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基于酵母产孢的syntaxin家族蛋白SNARE区域功能分析 被引量:3
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作者 王丽萍 邵侃凯 +1 位作者 高晓冬 中西秀树 《食品与生物技术学报》 CAS CSCD 北大核心 2015年第5期530-535,共6页
真核细胞中大部分膜融合过程由SNARE蛋白介导,但其功能和调节机制尚未完全清楚。酵母孢子形成是研究囊泡融合机制包括SNARE蛋白的理想模型系统。在该过程中涉及t-SNARE蛋白Sso1,它是突触囊泡融合所需蛋白syntaxin 1A的同源物,两者SNARE... 真核细胞中大部分膜融合过程由SNARE蛋白介导,但其功能和调节机制尚未完全清楚。酵母孢子形成是研究囊泡融合机制包括SNARE蛋白的理想模型系统。在该过程中涉及t-SNARE蛋白Sso1,它是突触囊泡融合所需蛋白syntaxin 1A的同源物,两者SNARE区域有51%的同源性。尽管如此,将SSO1的SNARE区域完全替换成syntaxin 1A,而构建的嵌合体却无法回补sso1△突变的产孢缺陷。为了确定哪些残基为Sso1功能所必须,作者进行了嵌合体和突变分析,发现Sso1/syntaxin 1A嵌合体中syntaxin 1A的SNARE区域的220位丙氨酸换成谷氨酸后获得产孢功能。另外,Sso1发生相应的突变-218位谷氨酸突变成丙氨酸后失去其功能。因此,218位谷氨酸残基为Sso1产孢功能所必须。 展开更多
关键词 SNARE 人源化 酿酒酵母 Sso1p syntaxin1a
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人SNARE复合物相关蛋白的表达与纯化
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作者 支旭勃 陈款民 +5 位作者 王昭维 李倩楠 孙雪嫄 张芷萌 杨影 李元 《生物技术》 CAS 2024年第4期407-411,438,共6页
[目的]构建人SNARE复合物中的SNAP25、Syntaxin 1a和VAMP1蛋白真核表达载体,并于体外进行表达纯化。[方法]将重组质粒转化至大肠杆菌DH10Bac感受态细胞,经抗性和蓝白斑筛选,获得重组杆粒;用脂质体转化法,将重组杆粒转染至Sf9昆虫细胞中... [目的]构建人SNARE复合物中的SNAP25、Syntaxin 1a和VAMP1蛋白真核表达载体,并于体外进行表达纯化。[方法]将重组质粒转化至大肠杆菌DH10Bac感受态细胞,经抗性和蓝白斑筛选,获得重组杆粒;用脂质体转化法,将重组杆粒转染至Sf9昆虫细胞中使其产生重组病毒,最后对病毒进行扩增后再感染Sf9昆虫细胞表达目的蛋白;所得蛋白进行Western Blot鉴定及纯化。[结果]测序结果表明SNAP25、Syntaxin 1a和VAMP1真核表达载体构建成功,Western Blot检测出三种蛋白的特异性条带,利用His标签蛋白纯化出Syntaxin 1a蛋白。[结论]利用杆状病毒表达系统成功表达出了SNARE复合物相关蛋白。 展开更多
关键词 SNARE复合物 SNAP25蛋白 Syntaxin 1a蛋白 VAMP1蛋白 重组杆粒 杆状病毒 Sf9昆虫细胞 蛋白纯化
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Targeting Syntaxin 1A via RNA interference inhibits feeding and midgut development in Locusta migratoria
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作者 Xiaojian Liu Ya Gao +1 位作者 Yao Li Jianzhen Zhang 《Insect Science》 2025年第2期385-397,共13页
Syntaxin 1A(Syx1A)has diverse and indispensable functions in animals.Pre-vious studies have mainly focused on the roles of Syx1A in Drosophila,and so how Syx1A operates during the development of other insects remains ... Syntaxin 1A(Syx1A)has diverse and indispensable functions in animals.Pre-vious studies have mainly focused on the roles of Syx1A in Drosophila,and so how Syx1A operates during the development of other insects remains poorly understood.This study in-vestigated whether disrupting LmSyx1A using RNA interference(RNAi)affects the growth and development of Locusta migratoria.LmSyx1A was expressed in all tissues tested,with the highest expression observed in the fat body.After 5th-instar nymphs were injected with double-stranded LmSyx1A(dsLmSyx1A),none of the nymphs were able to molt normally and all eventually died.The silencing of LmSyx1A resulted in the cessation of feeding,body weight loss,and atrophy of the midgut and gastric cecum in locusts.Hematoxylin and eosin(H&E)staining showed that the columnar cells in the midgut were severely dam-aged,with microvilli defects visible in dsLmSyx1A-injected nymphs.Secretory vesicles were observed with transmission electron microscopy(TEM).In addition,reverse tran-scription quantitative polymerase chain reaction(RT-qPCR)further indicates that silenc-ing LmSyx1A repressed the expression of genes involved in the insulin/mammalian target of rapamycin(mTOR)-associated nutritional pathway.Taken together,these results sug-gest that LmSyx1A significantly affects the midgut cell layer of locust nymphs,which was partially associated with the downregulation of the insulin/mTOR-associated nutritional pathway.Thus,we argue that LmSyx1A is a suitable target for developing dsRNA-based biological pesticides for managing L.migratoria. 展开更多
关键词 insulin/mTOR pathway Locusta migratoria midgut development RNAi Syntaxin 1A
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