香蕉MaBBI基因的克隆与功能初步探究

发布时间：2018-10-25 14:30

【摘要】：BBI(Bowman-Birk type protease inhibitor)是一种富含半胱氨酸的植物丝氨酸蛋白酶抑制剂,主要抑制胰蛋白酶、糜蛋白酶和弹性蛋白酶等。本实验从香蕉(Musa acuminata L.cv.Brazilian)中克隆了4个MaBBI基因(MaBBI1、MaBBI2、MaBBI3、MaBBI4),分别采用生物信息学、基因表达、酵母互补、转化拟南芥、亚细胞定位等方法对上述4个香蕉MaBBI个基因在胁迫响应中的功能进行了初步探究,主要结果如下:1)生物信息学分析表明MaBBI1、MaBBI2、MaBBI3、MaBBI4均含1个BBI结构域,每个BBI结构域中均包含2个蛋白酶结合位点。MaBBI1的BBI结构域中还含1个Bowman-Birk leg,而MaBBI2、MaBBI3、MaBBI4均不含有。2)基因表达分析显示MaBBI1、MaBBI2、MaBBI3在幼苗的根中的表达量最高,而MaBBI4则是在叶鞘中的表达量最大。上述MaBBI基因的表达量在不同激素处理和逆境胁迫下受到不同程度的影响,当中引起我们特别关注的是MaBBI1、MaBBI2、MaBBI3、MaBBI4在根中均受到JA的诱导,MaBBI1、MaBBI2、MaBBI4在叶中均受到甲基紫精(MV)的诱导,MaBBI1、MaBBI2、MaBBI3都响应重金属(CdCl2)的胁迫。这暗示上述MaBBI可能作为防卫应基因,参与香蕉对生物胁迫和非生物胁迫的应答。3)原核诱导表达后GST-MaBBI1、GST-MaBBI2、GST-MaBBI3、GST-MaBBI4重组蛋白均形成沉淀(包涵体),运用包涵体变性、复性的方法获得了可溶的重组蛋白,但在体外抑菌试验中复性后的可溶重组蛋白均没有对香蕉枯萎病4号生理小种表现出明显的抑菌活性。4)酵母互补试验表明MaBBI1、MaBBI2、MaBBI3、MaBBI4蛋白均具有一定的抗氧化能力,能使得氧化敏感型酿酒酵母菌株Δyap1、Δskn7的抗氧化能力得到不同程度的恢复。超量表达上述MaBBI基因的拟南芥植株也展现出比野生型更强的抗氧化能力。5)亚细胞定位显示MaBBI1、MaBBI2、MaBBI3、MaBBI4均广泛分布在细胞质中,且MaBBI3、MaBBI4可能在细胞核或某一细胞器的分布更为密集。
[Abstract]:BBI (Bowman-Birk type protease inhibitor) is a cysteine-rich plant serine protease inhibitor, which mainly inhibits trypsin, chymotrypsin and elastase. Four MaBBI genes (MaBBI1,MaBBI2,MaBBI3,MaBBI4) were cloned from banana (Musa acuminata L.cv.Brazilian and transformed into Arabidopsis thaliana by bioinformatics, gene expression and yeast complementation. The function of the four banana MaBBI genes in stress response was preliminarily investigated by subcellular localization. The main results were as follows: 1) bioinformatics analysis showed that MaBBI1,MaBBI2,MaBBI3,MaBBI4 all contained a BBI domain. Two protease binding sites were found in each BBI domain. The BBI domain of MaBBI1 contained 1 Bowman-Birk leg, and MaBBI2,MaBBI3,MaBBI4 did not. 2) the expression of MaBBI1,MaBBI2,MaBBI3 was the highest in the roots of seedlings, while MaBBI4 was the highest in leaf sheath. The expression of MaBBI gene was affected by different hormone treatments and stress. Among them, we pay special attention to the fact that MaBBI1,MaBBI2,MaBBI3,MaBBI4 is induced by JA in root, MaBBI1,MaBBI2,MaBBI4 is induced by (MV) in leaves, and MaBBI1,MaBBI2,MaBBI3 responds to CdCl2 stress. This suggests that MaBBI may be a defense response gene involved in banana responses to biological and abiotic stresses. 3) after prokaryotic expression, GST-MaBBI1,GST-MaBBI2,GST-MaBBI3,GST-MaBBI4 recombinant proteins are precipitated (inclusion bodies) and denatured by inclusion bodies. By renaturation, the soluble recombinant protein was obtained. However, the soluble recombinant proteins did not exhibit obvious bacteriostatic activity against the physiological race 4 of banana wilt disease in vitro. 4) yeast complementary tests showed that MaBBI1,MaBBI2,MaBBI3,MaBBI4 proteins had certain antioxidant activity. The antioxidant ability of 螖 yap1, 螖 skn7 could be recovered in different degree by the oxidation sensitive Saccharomyces cerevisiae strain 螖 yap1, 螖 skn7. Arabidopsis thaliana plants with overexpression of the MaBBI gene also showed stronger antioxidant activity than wild-type plants. 5) Subcellular localization showed that MaBBI1,MaBBI2,MaBBI3,MaBBI4 was widely distributed in cytoplasm and MaBBI3,MaBBI4 might be more densely distributed in the nucleus or a certain organelle.
【学位授予单位】：广州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S668.1;Q943.2

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