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ANALYSIS OF DWARF GENE(UZ)OF STRAIN UZU-AKASHINNRIKI OF BARLEY

大麦UZU-AKASHINRIKI矮化基因(UZ)分析

基本信息

批准号：
63540534
负责人：
SAKURAI Naoki
金额：
$ 1.15万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63540534/
关键词：
AUXIN BARLEY DWARF mRNA TRANSAMINASE RACEMASE 遺伝子オーキシントリプトファン C-DNA m-RNA

项目摘要

Normal barley strain (Nami Akashinnriki) and dwarf strain (uzu Akashinriki) were grown under the dark. The coleopitles were harvested on day 3, when the growth rate of the normal strain was ca. two times as fast as that dwarf strain. mRNA was extracted and purified from the coleoptile segments (1 cm in length, excised from 5 mm below the tip). mRNA thus obtained was introduced into the in vitro translation system using wheat germ extracts with radio labeled ^<35>S-methionine or ^3H-leucine. The transcripts were dissolved on two-dimensional gel electrophoresis. The comparison of the fluorogram of the normal strain with that of the dwarf strain revealed that there were 11 species of proteins distinctly appeared in the transcripts from mRNA of the nominal strain, but were not or little transcribed from mRNA of the dwarf strain.IAA was added to the in vitro translation system to see the effect of exogenously applied IAA on the translation process. IAA did not affect the translation in stra … More in uzu either using methionine or leucine, while in stmin Nami, IAA substantially inhibited the incorporation of labeled leucine into the proteins but not the incorporation of methilnine. The unexpected results remained to be answered.As reported previously, strain uzu produced less IAA than strain Nami. To see which step for IAA bioshynthesis was impeded in strain uzu, crude enzyme preparation was extracted from the coleoptile segments of uzu and Nami. IAA was produced from D-tryptophan in both strains. The production was inhibited by D-cycloserine (an inhibitor of D-transaminase), suggesting that IAA is produced through D-tryptophan in both strains. Next, racemase activity which converts L-trp to D-trp was assayed. pH optimum for the racemase of Nami was 7.6 while that of uzu was over 9.7. The difference of the optimum pH for the racemase between Nami and uzu strongly suggested that the amino acid composition of racemase of uzu was different from that of Nami, and DNA sequence for racemase of uzu was also different from that of Nami. The isolation of the racemase was now undertaken by ion- exchange and gel permeation chromatography. Purified enzyme and its amino acid composition will afford a great facility for the analysis of dwar gene of uzu. Less

普通大麦品系(Nami Akashinnriki)和矮秆品系(Uzu Akashinriki)在黑暗条件下种植。胚芽鞘在第3天收获，此时正常品系的生长速度是矮生品系的两倍左右。从胚芽鞘切段(长1 cm，从顶端以下5 mm处切下)中提取和纯化mRNA。将获得的mR NA导入带有放射性标记的小麦胚芽提取物的体外翻译系统中。S蛋氨酸或亮氨酸。转录本在双向凝胶电泳法上溶解。对正常品系和矮秆品系的荧光图谱进行比较，发现正常品系和矮秆品系的mRNA转录产物中有11种蛋白质明显存在，而矮秆品系的mRNA转录产物中没有或很少有IAA的转录。IAA不影响stra…中的翻译在UZU中，更多地使用蛋氨酸或亮氨酸，而在stmin Nami中，IAA基本上抑制标记的亮氨酸掺入蛋白质中，但不抑制甲氨酸的掺入。意想不到的结果仍有待回答。正如之前报道的那样，菌株UZU产生的IAA比菌株Nami少。为了了解菌株UZU抑制IAA生物合成的哪一步，从UZU和NAMI的胚芽鞘切段中提取粗酶制剂。两个菌株都是由D-色氨酸合成IAA的。D-环丝氨酸(一种D-转氨酶的抑制剂)抑制了IAA的产生，这表明两个菌株都是通过D-色氨酸产生IAA的。其次，测定了将L-色氨酸转化为D-色氨酸的外消旋酶活性。奈米的消旋酶最适pH为7.6，而乌苏的最适pH为9.7以上。两种消旋酶最适pH的差异强烈地表明，两种消旋酶的氨基酸组成不同，DNA序列也不同。用离子交换层析和凝胶渗透层析法分离外消旋酶。纯化的酶及其氨基酸组成为进一步分析UZU的DWAR基因奠定了基础。较少