水稻基因APRT的克隆及其与温敏核雄性不育的关系

在拟南芥中腺嘌呤磷酸核糖转移酶基因(APRT)突变导致植株雄性不育。本文首次报道从水稻(Oryza sativa subsp.indica)中克隆了基因APRT(GenBank登录号AY238894),并将其定位于水稻第4染色体的一个BAC克隆(AL606604)的58000bp至63000bp区域。该基因K4220bp(起始密码子至终止密码子),含7个外显子、6个内含子,编码的APRT蛋白长212个氨基酸残基,与其他物种来源的APRT序列存在很高的同源性。与大麦、小麦、拟南芥1型及其2型的该蛋白同源性分刖为54．9％、54．9％、49．6％和59．5％。经保守结构域搜索发现该蛋白中存在APRT催化结构域。从DNA、mRNA两个水平分析了该基因与水稻温敏核雄性不育(TGMS)的关系,结果表明：受温度诱导,水稻“安农S—1”APRT基因的表达变化可能与温敏核雄性不育表现型具相关性。     

水稻基因APRT的克隆及其与温敏核雄性不育的关系

在拟南芥中腺嘌呤磷酸核糖转移酶基因(APRT)突变导致植株雄性不育.本文首次报道从水稻(Oryza sativa subsp.indica)中克隆了基因APRT(GenBank登录号AY238894),并将其定位于水稻第4染色体的一个BAC克隆(AL606604)的58 000 bp至63 000 bp区域.该基因长4 220 bp(起始密码子至终止密码子),含7个外显子、6个内含子,编码的APRT蛋白长212个氨基酸残基,与其他物种来源的APRT序列存在很高的同源性.与大麦、小麦、拟南芥1型及其2型的该蛋白同源性分别为54.9%、54.9%、49.6%和59.5%.经保守结构域搜索发现该蛋白中存在APRT催化结构域.从DNA、mRNA两个水平分析了该基因与水稻温敏核雄性不育(TGMS)的关系,结果表明:受温度诱导,水稻"安农S-1"APRT基因的表达变化可能与温敏核雄性不育表现型具相关性.     

药用野生稻应答稻飞虱取食过程中基因表达的研究

褐飞虱和白背飞虱是水稻的刺吸式害虫。为了探讨水稻对虫害反应的分子机理,采用cDNA—AFLP(互补DNA扩增片段长度多态性)技术,研究了药用野生稻受这两种飞虱取食后基因表达的变化。通过调查约7％的虫．崮答后的药用野生稻转录本组,共分离了258个差异片段,测序112个;44个片段对应于已知或假定功能基因,其中35个基因的表达水平在白背飞虱为害后发生改变,24个基因在褐飞虱为害后改变。下调的基因比上调的基因多。有14个基因受这两种飞虱共同调节(2个上调,12个下调)。结果表明,药用野生稻对两种飞虱为害作出的反应有所不同。     

水稻MYB cDNA的克隆和表达分析

根据植物MYB类转录因子DNA结合功能域的保守区设计一对简并引物 ,以水稻根、小苗和未成熟种子中的RNA为材料 ,用RT PCR方法扩增出约 180bp的片段。序列分析表明 ,它们与MYB基因的保守区有很好的同源性。以未成熟种子中获得的这一 180bp片段作探针 ,从水稻未成熟种子cDNA文库中分离到 5个新的MYB基因家族成员 ,它们是OsMYB12、13、14、15和5 1。在酵母系统中证实OsMYB13、OsMYB15和Os MYB5 1蛋白具有转录激活功能。Northern印迹分析表明 ,OsMYB5 1主要在未成熟种子中表达 ,在根和小苗中表达水平较低。RT PCR分析表明 ,OsMYB15在根、茎、小穗、叶片和种子中有低水平的表达     

水稻双链RNA结合蛋白同源基因OsRBP的克隆及其表达的分析

在基因数据库中发现两个水稻EST片段与大白菜BcpLH基因的双链RNA结合结构域 (dsRBD)有同源的区域 ,根据同源片段的位置特征设计引物 ,用RT-RCR扩增粳稻 (Oryzasativasubsp .japonica)愈伤组织的cDNA ,得到大小为 1.8kb的DNA片段。该cDNA片段含完整的编码区 ,有两个典型的dsRBD ,分别与BcpLH的dsRBD在氨基酸水平上同源性为 75 %左右 ,故将其命名为OsRBP。RT -PCR表达分析显示该基因在未成熟的种子和愈伤组织中表达 ,在根、茎、叶、穗、成熟种子及胚芽鞘中没有表达信号 ,由此推测该基因的表达可能与种子和胚的早期发育相关。该研究首次从水稻中分离到双链RNA结合蛋白基因 ,并初步研究了其表达方式 ,为进一步探讨水稻重要器官的发育和植物中双链RNA结合蛋白的调节作用奠定了基础     

斑茅两个看家基因片段的克隆及其在基因芯片中的应用

根据已发表的同源基因序列,利用RT-PCR技术分离了斑茅(Erianthus arundinaceus)的GAPDH和APRT两个看家基因片段,用它们作为cDNA芯片阳性参照,以未经聚乙二醇(PEG)胁迫处理的斑茅叶片为对照,和PEG胁迫的4组材料同cDNA芯片进行杂交分析。杂交结果显示,GAPDH杂交的Cy5与Cy3平均信噪比(Signal/Noise,S/N)分别为56.12和60.8,APRT杂交的Cy5与Cy3平均信噪比分别为51.06和47.25,信噪比均很高;同时两个看家基因的杂交都显示出极强的信号,其中GAPDH的杂交信号值大于10000,APRT也在8000以上,杂交结果可靠。分析了PEG胁迫4个时段BADH与两个看家基因的表达,BADH的表达有明显变化,而看家基因表达均较稳定。上述结果表明所克隆的两个看家基因在斑茅中表达量高,且PEG胁迫下表达较为稳定,是基因芯片理想的阳性参照。     

基于mRNA水平的差异表达基因筛选技术

随着分子生物学技术的深入发展,基因组研究重点已经由基因组测序转向基因功能鉴定,即由结构基因组学向功能基因组学转变。研究获得不同处理下基因的差异表达谱是功能基因组学的重要一环,目前已经有多种检测基因差异表达的技术可供选用。在减法杂交技术和PCR基础上发展起来的DDRT—PCR、cDNA—RDA、cDNA—AFLP和SSH技术因其实用性而得到广泛的应用,并取得了令人满意的结果。     

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.     

水稻小G蛋白OsPra2基因的克隆及功能分析

利用micro array 技术对水稻幼苗在营养胁迫条件下根部基因表达的研究中发现: 一个与豌豆Pra2(小G蛋白)基因有同源性的基因的RNA水平在营养胁迫后再补充营养时, 表达量下降。用RT-PCR和PCR方法分别获得该基因的cDNA克隆—OsPra2和该基因翻译起始位点上游1 kb的启动子序列。OsPra2基因编码的蛋白质具有结合GTP/GDP的4个保守结构域和构成小G蛋白Rab家族的特有的结构域。该基因cDNA与GST蛋白基因融合表达载体在洋葱表皮细胞中的瞬间表达结果显示该蛋白定位在在细胞膜和细胞核上, OsPra2基因启动子与GUS报告基因融合表达转基因水稻显示该基因启动子驱动GUS在胚芽鞘和根中表达, 35S启动子驱动OsPra2基因过表达转基因水稻与野生水稻株型相比明显矮化, 类似BR缺陷型植物株型。本实验还对OsPra2和P450蛋白的相互作用及在BR代谢途径中的可能作用进行了分析。     

水稻OsMPK15的cDNA克隆和转录水平分析

MAPK基因参与水稻的生物与非生物胁迫应答及生长发育过程,通过克隆水稻OsMPK15并初步研究其表达特性,为后续功能研究奠定基础。采用RT-PCR技术从日本晴水稻的根组织中克隆OsMPK15的cDNA编码区,并采用实时荧光定量PCR(qRT-PCR)技术分析OsMPK15在不同组织中以及不同非生物胁迫下的表达特性。结果表明,从日本晴水稻根组织中克隆的OsMPK15的cDNA编码区序列与生物信息学预测的结果一致,OsMPK15的第13-304位为丝氨酸/苏氨酸激酶结构域,属于水稻MAPK家族E组。qRT-PCR结果表明,该基因在水稻幼穗期各组织表达存在差异,主要在叶和叶鞘中表达。干旱和盐胁迫均可诱导OsMPK15在水稻幼苗根中表达量的上调,但表达模式具有不同的规律。同时,OsMPK15对3种胁迫相关激素ABA、JA和SA处理均产生应答,其中以ABA诱导OsMPK15表达上调幅度最大。     

Mapping QTLs and candidate genes for rice root traits under different water-supply conditions and comparative analysis across three populations

To investigate the genetic factors underlying constitutive and adaptive morphological traits of roots under different water-supply conditions, a recombinant inbred line (RIL) population derived from a cross between the lowland rice variety IR1552 and the upland rice variety Azucena with 249 molecular markers, was used in cylindrical-pot experiments. Eighteen QTLs were detected for seminal root length (SRL), adventitious root number (ARN), and lateral root length (LRL) and lateral root number (LRN) on the seminal root at a soil depth of from 3 to 6 cm under flooding and upland conditions. One identical QTL was detected under both flooding and upland conditions. The relative parameters under the two water-supply conditions were also used for QTL analysis. Five QTLs for upland induced variations in the traits were detected with the positive alleles from Azucena. A comparative analysis was performed for the QTLs detected in this study and those reported from two other populations with Azucena as a parent. Several identical QTLs for root elongation were found across the three populations with positive alleles from Azucena. Candidate genes were screened from ESTs and cDNA-AFLP clones for comparative mapping with the detected QTLs. Two genes for cell expansion, OsEXP2 and endo-1,4--D-glucanase EGase, and four cDNA-AFLP clones from root tissues of Azucena, were mapped on the intervals carrying the QTLs for SRL and LRL under upland conditions, respectively.Communicated by H.C. Becker     

Induced activity of adenine phosphoribosyltransferase (APRT) in iron-deficiency barley roots: a possible role for phytosiderophore production

To isolate the genes involved in the response of graminaceous plants to Fe-deficient stress, a protein induced by Fe-deficiency treatment was isolated from barley (Hordeum vulgare L.) roots. Based on the partial amino acid sequence of this protein, a cDNA (HvAPT1) encoding adenine phosphoribosyltransferase (APRT: EC 2.4.2.7) was cloned from a cDNA library prepared from Fe-deficient barley roots. Southern analysis suggested that there were at least two genes encoding APRT in barley. Fe deficiency increased HvAPT1 expression in barley roots and resupplying Fe to the Fe-deficient plants rapidly negated the increase in HvAPT1 mRNA. Analysis of localization of HvAPT1-sGFP fusion proteins in tobacco BY-2 cells indicated that the protein from HvAPT1 was localized in the cytoplasm of cells. Consistent with the results of Northern analysis, the enzymatic activity of APRT in barley roots was remarkably increased by Fe deficiency. This induction of APRT activity by Fe deficiency was also observed in roots of other graminaceous plants such as rye, maize, and rice. In contrast, the induction was not observed to occur in the roots of a non-graminaceous plant, tobacco. Graminaceous plants generally synthesize the mugineic acid family phytosiderophores (MAs) in roots under Fe-deficient conditions. In this paper, a possible role of HvAPT1 in the biosynthesis of MAs related to adenine salvage in the methionine cycle is discussed.     

硝态氮（NO3^—）对水稻侧根生长及其氮吸收的影响

侧根是植物吸收利用土壤养分的重要器官 ,其生长发育受内部遗传因子和外部环境矿质养分的影响。通过琼脂分层培养发现 :局部供应NO-3 可以诱导水稻 (OryzasativaL .)主根或不定根上侧根的生长。为研究旱种条件下NO-3 对水稻侧根发育及其N吸收的影响 ,设置了 3个蛭石培养实验 :分根处理、全株缺N、全株供N处理。分根处理 (一半根系供应 3mmol/LKNO3,另一半根系供应 3mmol/LKCl)结果表明 :局部供应NO-3 能够促进水稻侧根生长。而在全株处理下 ,N饥饿诱导了侧根的伸长。水稻根系对NO-3 的这两种反应都存在着显著的基因型差异。同时对地上部N浓度、可溶性总糖含量及N含量分析表明 ,这些生理指标在分根处理与全株加N处理中的差异均不显著 ,表明分根处理也能基本满足植株正常生长对N的需求。在分根处理中 ,水稻的N含量与分根处理中供N一侧的平均侧根长度存在显著正相关 ,这表明在养分不均一的介质中 ,侧根长度对水稻N素吸收具有十分重要的作用。而在N素充足的条件下 ,两者之间的相关性并不显著 ,这暗示在养分充足的环境下 ,侧根长度可能并不是决定根系吸收N素的主要因素     

Differential expression of cell wall related genes in the elongation zone of rice roots under water deficit

Growth in the apical elongation zone of plant roots is central to the development of functional root systems. It has been known that rice seminal root elongation could be enhanced by water stress. In the present study, 17 cell-wall related genes were identified by cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. Five genes encoded cell-wall loosening enzymes and six genes were involved in the lignin biosynthesis. The six other genes were related to the metabolism of polysaccharide and protein matrices in cell wall. Northern blot analysis confirmed that they were differentially expressed in the elongation zone of rice seminal roots under water stress, and none of them was root-specific. The results indicated that the activity of cell-wall loosening enzymes was enhanced in the early stage (within 16 h), and some cell wall matrices were synthesized rapidly in the middle stages (from 16 to 48 h), while lignin biosynthesis was enhanced in the middle and late stages of water stress (from 48 to 72 h). Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 437–443. The text was submitted by the authors in English.     

Inhibition of ammonium assimilation restores elongation of seminal rice roots repressed by high levels of exogenous ammonium

Elongation of seminal and lateral roots of rice seedlings was markedly inhibited by high ammonium levels in growth medium. However, high exogenous nitrate concentrations had little inhibitory effect on root growth. The objective of this study was to elucidate the relationship between inhibition of rice root growth induced by high ammonium conditions and ammonium assimilation in the seedlings. Activity of glutamine synthetase (GS) was kept at a low level in the seminal roots of the seedlings grown under high nitrate levels. In contrast, high ammonium levels significantly enhanced the GS activity in the roots, so that Gln abundantly accumulated in the shoots. These results indicate that ammonium assimilation may be activated in the seminal roots under high ammonium conditions. Application of methionine sulfoximine (MSO), an inhibitor of GS, relieved the repression of the seminal root elongation induced by high ammonium concentrations. However, the elongation of lateral roots remained inhibited even under the same condition. Furthermore, MSO drastically increased ammonium level and remarkably decreased Gln level in the shoots grown under high ammonium conditions. These results show that, for rice seedlings, an assimilatory product of ammonium, and not ammonium itself, may serve as an endogenous indicator of the nitrogen status involved in the inhibition of seminal root elongation induced by high levels of exogenous ammonium.     

Differential expression of fungal genes at preinfection and mycorrhiza establishment between Terfezia boudieri isolates and Cistus incanus hairy root clones

Changes in gene expression by isolates of Terfezia boudieri during mycorrhization with Cistus incanus hairy roots were followed. Four fungus-hairy root clone combinations were cultivated under two sets of conditions, in which the root and the fungus were separated by a cellophane sheet or were allowed physical contact. One of the combinations produced endomycorrhizas, the other three solely ectomycorrhizas. Fragments isolated by cDNA-AFLP analysis from cellophane-separated cultures (preinfection) were used to identify differentially expressed genes by reverse Northern analysis. Genes showing no homology to known sequences constituted the largest group under both growth conditions. Some fungal genes were expressed transiently, while others exhibited altered expression patterns as conditions changed from individually growing through the preinfection stage to mycorrhizas. Genes expressed exclusively under combinations allowing either ectomycorrhiza or endomycorrhiza under a particular condition were detected. Our results point, for the first time, to some of the genes that might be involved in determining the type of association that will be formed: ecto- or endomycorrhiza.