Analysis of gene expression patterns with cDNA microarray during late stage of spermatogenesis in mice

The differentiation process of round spermatids to spermatozoa during the late stage of spermatogenesis is called spermiogenesis. To explore spermiogenesis-related genes, cDNA microarray was used to study expression patterns of 1176 genes in pachytene spermatocytes, round spermatids and elongating spermatids of Balb/c mice. The results showed that 208 genes were detected in all the three cell types. Most of them were down-regulated from pachytene spermatocytes to round spermatids and elongating spermatids. However, up-regulation of 7 genes expression in round spermatids and 3 genes in elongating spermatids were found. Expression of 7 differentially expressed genes in cDNA arrays was further confirmed by semi-quantitative RT-PCR study. The RT-PCR results indicated that the expression of 6 genes was consistent with that in cDNA arrays, only one gene did not show differential expression by RT-PCR. These results may provide important clues for studying of expression, regulation, and function of spermiogenesis-related genes.     

红鲫促性腺激素α亚基的cDNA克隆

从红鲫(Red crucian earp)脑垂体中提取总RNA，根据鱼类促性腺激素(Gonadotropin Hormone，简称GTH)α亚基的保守性，设计并合成了一对20个碱基长的简并引物，用RT-PCR方法扩增并克隆了红鲫GTH-α亚基的两种不同的cDNA，分别命名为GTH-α1和GTH-α2。GTH-α1和GTH-α2分别由363和366个核苷酸组成，分别编码117和118个氨基酸．这两种类型的cDNA的核苷酸和氨基酸的同源性非常高，分别达到了95.0％和96．6％．但是，它们之间也有4个氨基酸的差别，而且GTH-α1有一个三联体核苷酸组成的氨基酸的缺失。聚类分析表明，GTH-α亚基在鲤科鱼类中具有高度保守性。     

用加端PCR技术改造h－IGF－1cDNA

用ＤＮＡ合成仪合成了分别带有ＰｓｔＩ位点和ＳａｌＩ位点及终止密码子的２个用于扩增ｈＩＧＦ－１ｃＤＮＡ的ＰＣＲ引物．利用合成的引物，７００ｂｐ长的ｈＩＧＦ－１ｃＤＮＡ模板和Ｔａｑ聚合酶进行ＰＣＲ扩增．扩增产物经电泳鉴定后克隆进Ｍ１３ｍｐ１８载体，进行核苷酸序列分析．结果显示：ＰＣＲ产物含已发表的ｈＩＧＦ－１成熟蛋白的编码序列和５＇端的ＰＳｔＩ位点及３＇端的ＳａｉＩ位点及终止密码ＴＡＧ．用加端ＰＣＲ技术成功地扩增和改造了ｈＩＧＦ－１的编码序列．     

经大肠杆菌感染的中国家蚕cDNA文库的构建

用建立非表达文库的方法,从大肠杆菌感染后９ｈ的中国家蚕的ｍＲＮＡ合成ｃＤＮＡ片段,重组入噬菌体λｇｔ１０的ＥｃｏＲＩ的位点之间,所构成的基因非表达文库库容量为２×１０６重组子,经大肠杆菌Ｃ６００与Ｃ６００ｈｌｆ菌株平皿测定,重组率为７６％．     

大黄鱼凝血酶原类似基因的克隆及其表达分析

采用快速末端cDNA扩增法,首次从大黄鱼中克隆到全长为2 023 bp的凝血酶原类似基因cDNA,编码为617个氨基酸,其中包括80 bp的5′末端非编码区及89 bp包含poly(A)尾的3′末端非编码区。预测1~15位的氨基酸处存在1个信号肽。推导的氨基酸序列与哺乳动物及其他鱼类进行同源性比较,发现其与红鳍东方鲀有73%同源性,而与哺乳动物的同源性为50%~65%。凝血酶原类似基因虽然在大黄鱼的各个组织中组成型表达,但是在减毒鳗弧菌免疫的大黄鱼的脾脏和肾脏中表达明显上调,这表明凝血酶可能参与大黄鱼对细菌侵染的免疫应答。     

Cloning of a novel phosphateserine aminotransferase gene from a Triticum aestivum-Elytrigia elongatum alien substitution line with resistance to powdery mildew

Shannong 551, a T. aestivum-E, elongatum alien substitution line with resistance to powdery mildew, was inoculated with pathogenic spores of powdery mildew. The leaf samples were prepared 48 h after inoculation for scanning electron microscopy. The result showed that germination of spores and growth of young mycelia on leaves of Shannong 551 were suppressed at the early stage of infection. At the same time, RNAs were prepared from the leaves for the cloning of WRP1 and RPW2 by cDNA RDA and RACE technology. BLAST analysis of the sequences indicated that both WRP1 and RPW2 were novel genes. WRPI contains no complete ORE RPW2 contains the conserved structure domain of aminotransferase, and its DNA sequence shares high homology with genes of phosphateserine aminotransferase in many organisms. Therefore, it is speculated as a novel phosphateserine aminotransferase gene. The results of Northern blot suggested that expression of RPW2 occurred at the early stage of infection by powdery mildew. Southern blot using the probe of RPW2, in which there was strong hybridizing signals in both genome of Shannong 551 and E. elongatum, but not in those of Jinan 13 and Lumai No.5, indicated that RPW2 derived from the genome of E. elongatum.     

合系35号水稻Q酶基因的克隆

 支链淀粉含量是稻米品质的评价标准之一,控制支链淀粉合成的酶是淀粉分支酶.依据GenBank公布的日本水稻淀粉分支酶(Q酶)基因的cDNA序列合成相应引物,应用RT-PCR技术,SOE法成功地克隆到云南合系35号水稻Q酶基因的cDNA全长编码框2464bp.与日本水稻比较,合系35的Q酶基因有16个位点存在差异,并导致10个位点的氨基酸变化.