利用有序差异显示技术克隆受稻瘟病菌诱导表达的水稻cDNA的研究

利用有序差异显示技术(ODD)分析受稻瘟病菌诱导表达的水稻基因,通过反式RNA印迹进行辅助筛选,获得了37个在接种稻瘟病菌后表达量增强的水稻cDNA克隆.采用RNA印迹对其中5个克隆在接种稻瘟病菌后的表达分析表明,这些克隆在抗病以及感病的水稻株系中都具有诱导表达的特点.根据序列同源性分析,与这些克隆序列相应的同源基因可能涉及抑制病原菌生长、清除真菌毒素、传递抗病信号以及调节宿主生理状态等几方面的功能.     

稻瘟菌侵染诱导水稻凝集素基因的表达

利用mRNA差异显示技术(DDRT-PCR),从非亲和性稻瘟菌生理小种131侵染的水稻品种爱知旭(Oryza sati-va L. cv.Aichi-asahi)叶片中分离了8个诱导差异表达的cDNA片段.对这8个差示片段进行了回收、重扩增和克隆,以其中一个长度为321碱基并与甘露糖结合水稻凝集素和水稻盐诱导蛋白基因高度同源的差示片段为探针,筛选水稻非亲和性cDNA文库,获得12个阳性克隆.序列测定和数据库查询表明该基因的cDNA与水稻凝集素基因的cDNA及盐诱导蛋白基因的cDNA核苷酸同源性高达96%,推定的氨基酸序列与甘露糖结合水稻凝集素的氨基酸序列一致,与水稻盐诱导蛋白仅相差2个氨基酸.Southern杂交显示该基因在水稻基因组中有两个同源拷贝数,Northern杂交表明非亲和性稻瘟菌侵染可强烈诱导该基因表达.因此推测该基因参与了水稻对稻瘟菌侵染的防御反应.     

稻瘟病菌侵染诱导的水稻早期反应基因的cDNA片段克隆与序列分析

以稻瘟病菌感染水稻,利用ｍＲＮＡ 差异显示技术分离了稻瘟病菌侵染诱导的水稻早期反应基因ＥＲ１（ｅａｒｌｙ ｒｅｓｐｏｎｓｉｖｅ ｇｅｎｅ） 的ｃＤＮＡ 片段。Ｎｏｒｔｈｅｒｎ ｂｌｏｔ 分析表明,ＥＲ１ 基因在稻瘟病菌侵染水稻叶片６ ｈ 后开始表达,８ ｈ 最强,１０ ～１２ ｈ 开始减弱,１６ ｈ 消失。Ｓｏｕｔｈｅｒｎ ｂｌｏｔ 分析表明,ＥＲ１ 基因属于水稻基因组。对ＥＲ１ 基因片段（２１９ ｂｐ） 进行了克隆和序列分析。经查询,在ＧｅｎＢａｎｋ 中没有与ＥＲ１ 同源的基因序列。     

用同源序列的染色体定位寻找水稻抗病基因DNA片段

根据已知植物抗病基因的序列以及蛋白激酶序列中的高保守区域设计合成了特异性和简并引物,用聚合酶链反应从水稻（ＯｒｙｚａｓａｔｉｖａＬ．）ＤＮＡ中扩增同源片段,获约１００个大小不同的克隆。以这些克隆作探针进行限制性片段长度多态性（ＲＦＬＰ）分析,已将２６个克隆定位在两个水稻分子标记连锁图１２条染色体的３４个位点上。其中１０个克隆与８个已定位的水稻抗病基因在分子标记连锁图上的位置对应或毗邻。用其中部分与抗稻瘟病基因在染色体位置相对应的克隆作探针,分析抗稻瘟病近等基因系,ＲＦＬＰ带型在抗性基因系和感病亲本间表现出多态性,表明这些克隆与抗病基因在染色体位置上有较好的对应关系。     

稻瘟菌侵染诱导水稻凝集素基因的表达

利用mRNA差异显示技术（DDRT-PCR）,从非亲和性稻瘟菌生理小种131侵染的水稻品种爱知旭（Oryza sati-vaL.cv.Aichi-asahi)叶片中分离了8个诱导差异表达的cDNA片段,对这8个差示片段进行了回收,重扩增和克隆,以其中一个长度为321碱基并与甘露糖结合水稻凝集素和水稻盐诱导蛋白基因高度同源的差示片段为探针。筛选水稻非亲和性cDNA文库,获得12个阳性克隆。序列测定和数据库查询表明该基因的cDNA与水稻凝集素基因的cDNA及盐诱导蛋白基因的cDNA核苷酸同源笥高达96%。推定的氨基酸序列与甘露糖结合水稻凝集素的氨基酸序列一致。与水稻盐诱导蛋白仅相差2个氨基酸。Southern杂交显示该基因在水稻基因组中有两个同源拷贝数。Northern杂交表明非亲和性稻瘟菌侵染可强烈诱导该基因表达。因此推则该基因参与了水稻对稻瘟菌侵染的防御反应。     

稻瘟病菌侵染诱导的水稻早期反应基因的cDNA片段克隆与序列分析

以稻瘟病菌感染水稻,利用ｍＲＮＡ差汞显示技术分离了稻瘟病菌侵染诱导的水稻早期反应基因ＥＲ１的ｃＤＮＡ片段。Ｎｏｒｔｈｅｒｎ ｂｌｏｔ分析表明,ＥＲ１基因在稻瘟病菌侵染水稻叶片６ｈ后开始表达,８ｈ最强,１０￣１２ｈ开始减弱,１６ｈ消失。Ｓｏｕｔｈｅｍ ｂｌｏｔ分析表明,ＥＲ１基因属于水稻基因组。对ＥＲ１基因片段进行了克隆和序列分析。经查询,在ＧｅｎＢａｎｋ中没有与ＥＲ１同源的基因序列。     

水稻抗稻瘟病近等基因系的cDNA微阵列分析

应用cDNA微阵列对来源于中156／谷梅2号重组自交系的水稻抗稻瘟病近等基因系G205和G71的稻瘟病菌胁迫基因表达谱进行了分析,发现有3个cDNA克隆的表达仅在抗病基因系G205接种病原菌12h后受到诱导,其中两个为功能已知基因,另一个为功能未知的新基因。另有35个差异表达克隆在两个近等基因系中均检测到,其中17个克隆的表达在G205和G71均受到病原菌的诱导,另外18个克隆的表达则在G205和G71均受到病原菌原抑制。序列分析表明,这些稻瘟病菌应答基因分别与防卫反应,信号传递,逆境胁迫和光合作用及糖代谢等功能相关,为植物抗病机制提供了相关信息。另外,Northern还证实了编码富含甘氨酸蛋白基因（Glycinerich protein Grp)的表达受稻瘟病病原菌的诱导,是一个稻瘟病诱导相关基因。     

通过小麦Ms2近等基因系的抑制缩减杂交分析揭示小穗和花药中差异表达基因

以Ms2近等基因系处于减数分裂期的可育小穗cDNA作为驱动因子(driver),以同一时期的不育小穗cDNA作为测验因子(tester)进行缩减杂交(SSH),将扩增后的缩减杂交产物进行克隆,构建了一个包含882个重组克隆的SSH文库.分别以可育小穗和不育小穗的cDNA为探针与SSH文库克隆进行反式Northern杂交,结果显示接近90%的克隆在不育小穗中呈上调表达.对文库中21个克隆插入片段的序列相似性分析表明其中有18个与来源于穗部或减数分裂期的花药cDNA同源.13个克隆的编码产物与已知功能的蛋白质同源,其中5个参与碳代谢活动,4个参与胞内分子的运输,2个蛋白产物参与染色体的构成及染色体的结构变化,1个是生长素抑制蛋白,1个是转录因子.用中国春缺体四体材料对9个克隆进行了染色体定位,其中一个克隆定位于第四染色体同源群,与Ms2所在的染色体同属一个同源群.通过搜索水稻的同源BAC(bacterial artificialchromosome)和PAC(P1 artificial chromosome)克隆,推测另外11个克隆的染色体位置,其中4个克隆可能位于第四染色体同源群.用RNA点杂交对11个克隆进行表达谱分析,其中8个克隆在不育株的小穗和花药中呈上调表达.     

水稻线粒体丝氨酸羟甲基转移酶基因的电子克隆

采用基于EST的电子克隆方法得到了一段长1611bp的cDNA序列,以此为信息探针搜索HTGs数据库,找到一个与之高度匹配的基因组DNA序列——OSJNBa0057G07克隆。用FGENESH分析该克隆中的联配区域得到一个包含14个外显子和13个内含子的基因。该基因位于水稻第3染色体物理图谱的136．0～137．6cM区域。推导的ORF编码498个氨基酸,经BLASTP搜索SWISS-PROT数据库和蛋白序列的亚细胞定位显示,该基因编码水稻的线粒体丝氨酸羟甲基转移酶(mSHMT)。该基因受到EST序列的完全支持,其中不乏来自盐胁迫、稻瘟病菌侵染等逆境处理的EST序列,推测该基因与水稻对逆境的应答反应有关。     

稻曲病菌PMK1类同源基因克隆及在稻瘟病菌遗传互补中的功能验证

[目的]克隆稻曲病菌PMK1类MAPK(Mitogen-activated protein kinase)同源基因.[方法]根据丝状真菌MAPK蛋白保守性设计简并引物扩增稻曲病菌MAPK基因部分片段,进而利用TAIL-PCR进行染色体步移和RT-PCR获得UVMK1基因全长和cDNA全长.构建互补载体,交叉互补稻瘟病菌APMK1突变体菌株nn78进行功能验证,包括附着胞分化和致病性测定.[结果]UVMK1基因全长1435 bp,包含3个内含子,编码355氨基酸的蛋白.UVMK1推导蛋白与丝状真菌Magnaporthe grisea PMK1,Fusarium oxysporum FMK1,Fusarium solani FSMAPK,Colletotrichumlagenarium CMK1,Botrytis cinerea BMK1,Claviceps purpurea CMPK1等编码蛋白高度同源.转化稻瘟病菌菌株nn78,获得5个转化子.其中选取的转化子恢复了稻瘟病菌正常的附着胞分化和对大麦叶片的致病能力.[结论]本研究成功分离了首个稻曲病菌MAPK基因,而且UVMK1基因是稻瘟病菌PMK1的同源基因.     

Molecular Cloning of Early-expressed cDNAs from Rice in Response to Infection by Rice Blast Fungus Magnaporthe grisea

Compatible and incompatible reactions in rice plants (Oryza sativa L. cv. Shenxianggen No.4) were resulted from inoculation with two different virulent races of rice blast fungus (Magnaporthe grisea (Hebert) Barr), and thus an effective infecting system was established between rice plants and the rice blast pathogen. Two cDNA clones that showed induced and temporal patterns in expression in the very early stage in response to infection of the fungus were obtained from the plants by use of differential display. Of the two cDNA clones, Fastresp-a was induced to express in both compatible and incompatible interactions although it was expressed earlier in the former reaction. The second one, Fastresp-b, was only expressed in incompatible interaction. Southern blot analysis of the rice genomic DNA indicated that both of the two clones were from genome of the plant. No significant homology to the two genes was found from the rice gene database. This suggested that they were novel genes in rice and may play important roles in rice resistant response to infection of rice blast fungus.     

受稻瘟病原真菌侵染的水稻早期表达基因的cDNA克隆

以亲和性与非亲和性两个稻瘟病原真菌小种（Ｍａｇｎａｐｏｒｔｈｅ ｇｒｉｓｅａ（Ｈｅｂｅｒｔ）Ｂａｒｒ）感染同一水稻品种（Ｏｒｙｚａｓａｔｉｖａ Ｌ．ｃｖ．Ｓｈｅｎｘｉａｎｇｇｅｎｇ Ｎｏ．４）的植株产生明显不同的致病和抗病反应,由此建立了有效的感染系统。应用差异显示技术获得两个在侵染早期具有诱导表达特征的ｃＤＮＡ克隆,其中一个同时在致病和抗病反应中进行早期诱导表达,但在抗病反应中的诱导相对早于其在     

MGOS: A resource for studying Magnaporthe grisea and Oryza sativa interactions

The MGOS (Magnaporthe grisea Oryza sativa) web-based database contains data from Oryza sativa and Magnaporthe grisea interaction experiments in which M. grisea is the fungal pathogen that causes the rice blast disease. In order to study the interactions, a consortium of fungal and rice geneticists was formed to construct a comprehensive set of experiments that would elucidate information about the gene expression of both rice and M. grisea during the infection cycle. These experiments included constructing and sequencing cDNA and robust long-serial analysis gene expression libraries from both host and pathogen during different stages of infection in both resistant and susceptible interactions, generating >50,000 M. grisea mutants and applying them to susceptible rice strains to test for pathogenicity, and constructing a dual O. sativa-M. grisea microarray. MGOS was developed as a central web-based repository for all the experimental data along with the rice and M. grisea genomic sequence. Community-based annotation is available for the M. grisea genes to aid in the study of the interactions.     

Analysis of genes expressed during rice-Magnaporthe grisea interactions.

Expressed sequence tag (EST) analysis was applied to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe grisea and fungal genes involved in growth within the host during a compatible interaction. A total of 511 clones was sequenced from a cDNA library constructed from rice leaves (Oryza sativa cv. Nipponbare) infected with M. grisea strain 70-15 to generate 296 nonredundant ESTs. The sequences of 293 clones (57.3%) significantly matched National Center for Biotechnology Information database entries; 221 showed homologies with previously identified plant genes and 72 with fungal genes. Among the genes with assigned functions, 32.8% were associated with metabolism, 29.4% with cell/organism defense or pathogenicity, and 18.4% with gene/protein expression. cDNAs encoding a type I metallothionein (MTs-1) of rice and a homolog of glucose-repressible gene 1 (GRG1) of Neurospora crassa were the most abundant representatives of plant and fungal genes, comprising 2.9 and 1.6% of the total clones, respectively. The expression patterns of 10 ESTs, five each from rice and M. grisea, were analyzed. Five defense-related genes in rice, including four pathogenesis-related genes and MTs-1, were highly expressed during M. grisea infection. Expression of five stress-inducible or pathogenicity-related genes of the fungus, including two hydrophobin genes, was also induced during growth within the host. Further characterization of the genes represented in this study would be an aid in unraveling the mechanisms of pathogenicity of M. grisea and the defense responses of rice.