Targeting of the synthesized polypeptide in the cells is an important research field in modern cell biology. Cowpea trypsin inhibitor (cpti) gene has been modified and a fusion protein gene (sck) was produced by fusin...Targeting of the synthesized polypeptide in the cells is an important research field in modern cell biology. Cowpea trypsin inhibitor (cpti) gene has been modified and a fusion protein gene (sck) was produced by fusing a signal peptide sequence at cpti 5' end and an endoplasm reticulum (ER) retention signal peptide at cpti3' end respectively. The signal peptide can direct the newly synthesized polypeptide into ER, while ER retention signal can make the protein retained in the ER and its derivative protein body. ELISA test indicated that the accumulation level of foreign CpTI protein in sck transgenic tobacco (Nicotiana tabacum L.) was two times higher than cpti transgenic tobaccos and some individuals were four times higher. At the same time, sck transgenic tobacco has a high resistance to Lepidoptera pest due to the increased accumulation level of foreign CpTI protein. The strategy of foreign protein targeting can be used to increase the accumulation level of foreign protein in transgenic plants and can be widely applied to other related research field in plant genetic engineering.展开更多
In this study,cowpea trypsin inhibitor (CpTI) gene, an insecticidal gene,was introduced into two Populus tomentosa clones by gene transformation mediated by Agrobacterium tumefaciens. The transformed regeneration shoo...In this study,cowpea trypsin inhibitor (CpTI) gene, an insecticidal gene,was introduced into two Populus tomentosa clones by gene transformation mediated by Agrobacterium tumefaciens. The transformed regeneration shoots were obtained directly by leaf discs.In order to established selection condition,leaf discs were cultured in the medium with increasing concentration kanamycin.Kanamycin resistant(km r)plantlets were obtained by 3～4 cycles screening in selective condition.Then transformed shoots were rooted in the medium containing kanamycin 50?mg/L and transferred to greenhouse.The presence of CpTI gene in transgenic plants were confirmed by PCR and PCR\|Southern blot.展开更多
文摘Targeting of the synthesized polypeptide in the cells is an important research field in modern cell biology. Cowpea trypsin inhibitor (cpti) gene has been modified and a fusion protein gene (sck) was produced by fusing a signal peptide sequence at cpti 5' end and an endoplasm reticulum (ER) retention signal peptide at cpti3' end respectively. The signal peptide can direct the newly synthesized polypeptide into ER, while ER retention signal can make the protein retained in the ER and its derivative protein body. ELISA test indicated that the accumulation level of foreign CpTI protein in sck transgenic tobacco (Nicotiana tabacum L.) was two times higher than cpti transgenic tobaccos and some individuals were four times higher. At the same time, sck transgenic tobacco has a high resistance to Lepidoptera pest due to the increased accumulation level of foreign CpTI protein. The strategy of foreign protein targeting can be used to increase the accumulation level of foreign protein in transgenic plants and can be widely applied to other related research field in plant genetic engineering.
文摘In this study,cowpea trypsin inhibitor (CpTI) gene, an insecticidal gene,was introduced into two Populus tomentosa clones by gene transformation mediated by Agrobacterium tumefaciens. The transformed regeneration shoots were obtained directly by leaf discs.In order to established selection condition,leaf discs were cultured in the medium with increasing concentration kanamycin.Kanamycin resistant(km r)plantlets were obtained by 3～4 cycles screening in selective condition.Then transformed shoots were rooted in the medium containing kanamycin 50?mg/L and transferred to greenhouse.The presence of CpTI gene in transgenic plants were confirmed by PCR and PCR\|Southern blot.
文摘杨树受损伤后能诱导一些基因的表达,其编码的蛋白质可能在杨树的防卫反应中起一定作用。用PCR方法从新疆杨叶片中克隆出一个损伤诱导型Kun itz胰蛋白酶抑制剂基因PaTI1。序列分析表明:此基因不含内含子,其翻译起点上游具有‘TATA’和‘CCAAT’等转录控制元件,包含的阅读框架能编码一个长为213个氨基酸的多肽。此多肽与克隆自美洲山杨的PtTI2和PtTI1氨基酸序列同源性最高,分别为95%和80%,其N端存在一长度为27个氨基酸的信号肽。将此基因以融合蛋白的形式在大肠杆菌中进行表达,纯化后的融合蛋白对胰蛋白酶的活性有抑制作用,每8.5μg融合蛋白可完全抑制1μg牛胰蛋白酶的活性。W estern b lot分析表明融合蛋白与PtTI2特异的抗体之间有明显的血清学反应。
