地衣芽孢杆菌耐高温α-淀粉酶基因的克隆、烟草瞬时表达及转化拟南芥的研究

doi:10.3724/SP.J.1259.2015.00354

植物学报 ›› 2015, Vol. 50 ›› Issue (3): 354-362.DOI: 10.3724/SP.J.1259.2015.00354

地衣芽孢杆菌耐高温α-淀粉酶基因的克隆、烟草瞬时表达及转化拟南芥的研究

李潇¹, 孙海彦^1,^2,^*(), 阮孟斌², 王霈虹³, 彭明^2,^*()

¹海南大学, 海口 570228
²中国热带农业科学院热带生物技术研究所, 海口 571101
³北京市科学技术情报研究所, 北京 100048

收稿日期:2014-11-15 接受日期:2015-01-27 出版日期:2015-05-01 发布日期:2015-04-08
通讯作者: 孙海彦,彭明
作者简介:
? 共同第一作者
基金资助:
国家高技术研究发展计划重点项目(No.2012AA101204)、国家自然科学基金(No.31000029)、海南省自然科学基金(No.ZDFD 20120765)、海南省引进集成应用专项(No.YJJC2011004)、海南省研究生创新科研课题(No.Hyb2011-4)和海南省重大科技项目(No.ZDZX2013023-1)

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

Xiao Li¹, Haiyan Sun^{1, 2, *}, Mengbin Ruan², Peihong Wang³, Ming Peng^{2, *}

¹Hainan University, Haikou 570228, China
²Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
³Beijing Institute of Science and Technology Information, Beijing 100048, China

Received:2014-11-15 Accepted:2015-01-27 Online:2015-05-01 Published:2015-04-08
Contact: Sun Haiyan,Peng Ming
About author:
? These authors contributed equally to this paper

摘要/Abstract

摘要： 从地衣芽孢杆菌(Bacillus licheniformis)中克隆到耐高温α-淀粉酶基因全长, 构建了原核表达载体, 转入大肠杆菌(Escherichia coli)中, 使用IPTG于28°C诱导6小时后, 通过SDS-PAGE检测到目的蛋白, 分子量约为55 kDa, 并通过酶活力检测实验证明该蛋白具有耐高温α-淀粉酶活性。同时构建了该基因融合GFP的植物表达载体, 通过农杆菌(Agro- bacterium tumefaciens)介导瞬时转化烟草(Nicotiana tabacum)下表皮细胞并在荧光显微镜下观察, 发现在烟草下表皮细胞的细胞质和液泡中均有绿色荧光。使用I₂-KI溶液对乙醇脱色后的烟草叶片进行染色, 显色反应表明在烟草中表达的耐高温α-淀粉酶具有酶活性。最后, 采用农杆菌介导的花蕾浸泡法将重组载体转化到拟南芥(Arabidopsis thaliana)中, 筛选到稳定遗传的耐高温α-淀粉酶基因的拟南芥纯合子。研究结果为后期开展表达耐高温α-淀粉酶的转基因植物的相关研究奠定了实验基础。

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.

李潇, 孙海彦, 阮孟斌, 王霈虹, 彭明. 地衣芽孢杆菌耐高温α-淀粉酶基因的克隆、烟草瞬时表达及转化拟南芥的研究. 植物学报, 2015, 50(3): 354-362.

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. Chinese Bulletin of Botany, 2015, 50(3): 354-362.

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

图/表 3

图1 耐高温α-淀粉酶基因的PCR扩增及酶活性鉴定 (A) 地衣芽孢杆菌的淀粉水解圈; (B) 地衣芽孢杆菌耐高温α-淀粉酶基因的PCR扩增片段及重组质粒的酶切验证(M: 15 000-2 000 marker; 1: 耐高温α-淀粉酶基因; 2: pET-22b-α重组载体; 3: pET-22b-α酶切验证/BamHI/XhoI; 4: G1300-α重组载体; 5: G1300-α酶切验证/BamHI/XbaI); (C) BL21 (DE3)/pET-22b-α淀粉透明圈(1: 耐高温α-淀粉酶商品酶; 2: BL21 (DE3)/pET-22b-α; 3: BL21 (DE3)/pET-22b (CK)); (D) BL21 (DE3)/pET-22b-α诱导6小时后的SDS-PAGE (M: PageRulerTM Plus Prestained Protein Ladder; 1: 破碎前发酵液; 2: 破碎后上清; 3: 破碎后沉淀; 4: BL21 (DE3)/pET-22b菌体(对照))

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))

表1 高温处理后耐高温α-淀粉酶的酶活性变化

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

图2 耐高温α-淀粉酶在真核植物中的表达 (A) 绿色荧光融合蛋白在烟草表皮细胞中的表达(左图为LBA4404/G1300侵染烟叶(对照); 右图为LBA4404/G1300-α侵染烟叶); (B) 用I2-KI溶液对瞬时表达烟草叶片染色(上图为未染色叶片(1: LBA4404/G1300侵染烟叶(对照); 2: LBA4404/G1300-α侵染烟叶); 下图为I2-KI染色后叶片(1: LBA4404/G1300侵染烟叶(对照); 2: LBA4404/G1300-α侵染烟叶)); (C) 转基因拟南芥的筛选(上图为转基因拟南芥T1代, 下图为转基因拟南芥T3代); (D) 转基因纯合子株系cDNA的PCR检测及I2-KI溶液对叶片染色(上图为转基因纯合子株系cDNA的PCR检测(M: 2 000 marker; 1: 耐高温α-淀粉酶基因的目的条带; 2: Hyg基因的目的条带, 3: GFP基因的目的条带); 下图为野生型和转基因拟南芥叶片)

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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地衣芽孢杆菌耐高温α-淀粉酶基因的克隆、烟草瞬时表达及转化拟南芥的研究

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

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