小麦春化相关基因的克隆及其功能分析

一． 小麦相关基因ver203F cDNA全序列克隆与功能分析 　　根据本实验室通过差异筛选技术克隆到的与春化相关的基因cDNA verc203的序列，设计PCR 5’端PCR引物，利用RACE（rapid amplification of cDNA ends）克隆策略，得到春化相关基因ver203基因的同源基因ver203F cDNA的3’端序列，长度为1,197bp。Northern分析表明ver203F全长约为1.5 kb，且其表达具有春化处理的特异性。根据3’RACE克隆的ver203F 3 ’端核苷酸序列设计了3’端PCR引物，利用5’RACE克隆到该基因的5’端片段，经DNASIS核酸分析软件分析将5’45RACE和3’RACE DNA序列拼接合并，得到ver203F全长cDNA，从TdT加尾5’末端到poly A全长为1,561 bp，5’端起始密码子ATG上游非编码区－1～－192共了192bp，终止密码子TGA到poly A的非编码区有253bp，cDNA编码区全长1,119 bp，推测编码373氨基酸残基。国际基因序列数据库检索表明该基因序列（GenBank/EMBL/DDBJ：AB012013）与大麦茉莉酸诱导基因有部分同源性。因上推测该基因在调控开花过程中可能参与茉莉酸介导的信号传导途径，ver203F作用的发挥可能需要其它蛋白的参与，或ver203F本身就是一个受体蛋白。 　　为了研究ver203F基因的功能，将通过3’RACE克隆到的ver203F 3’端序到分别构建正义和反义植物表达载体，通过花粉管通道法、农杆菌介导的叶圆片法以及农杆菌介导的真空转化法分别转化小麦、烟草和拟南芥菜。获得转基因植株后，PCR、DNA Dob Blot、Southern Blot分析以及GUS活性检测证明外源基因已整合到转基因植株中，并得到表达。在获得的小麦、烟草和拟南芥菜转基因植株中，它们开花时间都相应地推迟，表明正常植物体内该基因在控制营养生长向生殖生长的转变中起作用。ver203F可以影响小麦和拟南茶菜花序的发育，首先无论正义还是反义都使得花序的发育受到抑制，在小麦中表现为顶部小穗退化，在拟南芥菜中表现为顶花。其次在转化正义基因的转基因拟南芥菜中，观察到产生的顶花为对称的两朵或以对称的两朵顶花基部为生长点长出丛生花，这种对称花的麦型小麦小穗中小花的表型相类似，说明ver203F基因可能在小麦小花的发育过程中也起着重要作用。 二． 春化相关基因ver17在开花过程中功能的分析 以春化处理冬小麦（京冬1号）幼苗cDNA为材料，通过减法杂交与差异筛选得到春化相关cDNA克隆verc17。为了研究该基因的功能，以包含CaMV 35S启动子的pBI221为载体，将ver17cDNA片段分别从两个方向插入pBI221的BamH I－Sma I, Xba I－BamH I间，构建正义和反义表达质粒：p17S和p17X，通过花粉管通道法转化小麦。对T0和T1两代转基因小麦的观察发现，在转化反义基因p17X的转基因小麦首先表现为抽穗推迟，其次穗的顶部和基部小穗严重退化，另外还发现转化反义基因的小麦败育现象严重（主要是花粉败育），因此推测ver17基困能可具有以下几方面的特点：a．春化诱导型表达;b．促进植物开花;c．促进穗顶端和基部花的发育，减少其退化;d．影响雄蕊的发育。

Identification of a C1q family member associated with cortical granules and follicular cell apoptosis in Carassius auratus gibelio

C1q family proteins with C1q domain have been reported in vertebrates, but their biological roles are currently unknown. In this study, a C1q-like factor, designated Carassius auratus gibelio ovary-specific C1q-like factor (CagOC1q-like), was identified as a cortical granules component. Immunofluorescence localization revealed that the C1q family member was specifically expressed in follicular epithelial cells, and associated with cortical granules in fully grown oocytes. Moreover, it was discharged to the perivitelline space and egg envelope upon fertilization. As it is the first identified C1q family member that is expressed in follicular cells that surround oocyte, CagOC1q-like was applied to detection of follicular cell apoptosis and deletion. The entire cytological process of follicular cell apoptosis and deletion was clearly seen from double visualizations of follicular cells with CagOC1q-like immunofluorescence and apoptotic follicular cells labeled by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) during oocyte maturation and ovulation. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Programmed cell death in Laminaria japonica (Phaeophyta) tissues infected with alginic acid decomposing bacterium

TdT-mediated dUTP-biotin nick end labeling (TUNEL) is a sensitive and valid method for detecting DNA cleavage in programmed cell death (PCD). Using this method, DNA cleavage was observed in Laminaria japonica sporophytic tissues, which were infected with alginic acid decomposing bacterium. It was found that DNA cleavage occurred 5 min after the infection, the fragments with 3'-OH groups of cleaved nuclear DNA increased with time of infection and spread from the infection site. Although no typical DNA ladder (200 bp/ 180 bp) was detected by routine agarose gel electrophoresis, the cleavage of nuclear DNA fragments of 97 similar to 48.5 kb could be detected by pulsed field gel electrophoresis (PFGE). By using CaspGLOW(TM) fluorescein active caspase-3 staining method, caspase-3 activity has been detected in response to the infection of alginic acid decomposing bacterium. Our results are similar to the observations in hypersensitive response (HR) of higher plant, suggesting that the rapid cell death of L. japonica infected by alginic acid decomposing bacterium might be involved in PCD, and indicating that the occurrence of PCD is an active defense process against the pathogen's infection.