Transgenic tobacco cell cultures expressing a Trichoderma harzianum endochitinase gene release the enzyme into the medium
Transgenic tobacco cell cultures expressing a Trichoderma harzianum endochitinase gene release the enzyme into the medium
复制标题
表达哈茨木霉内切几丁质酶基因的转基因烟草细胞培养物将酶释放到培养基中
DOI：
10.1007/s002990000286
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发表时间：
2001
影响因子：
6.2
通讯作者：
Elizabeth D. Earle
中科院分区：
文献类型：
--
作者：
A. Brants;Elizabeth D. Earle
用哈氏菌内生几丁质酶编码cDNA转化烟草愈伤组织和细胞悬浮液。来自四个初级转化体的愈伤组织具有高水平的内生几丁质酶活性，就像它们的来源一样。在愈伤组织周围的培养基和转基因细胞悬浮液的培养基中也检测到几丁质内质酶活性。Western blot显示预期的40-kDaT的存在。在转基因样本中发现了哈氏菌蛋白，而在对照组中没有。这些结果表明，真菌酶在植物细胞中有分泌，cDNA结构中的真菌信号肽在植物细胞中起作用。通过硫酸铵沉淀使蛋白质浓度增加到34倍的细胞悬浮培养基抑制了数字青霉孢子的萌发。对照悬浮液在浓缩培养基中对孢子萌发也有一定的抑制作用，这可能与内源酶的分泌和浓度有关。
Calli and cell suspensions were obtained from tobacco plants transformed with an endochitinase-encoding cDNA from the biocontrol fungusTrichoderma harzianum.Calli from four primary transformants had high levels of endochitinase activity, like the plants from which they were derived. Endochitinase activity was also detected in the medium surrounding the calli and in the medium from transgenic cell suspensions. Western blots demonstrated the presence of the expected 40-kDaT. harzianumprotein in transgenic samples but not in controls. These results indicate that the fungal enzyme is secreted and that the fungal signal peptide in the cDNA construct functions in plant cells. A cell suspension medium in which the protein concentration was increased up to 34-fold by ammonium sulfate precipitation inhibited germination ofPenicillium digitatumspores. Some inhibition of spore germination was also observed in concentrated medium from control suspensions, probably due to the secretion and concentration of endogenous enzymes.