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Analysis of the gene of lysine-synthesizing enzyme in rumen protozoa

瘤胃原虫赖氨酸合成酶基因分析

基本信息

批准号：
05454113
负责人：
ONODERA Ryoji
金额：
$ 4.8万
依托单位：
MIYAZAKI UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

项目摘要

Gene analysis of rumen protozoa has not been explored. The happening of some unexpeted problems in the present study has delayd the accomplishment of our research plan. As a result, however, the present study reached for sure at the research field of gene analysis and a part of the gene of lysine-synthesizing enzyme (lysA : diaminopimelate decarboxylase) of Entodinium caudatum has been determined for the first time in the world. The results seemed meaningful as fundamental data for continuing the gene analysis of the rumen protozoa in future. The first problem we met at the beggining of this study was the origin of the protozoon to be used for purifying mRNA.The protozoon derived from an agnotobiotic culture gave us too small amount of mRNA to make cDNA.The protozoon collected from a monofaunated goat, to which only E.caudatum had been inoculated, gave us an enough amount of mRNA.After making cDNA library, screening of lysA gene was carried out at first with lysine-requiring mutant of E.coli. But the method was interrupted by disappearance of the response of the bacterium to lysin-deficiency. This was left as a pending issue. Then, considering the fact that the codon usage (especially, stop codon) of protozoa (ciliates) in general is different from the universal one, lysA gene of the protozoon was screened from the cDNA library using a probe with a sequence 5'TTAAATCTTGGAGGCGGATTC3', which had a high homology in lysA gene of several bacteria and labelled with digoxigenin, and positive plaques were detected. One of these plaques was cloned and the DNA sequence of the insert was determined by the chain termination method. The protozoon, E.caudatum, seemed to use TAA as a stop codon like Euplotes (one of ciliates) and different from other ciliated protozoa, and to use TGC for recognizing cysteine like other organisms and different from Euplotes which had known to use TGA for cysteine.

瘤胃原生动物的基因分析尚未探索。由于本研究中一些未预料到的问题的发生，耽误了我们研究计划的完成。然而，结果，本研究确实达到了基因分析的研究领域，并且在世界上首次确定了尾甲藻赖氨酸合成酶（lysA：二氨基庚二酸脱羧酶）的一部分基因。本研究结果为进一步开展瘤胃原虫基因分析奠定了基础。在本研究开始时，我们遇到的第一个问题是用于纯化mRNA的原生动物的来源。来自无菌培养物的原生动物提供的mRNA量太少，无法制备cDNA。从一只只接种了尾状艾美耳球虫的单底栖山羊中收集的原生动物提供了足够的mRNA量。制备cDNA文库后，首先用大肠杆菌的赖氨酸需要突变株进行lysA基因的筛选。但是这种方法由于细菌对溶素缺乏的反应消失而中断。这是一个悬而未决的问题。然后，考虑到原生动物（纤毛虫）的密码子使用（特别是终止密码子）与通用密码子不同，使用与几种细菌的lysA基因具有高度同源性的序列为5 'TTAAATCTTGGAGGCGGATTC 3'的探针，用地高辛标记，从cDNA文库中筛选原生动物的lysA基因，并检测到阳性噬斑。克隆其中一个空斑，并通过链终止法测定插入片段的DNA序列。尾状埃立克体（E.caudatum）与游仆虫（Euplotes，纤毛虫之一）一样，以TAA为终止密码子，与其他纤毛虫不同;与游仆虫（Euplotes）一样，以TGC识别半胱氨酸，与已知的游仆虫以TGA识别半胱氨酸不同。