烟草花粉发育过程及不同组织中的内源GUS活性

INTRINSIC GUS ACTIVITY IN VARIOUS TISSUES AND DURING POLLEN DEVELOPMENT OF NICOTIANA TABACUM

以 X- gluc作为葡糖苷酸酶 ( GUS)酶反应底物 ,用酶组织化学方法检测了烟草 ( Nicotianatabacum L.)不同组织细胞的内源 GUS。在幼苗的根、茎、叶不同发育时期的花药壁、柱头、胚珠分离的生殖细胞和胚囊中 ,均未检测到内源 GUS活性。不同发育时期的烟草花粉内源GUS活性存在差异 ,在小孢子分裂前后和成熟花粉萌发前后有两个活性高峰。检测花粉内源GUS活性的适宜 p H为 5 .0 ;p H7.0的条件下未检测到内源 GUS。用 2 0 %甲醇或 0 .2mmol/L葡糖二酸内酯处理不能完全抑制内源

The β glucuronidase (GUS) gene has been widely used as a reporter gene in the study of plant molecular biology and genetic engineering.One of the major reasons leading to the popularity of GUS fusion system was the belief that there was no detectable intrinsic GUS activity in plant tissues.However, investigators have been troubled by the “false positive” results or “background” activities when GUS assays were performed.In the present experiment, histochemical observations of intrinsic GUS activity in various tissues and during pollen development of tobacco (Nicotiana tabacum L.) was carried out using 5 bromo 4 chloro 3 indolyl β D glucuronic acid (X gluc) as a substrate for overnight incubation of the treated tissues at 37℃.No detectable intrinsic GUS activity was found in seedling root, stem, leaf, anther wall and stigma of different stages, ovule, as well as isolated generative cell and embryo sac.During pollen development, two peaks of intrinsic GUS activity appeared, one, close to the microspore mitosis and the other from the full maturation of pollen lasting to the post germination pollen tube stage, no or weak activity was found at other pollen developmental stages.GUS was located in the cytoplasm of the pollen.The pH value of staining solution strongly influenced the experimental results.Blue color was visualized at pH 5, even when 20% methanol or 0 2 mmol/L glucaric acid 1 4 lactone (GAL, a specific GUS inhibitor) were added.At pH 7, no detectable reaction was found at all.The aforementioned results indicate that when using tobacco pollen as the target of GUS gene transformation, the assay should be strictly controlled to neutral condition for avoiding false positive results.