【摘要】 采用盐析、凝胶过滤和离子交换层析等方法从甜荞中纯化出荞麦胰蛋白酶抑制剂 buckwheattrypsinin-hibitor,BTI ,经活性鉴定该抑制剂属丝氨酸类蛋白酶抑制剂家族.为了获得BTI的基因序列,并弄清其在体内的表达调控机制,应用RT-PCR和3′-RACE等方法,直接体外扩增该抑制剂基因,首次获得总长为361bp的DNA片段 GenBank登录号为AY335158 ,并命名为BTI-W1.该片段包括一个183bp的开放阅读框,编码61个氨基酸.由该基因推导的氨基酸序列与已报道的荞麦胰蛋白酶抑制剂BTI-2的氨基酸序列的同源性达100%.BTI-W1基因的获得,对于深入开展荞麦胰蛋白酶抑制剂结构与功能关系的研究具有重要意义,也为荞麦植物资源的开发利用建立了前期研究基础.更多还原

【Abstract】 A buckwheat(Fagopyrum esculentum) trypsin inhibitor (BTI) has been purified by using salting out,gel filtration and ion exchange chromatography to homogeneity and identified as one of the serine proteinase inhibitors.In order to obtain the structure gene sequence of buckwheat proteinase inhibitor and to further analyze its expression and regulation mechanism,specific primers were designed according to the reported amino acid sequences of proteinase inhibitors from common buckwheat and other kinds of plants.Using the cDNA fragments as the templates,part of the structure gene sequence and 3′-end nucleotide sequence were amplified by RT-PCR and 3′-Rapid Amplification of cDNA Ends (3′-RACE),respectively.The total amplified DNA fragment contained 361 base pairs including a translation region of 183 base pairs encoding 61 amino acids and an untranslated region of 202 base pairs rich in 70% AT at the 3′ end.It was named BTI-W1.Comparing with the reported amino acid sequences in BLAST database,the deduced amino acid sequence from target gene fragment had 100% homology with BTI-2.This is the first report of the nucleotide sequence of buckwheat proteinase inhibitor.更多还原