95%烯丙异噻唑原药

烯丙异噻唑;是防治水稻稻瘟病的药剂,采用糖精、烯丙醇氯化亚砜等原料,经氯化醚化反应得到烯丙异噻唑原药总收率为82%以上,含量在95%以上,其工艺可行,设备选型合理,产品属国内领先.     

重金属镉处理的水稻幼根cDNA抑制差减杂交文库的构建

重金属污染是影响水稻生长和稻米品质的重要因素,其中又以镉(Cadmium,Cd)污染最为严重,然而目前对其分子机制以及解决措施却了解甚少。本研究以水稻品种日本晴为材料,采用0.5 mmol/L镉处理水稻幼根48 h,通过运用结合分子镜像选择(mirror orientation selection,MOS)技术的抑制差减杂交(suppression subtractive hybridization,SSH)方法,构建cDNA抑制差减杂交文库,并进行microarray筛选,用于分离应答镉胁迫的差异表达基因。揭示水稻应对镉胁迫处理的分子机理,为水稻耐受镉胁迫基因的分离奠定了基础。     

大蕉冷诱导差减文库的构建与分析

以低温处理的大蕉幼苗cDNA为检测子,未处理的大蕉幼苗cDNA为驱赶子,利用抑制性差减杂交技术(suppression subtractive hybridization,SSH)构建了大蕉幼苗冷诱导差减cDNA文库,通过点杂交差异筛选cD-NA文库,得到约50个低温下表达增强的候选克隆,对其进行DNA测序和同源性比较,发现获得的ESTs在功能上主要涉及信号传导、表达调控、非生物胁迫防御、蛋白质加工和能量代谢等方面。该SSH文库的构建为克隆大蕉抗寒相关基因奠定了基础。     

用差减cDNA文库筛选由热激导致的小鼠睾丸中差异表达的基因

采用抑制性差减杂交（Suppression Subtractive Hybridization, SSH）方法,构建正常和热激小鼠睾丸组织的差减 cDNA 文库,以期 筛选出小鼠生精过程中对热敏感的基因。分别从正常和热激小鼠睾丸组织提取总RNA,反转录成cDNA,以正常小鼠睾丸组织cDNA作为待检组织(tester),以热激小鼠睾丸组织cDNA作为驱动组织(driver), 经过2轮杂交和抑制性PCR扩增,产物与T载体连接,经蓝白斑筛选,提取质粒,经EcoRI酶切鉴定插入片段并测序,由此构建了2种组织间差异表达基因的差减cDNA文库。用半定量RT-PCR方法,进一步验证 了该文库中差减出的基因基本上为差异表达的基因。对随机挑选其中的932个克隆测序,对有效测序的565个基因序列与GenBank数据库中发布的序列进行同源性比较,大部分片段都可以检索到同源序列。 结果显示,cPGES/p23等13个基因热激后表达量上调;septin2等120个基因热激后表达量下调。其中cPGES/p23为本研究中首次发现的小鼠生精过程中对热敏感的基因。     

铝诱导普通野生稻(Oryza rufipogon L.)光合系统相关基因表达

采用抑制差减杂交技术对正常与铝毒处理的普通野生稻植株进行表达基因差异性分析，通过对差异表达基因的PCR选择性研究，阳性表达基因的测序、Northern杂交的进一步分析，得出3个cDNAs在铝毒处理的叶片中高效表达，其中2个与光合系统代谢相关，1个与花的发育有关，研究结果为更好地理解植物在铝毒胁迫下叶片相关基因表达提供思路，同时也为在野生稻这个丰富基因库中筛选出耐铝毒的重要基因，以便改良亲源较近的水稻适应日益酸化的土壤具有一定的意义。     

Analysis of Salicylic Acid Induced Proteins in Rice

ＳｉｎｃｅＷｈｉｔｅｏｂｓｅｒｖｅｄｔｈａｔｓｐｒａｙｉｎｇｔｏｂａｃｃｏｐｌａｎｔｓｗｉｔｈｓａｌｉｃｙｌｉｃａｃｉｄ (ＳＡ)ｏｒａｃｅｔｙｌｓａｌｉｃｙｌｉｃａｃｉｄｉｎｄｕｃｅｄｒｅｓｉｓｔａｎｃｅｔｏｉｎｆｅｃｔｉｏｎｗｉｔｈｔｏｂａｃｃｏｍｏｓａｉｃｖｉｒｕｓ[1] ,ｍａｎｙｅｘｐｅｒｉｍｅｎｔｓｈａｖｅｄｅｍｏｎｓｔｒａｔｅｄｔｈａｔＳＡｐｌａｙｓａｎｉｍｐｏｒｔａｎｔｒｏｌｅｉｎｐｌａｎｔｄｉｓｅａｓｅｒｅｓｉｓｔａｎｃｅ ,ａｃｔｉｎｇａｓａｎｅｎｄｏｇｅｎｏｕｓｓｉｇｎａｌｍｏｌｅｃ…     

抑制性差减杂交方法克隆重力适应相关基因

采用抑制性差减杂交方法,分别从经重力环境改变适应性锻炼（实验组）和未经重力环境改变适应性锻炼（驱动组）的两组SD大鼠的淋巴细胞中分别提取RNA和mRNA,并经逆转录酶合成dscDNA,经Rsa Ⅰ酶切后将实验组cDNA分为2组,分别与两种不同的接头街接,再与驱动组cDNA进行2次消减杂交及2次抑制性PCR,将产物与T／A载体连接构建cDNA消减文库,并转染大肠杆菌进行文库扩增。随机挑选80个克隆,通过斑点杂交,初步筛选出20个阳性克隆。     

Isolation of cDNA fragment of fertility-related gene in photoperiod-sensitive genic male sterile rice

The photoperiod_sensitive genic male sterile rice (PGMR) is particularly useful to take advantage of heterosis in rice. mRNA differential display was used to isolate the fertility_relative genes in rice. After establishing an optimized mRNA differential display system, one of the differential cDNA fragments that maybe related to the development and maturation of rice panicle was cloned from a PGMR Nongken 58S.     

籼稻与粳稻品种间叶绿体DNA的RFLP差异

用ＥｃｏＲＩ与ＡｖａＩ对栽培稻的７个籼稻品种和４个粳稻品种进行叶绿体ＤＮＡ（ｃｐＤＮＡ）的ＲＦＬＰ分析，结果显示籼稻品种的ｃｐＤＮＡ具有较高的多态性，粳稻品种的则较为均一．另外，栽培稻的ｃｐＤＮＡ并无严格的亚种特异性．     

水稻小GTP蛋白OsRab5A的表达、纯化和GTP结合分析

小GTP结合蛋白是一类在细胞内的运输过程中重要作用的蛋白。它们通过结合子GTP而激活 ,当GTP被水解为GDP后处于非活性状态。哺乳动物中的研究表明 ,Rab5A蛋白是细胞内的形成早期内吞体 (earlyendosome)的限速因子。证实了所克隆的水稻rab5A基因编码的蛋白具有GTP结合功能。将Osrab5A基因的编码序列按正确读码框重组到pGEX4T1载体中 ,转化大肠杆菌BL2 1(DE3) ,电泳判定插入片段大小无误后 ,进一步测序鉴定其读码也无误。将该克隆命名为pG_5AE。以IPTG诱导 pG_5AE所编码的融合蛋白GST_OsRab5A的表达。SDS_PAGE电泳与无外源质粒和表达谷胱甘肽硫转移酶 (GST)的对照相比 ,得到了预计大小的融合蛋白。以GSTrapTM亲和柱纯化融合蛋白。在不同的泳道分离纯化的融合蛋白和作为对照的含GST以及无外源蛋白的细菌总蛋白 ,电转移到硝酸纤维膜上。将该膜与α_3 2 PGTP温育 ,洗膜 ,放射自显影后 ,获得了融合蛋白结合GTP的结果 ,这表明在原核中表达出的水稻Rab5A蛋白能在体外结合GTP。     

Identification of differentially expressed genes in esophageal cancer through SSH in combination with high throughput reverse Northern screening

To understand the molecular mechanisms of carcinogenesis of esophagus and to isolate genes with different expression levels in esophageal cancer, suppression subtractive hybridization (SSH) was combined with PCR-based cDNA synthesis and reverse Northern on the cancer tissues and matched almost normal mucosa using 5 microgram of total RNA as starting marterial. Eight genes were found expressed differentially in esophageal cancer, in which 5 were known genes and 3 were novel ones; and 6 were down-regulated in cancer tissues, while 2 were up-regulated; 6 were of mid-high abundance and 2 were of low abundance in esophagus. The results revealed that alteration in expression level of multiple genes underlied the initiation and development of esophageal cancer. The differentially expressed genes identified in this study such as liporcotinⅠ, cystatin A, cystatin B, cytokeratin 13 may play roles in dedifferentiation, transformation and malignant proliferation of esophageal cancer. The combination of SSH with PCR-based double- strand cDNA synthesis and high throughput reverse Northern screening is an efficient way to isolate differentially expressed genes from microgram of total RNA.