit the degradati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/81C12N15/29C12N15/81A01N57/16A01N47/44A01P21/00A01P1/00C12R1/865
CPCA01N47/44A01N57/16A01N63/30C07K14/81
Inventor 杨礼香黄司法唐志敏
Owner GUANGZHOU UNIVERSITY
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