Changes of DHN1 expression and subcellular distribution in A. delicisoa cells under osmotic stress

The changes of DHN1 expression and subcellular distribution in A. delicisoa cells under osmotic stress were studied by using GFP as a reporter molecule. Through creating the Xba I and BamH I restriction sites at the ends of dhn1 by PCR, the expression vector for the fusion protein DHN1-mGFP4 was constructed by cloning dhn1 into plasmid pBIN-35SmGFP4. Then the DHN1-mGFP4 expression vector was transformed into A. delicisoa suspension cells by micropro-jectile bombardment method. Bright green fluorescence of GFP which shows the high-level expression of DHN1-mGFP4 was visualized after culture for 10 h. However, the green fluorescence was only located within the nucleus. By increasing the culture medium osmotic potential, the green fluorescence was visualized in the cytoplasm (mainly around the plasma membranes). The generation of GFP fluorescence in the cytoplasm was also promoted by increasing the medium osmotic potential. Moreover, GFP green fluorescence was abolished by protein synthesis inhibitor dicyclo     

渗透胁迫下脱水蛋白DHN1在猕猴桃细胞中表达及亚细胞分布的动态变化

以绿色荧光蛋白(GFP)为报告分子, 通过构建DHN1-mGFP4融合蛋白表达载体, 研究了渗透胁迫条件下脱水蛋白DHN1的表达及其亚细胞分布的动态变化. 采用PCR方法在脱水蛋白基因dhn1两端引入XbaⅠ和BamHⅠ限制性内切酶位点, 克隆到质粒pBIN-35S mGFP4, 构建DHN1-mGFP4融合蛋白表达载体, 并采用基因枪转化猕猴桃(A. delicisoa)悬浮细胞. 培养10 h后观察到高效表达的GFP绿色荧光, 绿色荧光只出现在细胞核内. 提高培养介质渗透势后, 可以诱导脱水蛋白向细胞质分布(主要集中在质膜周围), 并且增加介质渗透势可以明显缩短细胞质出现绿色荧光的时间. 蛋白合成抑制剂环己亚胺能够抑制细胞质绿色荧光的出现, 暗示细胞质出现的脱水蛋白是诱导产生的结果. ABA可以明显促进细胞质绿色荧光的出现, 并且随着介质渗透势的升高迅速缩短细胞质绿色荧光的出现时间.     

Changes of DHN1 expression and subcellular distribution in A. delicisoa cells under osmotic stress

The changes of DHN1 expression and subcellular distribution in A. delicisoa cells under osmotic stress were studied by using GFP as a reporter molecule. Through creating the Xba I and BamH I restriction sites at the ends of dhn1 by PCR, the expression vector for the fusion protein DHN1-mGFP4 was constructed by cloning dhn1 into plasmid pBIN-35SmGFP4. Then the DHN1-mGFP4 expression vector was transformed into A. delicisoa suspension cells by microprojectile bombardment method. Bright green fluorescence of GFP which shows the high-level expression of DHN1-mGFP4 was visualized after culture for 10 h. However, the green fluorescence was only located within the nucleus. By increasing the culture medium osmotic potential, the green fluorescence was visualized in the cytoplasm (mainly around the plasma membranes). The generation of GFP fluorescence in the cytoplasm was also promoted by increasing the medium osmotic potential. Moreover, GFP green fluorescence was abolished by protein synthesis inhibitor dicyclohexylcarbodiimid, indicating that the cytoplasmic DHN1 was newly synthesized under osmotic stress. Furthermore, ABA promoted the presence of green fluorescence in the cytoplasm, and the GFP fluorescence was visualized within a shorter time under a higher osmotic potential.     

玉米原生质体的植株再生

以玉米花粉诱导产生的胚性愈伤组织,在 N6基本培养基附加激动素2 mg/l,6-苄基氨基嘌呤1mg/l,2,4-D 0.3 mg/l,水解酪蛋白500 mg*l 及谷酰胺250 mg/l 的培养基上进行转代培养。用转代培养一年半后的胚性愈伤组织分离原生质体,原生质体培养在附加激动素0.2 mg/l,6-苄基氨基嘌呤0.1 mg/l,2,4-D 0.5 mg/1,水解酪蛋白200 mg/l,谷酰胺100 mg/l及椰乳296的原生质体培养基 Z_2中。培养4—6天后,原生质体的再生细胞进行第一次分裂;培养3星期后发育成肉眼可见的小愈伤组织。此后,需添加降低糖浓度的同样原生质体培养基 Z_2共两次。待再生愈伤组织长到直径2—4 mm 大小时,把它们先后转经第一及第二(即Z_3及 Z_4)分化培养基上诱导器官分化。最后在 Z_4分化培养基上同时有胚状体的发生及植株的分化。