Studies of the Transient Expression and Transformation of Cloned Thermostable α-Amylase Genes from Bacillus licheniformis in Tobacco and Arabidopsis

doi:10.3724/SP.J.1259.2015.00354

Abstract

Abstract: The full-length thermostable α-amylase gene from a Bacillus licheniformis strain was cloned in a prokaryotic expression vector and a plant expression vector with GFP as reporter gene. Thermostable α-amylase was overexpressed in Escherichia coli by IPTG induction for 6 h at 28°C, protein bands at 55 kDa were detected with SDS-PAGE, and activity of the enzyme was confirmed by amylolysis experiments. The plant expression vector was transformed into tobacco via Agrobacterium tumefaciens mediated transformation. GFP expression was observed both in cytoplasm and vacuoles of the lower epidermis of tobacco by fluorescence microscopy. Staining of the tobacco leaf by I₂-KI revealed that the thermostable α-amylase expressed in tobacco leaf had enzyme activity. Finally, the gene was introduced into Arabidopsis by the floral dip method and homozygous transgenic plants expressing α-amylase gene were identified. The results of this research provide further information for overexpression of thermostable α-amylase in transgenic plants.

Xiao Li, Haiyan Sun, Mengbin Ruan, Peihong Wang, Ming Peng. Studies of the Transient Expression and Transformation of Cloned Thermostable α-Amylase Genes from Bacillus licheniformis in Tobacco and Arabidopsis[J]. Chinese Bulletin of Botany, 2015, 50(3): 354-362.

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URL: https://www.chinbullbotany.com/EN/10.3724/SP.J.1259.2015.00354

https://www.chinbullbotany.com/EN/Y2015/V50/I3/354

Figures/Tables 3

Figure 1 PCR product of thermostable α-amylase from Bacillus licheniformis and identification of the activity of amylase (A) The transparent zone of Bacillus licheniformis on an Antibiotic Medium 3 plate containing 1% soluble starch; (B) PCR product of thermostable α-amylase from Bacillus licheniformis and identification of the recombinant plasmid (M: 15 000-2 000 marker; 1: The gene of thermostable α-amylase; 2: Recombinant plasmid pET-22b-α; 3: Identification of enzyme digestion of pET-22b-α by BamHI and XhoI; 4: Recombinant plasmid G1300-α; 5: Identification of enzyme digestion of G1300-α by BamHI and XbaI); (C) The transparent zone of BL21 (DE3)/pET-22b-α on a LB plate containing 1% soluble starch (1: Commercial thermostable α- amylase; 2: BL21 (DE3) with pET-22b-α; 3: BL21 (DE3) with pET-22b (CK)); (D) SDS-PAGE analysis of BL21 (DE3)/pET-22b-α after induction for 6 hours (M: PageRulerTM Plus Prestained Protein Ladder; 1: Untreated fermentation liquid; 2: Superna- tant after ultrasonication; 3: Precipitate after ultrasonication; 4: Bacterium solution of BL21 (DE3)/pET-22b (CK))

Table 1 The change of enzyme activity of thermostable α-amylase after high-temperature treatments

Treatment time (min)	0	10	20	30	40	50	60
Enzyme activity (U·mL^-1)	1 533	1 538	1 528	1 509	1 489	1 458	1 373

Figure 2 The expression of thermostable α-amylase in plants (A) Expression of fluorescent fusion proteins in tobacco epidermal cells (Tobacco epidermal cells infected by LBA4404/G1300 (CK) (left); Tobacco epidermal cells infected by LBA4404/G1300-α (right); (B) I2-KI dyeing of the tobacco leaf of transient expression with I2-KI solution (Leaf without dyeing (above) (1: Tobacco leaf infected by LBA4404/G1300 (CK); 2: Tobacco leaf infected by LBA4404/G1300-α); Leaf dyed with the I2-KI solution (below) (1: Tobacco leaf infected by LBA4404/G1300 (CK); 2: Tobacco leaf infected by LBA4404/G1300-α)); (C) Screening of transgenic Arabidopsis (Transgenic Arabidopsis of T1 generation (above); Transgenic Arabidopsis of T3 generation (below); (D) The analysis of PCR amplification for the cDNA of homozygous transgenic lines and the leaf of Transgenic Arabidopsis dyed with I2-KI solution (The analysis of PCR amplification for the cDNA of homozygous transgenic lines (above) (M: 2000 marker; 1: The objective band of thermostable α-amylase genes; 2: The objective band of Hyg; 3: The objective band of GFP); The leaf of wild type and transgenic Arabidopsis dyed with I2-KI (below))

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