TRANSFORMATION OF BIPOLARIS-SOROKINIANA WITH THE GUS GENE AND USE FOR STUDYING FUNGAL COLONIZATION OF BARLEY ROOTS
TRANSFORMATION OF BIPOLARIS-SOROKINIANA WITH THE GUS GENE AND USE FOR STUDYING FUNGAL COLONIZATION OF BARLEY ROOTS
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DOI：
10.1094/phyto-83-1484
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发表时间：
1993-12-01
期刊：
影响因子：
3.2
通讯作者：
SCHAFER, W
中科院分区：
文献类型：
--
作者：
LILJEROTH, E;JANSSON, HB;SCHAFER, W
利用含有GUS基因的质粒pGUS 5，将GUS基因导入小麦根际病原菌---小麦根际小斑病菌（Bipolaris sorokiniana），并在该质粒上插入了一个组成型启动子GPD-1和一个潮霉素抗性基因（hph）。潮霉素抗性用作选择标记。在含潮霉素的培养基上生长的菌落以每个活真菌原生质体6.5 × 10 ~（-7）的频率获得。20个测试的潮霉素抗性菌落中有19个也呈GUS阳性。在非选择性培养基上生长几个月后，经过几个分生孢子循环和几个月的生长，可以选择表现出稳定表达GUS活性和潮霉素抗性的转化子。然而，一些转化子在非选择性培养基上经过两个分生孢子循环后失去了GUS活性和潮霉素抗性。稳定的转化体在生长速率或致病性方面与野生型菌株没有差异。一个显着的，正相关的GUS活性和麦角甾醇含量和GUS活性和病变的大小在大麦根感染转化株。在发病前的根组织中检测到GUS活性，而在感染野生型真菌的根中仅检测到可忽略的背景GUS活性。sorokiniana可用于研究植物组织中的真菌定殖和生长。
Bipolaris sorokiniana, a fungal pathogen of cereals, was transformed with the GUS (beta-glucuronidase) gene, using a plasmid (pGUS5) containing GUS A with the constitutive promotor GPD-1 and a hygromycin-resistance gene (hph) under the control of the same promotor. Hygromycin resistance was used as the selectable marker. Colonies growing on medium with hygromycin were obtained at a frequency of 6.5 X 10(-7) per viable fungal protoplast. Nineteen out of 20 tested hygromycin-resistant colonies were also GUS positive. Transformants that showed a stable expression of GUS activity and hygromycin resistance after several conidiation cycles and several months of growth on nonselective medium could be selected. However, some transformants lost their GUS activity and hygromycin resistance after two conidiation cycles on nonselective medium. The stable transformants did not differ from the wild-type strain in growth rate or pathogenicity. A significant, positive correlation was found between GUS activity and ergosterol content and between GUS activity and lesion size in barley roots infected with a transformed strain. GUS activity could be detected in the root tissue before disease symptoms appeared, and only negligible background GUS activity was found in roots infected with the wild-type Fungus, These results indicate that GUS-transformed strains of B. sorokiniana can be used for studying fungal colonization and growth in plant tissue.