苏云金芽孢杆菌几丁质酶的研究进展

苏云金芽孢杆菌制剂作为无公害农药已经得到社会的认可,如果再开发其几丁质酶抑制真菌和杀虫增效功能,不仅可充分利用这一农业微生物菌种资源,也将给予传统生物农药以新的生命力。综述了苏云金芽孢杆菌几丁质酶方面研究的国内外最新进展。     

Isolation and characterization of chitinase from a marine bacterium Vibrio sp.

A chitinase was separated from the culture broth of Vibrio sp. 11211 isolated from sediment from the South China Sea. The chitinase was purified 18.3-fold with 33% recovery by ammonium sulphate precipitation and chromatography. The subunit molecular weight of the enzyme was estimated by SDS-PAGE to be about 30kDa. The enzyme showed optimum pH at 6.5 and optimum temperature at 50^°C, and was stable in the pH range of 4 to 9 and at the temperature below 40^°C.     

产气肠杆菌几丁质酶的分离纯化及性质研究

从自然罹病死亡的草原毛虫(Gynephorap ruoergnesis)体内分离到一株产气肠杆菌(Enterobacter aerogenes),它在几丁质的诱导下能产生较高活性的几丁质酶。发酵液经硫酸铵盐析、DEAE纤维素柱层析和Sephadex G-100柱层析分离出几丁质酶。用SDSPAGE测得该酶的分子量为425kD。水解几丁质的Km值为2.88mg/mL-1。酶反应的最适温度为55℃,最适pH值为60,金属离子对几丁质酶活性影响较大,其中Zn2+、Ba2+、Ca2+和Mn2+对酶有较强的激活作用,而Hg2+、Co2+和Mg2+则有较强的抑制作用。     

Enhanced resistance to blast (Magnaporthe grisea) in transgenic Japonica rice by constitutive expression of rice chitinase

Rice blast is the most devastating plant disease in Japan. Our goal is to create new rice varieties which show enhanced resistance against blast, regardless of the race of blast. By an Agrobacterium-mediated transformation method, we reintroduced a rice class-I chitinase gene, Cht-2 or Cht-3, under the control of the enhanced CaMV 35S promoter and a hygromycin phosphotransferase gene, as a selection marker into the Japonica rice varieties Nipponbare and Koshihikari, which have retained the best popularity over a long period in Japan. In regenerated plants (R₀), the Cht-2 product was found to accumulate intracellularly whereas the Cht-3 product was found to be targeted extracellularly. The transgenic rice plants which constitutively expressed either chitinase gene showed significantly higher resistance against the rice blast pathogen Magnaporthe grisea races 007.0 and 333. Both high-level expression of the chitinase and blast-resistance were stably inherited by the next generation in several lines. Received: 16 November 1998 / Accepted: 30 January 1999     

Genetic engineering of wheat for increased resistance to powdery mildew disease

 Fungal wheat (Triticum aestivum) diseases greatly affect crop productivity and require the economically and ecologically undesirable application of fungicides in wheat agriculture. We have generated transgenic wheat plants constitutively expressing an antifungal barley-seed class II chitinase. The transgene was stably expressed and the chitinase properly localized in the apoplast of the transgenic lines. The engineered wheat plants showed increased resistance to infection with the powdery mildew-causing fungus Erysiphe graminis. Received: 20 October 1998 / Accepted: 26 October 1998     

聚丙烯酰胺凝胶电泳配合荧光增白剂显色检测木霉几丁质酶同工酶谱

为提高木霉几丁质酶检测方法的准确性和灵敏度,建立一种快速检测几丁质酶同工酶的方法。采用活性凝胶电泳、变性凝胶电泳、原位显色凝胶电泳结合荧光增白剂（Calcofluor white M2R）显色从绿色木霉LTR-2发酵产物中检测几丁质酶同工酶。活性凝胶电泳在粗酶液浓缩5倍时显示两条活性谱带,变性凝胶电泳在浓缩10倍时显示一条活性谱带,原位显色凝胶电泳在浓缩20倍时显示两条不清晰的活性谱带,SDS-PAGE显示这两条活性谱带的分子量分别为65kDa和42kDa。结果表明活性聚丙烯酰胺凝胶电泳和Calcofluor white M2R显色相结合的方法在几丁质酶上样量为0.47U时具有较好的分辨能力,是检测木霉几丁质酶同工酶的有效的方法。     

棉铃虫单核衣壳核多角体病毒几丁质酶基因的克隆与表达

根据棉铃虫单核衣壳核多角体病毒(HaSNPV)几丁质酶基因的序列,设计引物,引入适当的酶切位点,利用PCR扩增出不含N末端信号肽以及C末端内质网定位肽的几丁质酶基因片段.将该基因片断克隆至原核表达载体pProEXHTb,经IPTG诱导,在大肠杆菌DH5α中获得了高效表达,表达产物的大小为60kD,含量占菌体总蛋白量的40%.利用来源于AcMNPV 几丁质酶的抗体对表达蛋白进行检测,获得特异性的显色信号,证实所获原核表达产物与杆状病毒的几丁质酶具有同源性.     

几丁质酶和壳聚糖酶对部分乙酰化壳聚糖作用方式的比较

通过对几丁质酶和壳聚糖酶降解部分乙酰化壳聚糖的作用方式的比较,得到几丁质酶切断壳聚糖的ＧｌｃＮＡｃ－ ＧｌｃＮＡｃ 和ＧｌｃＮＡｃ－ ＧｌｃＮ 或ＧｌｃＮ－ ＧｌｃＮＡｃ 糖苷键,而壳聚糖酶切断壳聚糖的ＧｌｃＮ－ ＧｌｃＮ 和ＧｌｃＮ－ ＧｌｃＮＡｃ 或ＧｌｃＮＡｃ－ ＧｌｃＮ 糖苷键,为得到较高聚合度的壳寡糖提供理论基础。     

免疫亲和层析法纯化苦瓜几丁酶

用扁豆几丁酶免疫家兔,获得抗扁豆几丁酶的抗体,将此抗体与Ｓｅｐｈａｒｏｓｅ ４Ｂ偶联,制备免疫亲和吸附剂,用以纯化苦瓜几丁酶．苦瓜叶片的粗提液经过免疫亲和吸附柱后,可获得电泳纯的几丁酶,其分子量为３５ ｋＤ,与用几丁质凝胶为亲和吸附剂的纯化结果一致．表明利用植物几丁酶在结构上的保守性,用免疫亲和法可纯化不同植物的同类几丁酶．与几丁质凝胶亲和柱相比,免疫亲和法纯化植物几丁酶具有快速、亲和柱可重复使用等的优点．利用免疫亲和层析获得的纯化样品,研究了苦瓜几丁酶对真菌的抑制试验,研究结果表明,苦瓜几丁酶能分解棉花枯萎病菌的菌丝体细胞壁制备物,并对其孢子芽管的伸长有一定抑制作用．