水稻AtpH基因的表达受冷抑制

采用RT-PCR差异显示法,从水稻（Oryza sativaL.)幼苗克隆了1个受冷抑制表达的cDNA片段。该片段序列与水稻叶绿体基因组编码ATP合酶CF0Ⅲ亚基的atpH基因完全同源,且覆盖了atpH基因编码区。以Northern 杂交分析了水稻幼苗在冷处理不同时间后的atpH基因转录水平,结果表明,atpH基因的转录受冷抑制,在冷处理第1天就明显下降,第2天以后完全受抑制。     

冷胁迫诱导、咖啡因抑制的水稻根新基因片段的分离及表达分析

利用差异显示（Differential display）及H.Y-Yellow琼脂糖凝胶分离等方法,从冷胁迫和甘露醇共同处理的冷敏感水稻（Wase toitsu）根的总RNA中,分离出冷胁迫诱导的cDNA片段,经过测序和同源比较,发现它是一尚未报道的新基因片段。这一基因的表达对温度具有敏感的反应特性,4℃时表达水平明显增加,在正常温度（25℃）下,其表达水平又可快速降低。当幼苗根用0.5mol/L甘露醇预处理2h后,转入冷胁迫,其低温诱导的表达水平增加更为明显。在甘露醇预处理之前加入咖啡因处理根1h,其表达水平明显降低,几乎与正常温度下的对照相同。据此推测,这一基因可能参与了冷胁迫或水分胁迫诱导的耐冷过程中咖啡因敏感信号的传导。      

RsICE1基因表达和转基因水稻抗冷通路研究

以萝卜幼苗和水稻T1代转RsICE1基因(从抗寒萝卜植株中分离的一个编码碱性螺旋-环-螺旋低温胁迫转录因子,HQ891287)株系为材料,通过半定量RT-PCR及实时定量PCR等方法,分析RsICE1基因的表达、水稻转基因株系中RsICE1基因的遗传及冷诱导基因的表达情况.结果显示:(1)半定量RT PCR分析表明,RsICE1基因在萝卜的根、茎、叶中为组成型表达,在幼苗根和茎中表达量较强;RsICE1基因的表达水平能够被冷处理和NaCl处理诱导上调表达,但ABA和脱水处理无上调作用.(2)卡方测验表明,水稻转基因T1代潮霉素抗性发生了3∶1分离模式;Southern和Northern杂交结果表明,4个抗冷转基因株系中RsICE1基因均以单拷贝、单位点整合到水稻基因组并正常表达.(3)实时定量PCR分析表明,冷胁迫下,RsICE1基因超表达但对水稻OsDREB1A(AF300970)、OsDREB1B(AF300972)、OsDREB1F(AY785897)基因表达没有影响,表明RsICE1基因对转基因水稻抗寒性的影响不依赖OsDERB1冷反应通路.     

水稻花粉[肌动蛋白]抑制蛋白基因的克隆和表达分析

[肌动蛋白]抑制蛋白（profilin）是一种低分子量、与肌动蛋白结构的蛋白质。通过筛选水稻成熟花粉的cDNA文库,获得了两个全长cDNA片段,序列分析结果表明,两个cDNA片段长度分别为821bp和805bp;共同拥有一个由131个氨基酸组成的开放密码框、5′末端翻译区和一个带有poly(A)的3′区域。[肌动蛋白]抑制蛋白与玉米、C.dactylon、H.brasiliensis、P.pratense中的该蛋白质的同源性分别为89％、87％、83％、89％。Southern杂交分析显示,在基因组至少有两个基因存在。Northern杂交和RT-PCR结果显示它在花粉和花粉中特异表达。     

抑制表达谷氨酸合酶基因对水稻碳氮代谢的影响

提高水稻的氮素利用率对农业生产极为重要,水稻谷氨酸合酶（GOGAT,EC1．4．1．14）被认为具有提高水稻氮素利用率的潜力,但该酶在水稻中的功能以及其对碳氮代谢的影响一直未被系统的报道．本研究以抑制表达谷氨酸合酶基因的转基因水稻为材料,结合谷氨酸合酶基因家族全生育期表达谱数据,研究该基因家族在水稻体内的功能及其对碳氮代谢的影响．结果表明,谷氨酸合酶家族基因成员的表达模式各不相同,具有明显的组织和器官特异性,表明其在体内行使着不同的功能．与野生型相比,抑制表达谷氨酸合酶基因的转基因植株的分蘖数、地上部干重以及单株产量显著下降．同时,转基因植株叶片中的硝酸盐、部分游离氨基酸、叶绿素、糖、糖磷酸以及吡啶核苷酸含量也显著降低,但游离铵、仅一酮戊二酸以及异柠檬酸含量上升．分析表明,谷氨酸合酶在水稻的碳氮代谢过程中扮演着重要角色,是水稻氮素高同化过程中必不可少的因子．     

水稻OsEBP-89基因的诱导表达

OsEBP-89基因是水稻中一个EREBP(ethyleneresponsive elements binding protein)类转录因子.Northern杂交检测出OsEBP-89基因的表达受激素及其类似物ACC、2,4-D、ABA、BR、JA、GA和6-BA的诱导,此外也受盐胁迫的诱导.通过序列比较在OsEBP-89基因启动子区找到一个类似乙烯应答元件(ethvlene responsive element,ERE),称为IVC box.实验表明IVC box能够和水稻未成熟胚乳核蛋白特异性结合;用IVC box和35S(-46)mini-promoter/GUS的融合结构转化水稻,在转基因水稻愈伤细胞中GUS基因的表达能被ACC诱导.然而,OsEBP-89基因5′调控区失去了IVC box及其上游序列后,受ACC诱导而表达的能力有所降低,但降低的幅度不大.推测OsEBP-89基因可能有不止一个受乙烯调控的位点.     

PNAS：发现抑制镉蓄积水稻基因

日本冈山大学的研究人员目前在美国《国家科学院院刊》上报告说,他们发现一种能抑制重金属镉在稻米中蓄积的水稻基因,该基因能把从土壤中吸收的镉封闭在水稻根部细胞内。这一发现为培育难以蓄积镉的水稻品种开辟了道路。     

水稻中一个人类肿瘤抑制基因同源物的克隆与表达分析

用同源筛选方法,从水稻（Oryza sativa L.）基因组库中分离到一个与人类肿瘤抑制基因QM具有同源性的基因,命名为OSQM1,该基因包括4个外显子和3个内含子,编码219个氨基酸,其中有46个碱性氨基酸,其等电点高达11.02。同源性搜寻发现此基因存在于真核生物中保守性较强,表明它可能具有重要的作用,North-ern分析结果表明,它在不同的水稻器官中都有表达,但在花和愈伤组织中的表达水平明显低于其他营养器官,它在根和叶中的表达水平受环境因素的影响。     

水稻rbcS启动子控制的外源基因在转基因水稻中的特异性表达

为将不同启动子用于转基因水稻的研究,从武运粳8号水稻中克隆了Rubisco小亚基基因(rbcS)的5'上游调控区,构建了由rbcS启动子引导的GUS融合基因,并经农杆菌介导导入到水稻中.对转基因水稻植株中GUS活性的定性与定量测定结果表明,rbcS启动子可驱动GUS报告基因在转基因水稻植株叶片和叶鞘内的叶肉细胞中特异性高效表达,而在茎、根和种子等器官中不表达或表达活性极弱,表现出明显的组织与细胞特异性.结果还表明,光诱导处理可明显提高rbcS启动子启动的外源基因的表达量.     

水稻CesA4基因启动子与GUS基因融合表达

目的:利用GUS报告基因,研究水稻CesA4基因在水稻组织和器官的定位.方法:克隆水稻的CesA4基因的启动子并用GUS组织化学染色检测启动子与GUS基因融合表达情况.结果:成功克隆了水稻的CesA4基因的启动子,并发现水稻的CesA4基因在水稻的根、茎、叶、鞘、穗的纤维均有表达.结论:通过将水稻CesA4基因的启动子与GUS基因融合来分析水稻此基因的表达部位,是一种简便和有效方法.     

热激对水稻幼苗耐冷性及热激蛋白合成的诱导

萌发的水稻种子经42℃热激处理后其幼苗的耐冷性明显增强,膜伤害程度降低,脯氨酸含量增加,超氧物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)活性和抗氧化物质抗坏血酸含量增加,而膜脂过氧化的关键酶脂氧合酶(LOX)活性及其产物丙二醛(MDA)含量下降.并且热激诱导萌发的水稻胚合成78、70、64、60、46、38、24、17、16kD的热激蛋白(HSP),其中属于HSP70的内质网结合蛋白(BiP)的合成与水稻幼苗耐寒性的提高有关.     

Characterization of a Rice Gene Family Encoding Type-A Diterpene Cyclases

We have previously isolated and characterized the rice (Oryza sativa) cDNAs, OsCyc1/OsCPS4, OsCyc2/OsCPS2, OsKS4, OsDTC1/OsKS7, OsDTC2/OsKS8 and OsKS10, which encode cyclases that are responsible for diterpene phytoalexin biosynthesis. Among the other members of this gene family, OsCPS1 and OsKS1 have been suggested as being responsible for gibberellin biosynthesis, OsKSL11 has recently been shown to encode stemodene synthase, and the functions of the three other diterpene cyclase genes in the rice genome, OsKS3, OsKS5 and OsKS6, have not yet been determined. In this study, we show that recombinant OsKS5 and OsKS6 expressed in E. coli converted ent-copalyl diphosphate into ent-pimara-8(14),15-diene and ent-kaur-15-ene, respectively. Neither product is a hydrocarbon precursor required in the biosynthesis of either gibberellins or phytoalexins. OsKS3 may be a pseudogene from which the translated product is a truncated enzyme. These results suggest that the diterpene cyclase genes responsible for gibberellin and phytoalexin biosynthesis are not functionally redundant.     

Agrobacterium-Mediated Multiple Gene Transformation in Rice Using a Single Vector

The homodimeric hemoglobin gene (VHb), the trans-zeatin synthetase gene (tzs), the modified 5-enolpyruvylshikimate-3-phosphate synthase gene (EPSPS), a selectable marker gene (hpt), and a reporter gene (gus), as linked expression cassettes, were stacked into the T-DNA region of a binary vector and introduced simultaneously into immature embryos of the rice (Oryza sativa L.) varieties Xiushui-11, Qiufeng,Youfeng, and Hanfeng by Agrobacterium tumefaciens. A total of 1 153 transgenic lines was obtained through selection for hygromycin B resistance. Approximately 90.2% of the transgenic lines harbored all the transgenes. Integration of multiple transgenes occurred at one to three genetic loci. Expression analysis revealed that the transgenes were coexpressed and inherited in a simple Mendelian fashion in transgenic plants and the frequency of coexpression was approximately 85%. On the basis of the cointegration and coexpression of the transgenes, most transgenic families were considered to be useful in a breeding program.     

Agrobacterium-Mediated Multiple Gene Transformation in Rice Using a Single Vector

The homodimeric hemoglobin gene (VHb), the trans-zeatin synthetase gene (tzs), the modified 5-enolpyruvylshikimate-3-phosphate synthase gene (EPSPS), a selectable marker gene (hpt), and a reporter gene (gus), as linked expression cassettes, were stacked into the T-DNA region of a binary vector and introduced simultaneously into immature embryos of the rice (Oryza sativa L.) varieties Xiushui-11, Qiufeng,Youfeng, and Hanfeng by Agrobacterium tumefaciens. A total of 1 153 transgenic lines was obtained through selection for hygromycin B resistance. Approximately 90.2% of the transgenic lines harbored all the transgenes. Integration of multiple transgenes occurred at one to three genetic loci. Expression analysis revealed that the transgenes were coexpressed and inherited in a simple Mendelian fashion in transgenic plants and the frequency of coexpression was approximately 85%. On the basis of the cointegration and coexpression of the transgenes, most transgenic families were considered to be useful in a breeding program.     

光照条件下低温对水稻籼粳亚种幼苗抗氧化物质含量的影响

与对低温不敏感的粳稻台北309和武育粳相比,对低温敏感的籼稻IR64、CA212和Pusa经光照条件下8℃处理后最大光合速率(Pmax)和原初光化学效率(Fv/Fm)下降较多,出现了O2-·、过氧化氢、氧化型谷胱甘肽(GSSG)和氧化型抗坏血酸(DHA)的大量累积,其GSSG和DHA的含量分别与叶绿素含量的下降呈极显著负相关,表明光照条件下低温胁迫下,还原态的谷胱甘肽(GSH)和抗坏血酸的再生受阻,不能有效地清除活性氧,导致其叶绿素含量降低和光合能力受抑,而汕优63的变化位于上述两种类型之间。其中AsA/DHA和GSH/GSSG的变化与叶绿素含量的变化呈极显著正相关。     

一个受褐飞虱取食诱导的水稻GST类似蛋白基因的克隆

BpHi006AcDNA长度为1943bp,具有一个795bp组成的完整的读码框,其表达受褐飞虱取食的诱导。BpHi006A蛋白含有谷胱苷肽S转移酶(glutathione Stransferase)的N末端结构域和C末端结构域,为谷胱苷肽S转移酶超家族的成员。BpHi006A蛋白与拟南芥四氯氢醌还原性脱卤素酶相关蛋白有61％的一致性,序列分析表明这两种蛋白质构成一类新的植物GST。     

水稻幼苗的低温伤害及其与腺苷酸代谢的关系

抗寒性强的早二六14和抗寒性弱的二九丰水稻幼苗,经2～4℃低温处理后,细胞膜透性增加,质膜ATPase水解活性大大提高,而线粒体ATPase磷酸化活性和丙酮酸激酶活性明显下降。从而导致腺苷酸水平尤其是ATP水平急剧下降,能荷值降低.幼苗低温处理4d后回到30℃,早二六14能很快转绿恢复生长,二九丰不能转绿,逐渐死亡。这时,腺苷酸水平及其上升幅度表现出前者高于后者。腺苷酸代谢的失调可能是水稻幼苗低温伤害的一个重要原因。     

Expression Enhancement of a Rice Polyubiquitin Gene Promoter

An 808 bp promoter from a rice polyubiquitin gene, rubi3, has been isolated. The rubi3 gene contained an open reading frame of 1140 bp encoding a pentameric polyubiquitin arranged as five tandem, head-to-tail repeats of 76 aa. The 1140 bp 5′ UTR intron of the gene enhanced its promoter activity in transient expression assays by 20-fold. Translational fusion of the GUS reporter gene to the coding sequence of the ubiquitin monomer enhanced GUS enzyme activity in transient expression assays by 4.3-fold over the construct containing the original rubi3 promoter (including the 5′ UTR intron) construct. The enhancing effect residing in the ubiquitin monomer coding sequence has been narrowed down to the first 9 nt coding for the first three amino acid residues of the ubiquitin protein. Mutagenesis at the third nucleotide of this 9 nt sequence still maintains the enhancing effect, but leads to translation of the native GUS protein rather than a fusion protein. The resultant 5′ regulatory sequence, consisting of the rubi3 promoter, 5′ UTR exon and intron, and the mutated first 9 nt coding sequence, has an activity nearly 90-fold greater than the rubi3 promoter only (without the 5′ UTR intron), and 2.2-fold greater than the maize Ubi1 gene promoter (including its 5′ UTR intron). The newly created expression vector is expected to enhance transgene expression in monocot plants. Considering the high conservation of the polyubiquitin gene structure in higher plants, the observed enhancement in gene expression may apply to 5′ regulatory sequences of other plant polyubiquitin genes.