Banana antioxidant associated protein MaBBI1 and new application of gene of protein

Abstract

The invention discloses banana antioxidant associated protein MaBBI1 and application of an encoding gene thereof to improvement of oxidative stress tolerance of plants and bacterium resistance .The expression of the gene in engineering bacteria brewer's yeast can improve the tolerance ability of the yeast to H2O2 stress, it shows that the protein MaBBI1 has an antioxidant ability, meanwhile, the protein MaBBI1 subjected to induction, separation and purification has an effect of restraining pathogenic bacterium growth in vitro, and it shows that the protein MaBBI1 has an antibacterial peptide function .The gene has a potential for being applied to disease and damage resistant plant genetic engineering breeding, and by regulating the expression of the gene in the plants, the tolerance of transgene plants to pathogenic bacteria can be changed.

Description

technical field

[0001] The invention belongs to the field of biological genetic engineering, in particular to banana antioxidant-related protein MaBBI1 (Musaacuminata Bowman-Birkinhibitor 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-Birkinhibitor) is a plant protease inhibitor rich in cysteine, which can inhibit serine proteases such as trypsin, chymotrypsin and elastase. BBI can inhibit the degradation...

Examples

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 viologen (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. The extraction of RNA was carried out according to the specification of HiPurePlantRNAKits (R4151) of 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...

Embodiment 2

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

[0049] Take the leaves of the Brazilian banana seedlings, extract the RNA of the young leaves, and reverse-transcribe them into cDNA, and use the cDNA as a template to design primers MaBBI1YEF: 5'-TACCGAGCTCGGATCCATGAGGTACAACATGGTGG-3' and MaBBI1YER: 5'-TAGATGCATGCTCGAGCTACTGGGCGAGGAGCG-3' (SEQ ID NO .7 and SEQIDNO.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 PrimeSTARHSDNAPolymerasewithGCBuffer from TaKaRa Company. The amplified DNA fragment was in accordance with the instructions of HiPureGelPureDNAKits of 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 plasmid. The conc...

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-fra nkfurt.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, SDmedium) 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 s...

technical field

[0001] The invention belongs to the field of biological genetic engineering, in particular to banana antioxidant-related protein MaBBI1 (Musaacuminata Bowman-Birkinhibitor 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-Birkinhibitor) is a plant protease inhibitor rich in cysteine, which can inhibit serine proteases such as trypsin, chymotrypsin and elastase. BBI can inhibit the degradation...

Examples

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 viologen (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. The extraction of RNA was carried out according to the specification of HiPurePlantRNAKits (R4151) of 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...

Embodiment 2

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

[0049] Take the leaves of the Brazilian banana seedlings, extract the RNA of the young leaves, and reverse-transcribe them into cDNA, and use the cDNA as a template to design primers MaBBI1YEF: 5'-TACCGAGCTCGGATCCATGAGGTACAACATGGTGG-3' and MaBBI1YER: 5'-TAGATGCATGCTCGAGCTACTGGGCGAGGAGCG-3' (SEQ ID NO .7 and SEQIDNO.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 PrimeSTARHSDNAPolymerasewithGCBuffer from TaKaRa Company. The amplified DNA fragment was in accordance with the instructions of HiPureGelPureDNAKits of 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 plasmid. The conc...

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-fra nkfurt.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, SDmedium) 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 s...

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