水稻OsRhoGDI2基因RNA干扰载体的构建

水稻OsRhoGDI2是通过酵母双杂交筛选到的小G蛋白Rho家族成员OsRacD互作蛋白的编码基因,为了研究OsRhoGDI2与OsRacD的相互作用及其在水稻发育中的功能联系,本研究基于序列比对的提示,选择了OsRhoGDI2基因长度为285 bp的特异区段,分别以正向和反向插入中间载体pKANNIBAL中,并亚克隆...     

osRACD基因表达与光敏核不育水稻光周期育性转换的相关性

为检测水稻低分子量GTP结合蛋白编码基因osRACD与光敏核不育水稻农垦58S光周期育性转换的相关性,用农杆菌介导转化和潮霉素抗性筛选获得了大量的转基因水稻植株.通过PCR、Southern杂交、RT-PCR和Northern检测等,证明反义osRACD基因和正义osRACD基因均已分别在转基因农垦58N和58S水稻植株的幼穗中得到了稳定的表达.成熟转基因植株的自交结实率统计分析表明,反义osRACD基因的表达大大降低了转基因58N水稻植株的育性,其平均结实率明显低于对照植株;而正义osRACD基因在转基因58S水稻植株的幼穗中表达后,使得长日下生长的、本来不育的58S植株的育性得到一定程度上的恢复,有的植株的结实率还高于短日下生长的对照植株;而且,osRACD基因表达水平越高的转基因58S植株,其对应的结实率也越高.降低幼穗中内源osRACD基因的表达水平会导致转基因58N水稻植株的育性降低,恢复长日下生长的转基因58S水稻植株幼穗中osRACD基因的表达,则可恢复其育性.这表明,osRACD基因的表达参与了农垦58S光周期育性转换的调控过程.这是第一个报道的参与光信号传导与光敏核不育水稻光周期育性调控的低分子量GTP结合蛋白的编码基因.     

野栽远缘杂交来源的反向温敏核雄性不育系不育性遗传研究

利用3个来源于野生稻与栽培稻杂交后代的反向温敏不育系R6S、N13S、Tb7S为材料,开展了反向温敏不育系不育性遗传研究,结果表明反向温敏不育系N13S的育性是细胞核内1对隐性基因控制的;Tb7S的育性是受隐性核基因控制的,F2代的育性分离比为9∶7,不育性状表现为2对基因的独立遗传;R6S的F2代育性分离比为37∶27,受细胞核内的3个隐性基因位点分别位于不同染色体上而通过互补作用的独立遗传.为进一步分离、定位及克隆有关这些反向温敏不育基因及发展分子标记辅助选择选育反向温敏不育系奠定了良好的遗传学基础.     

利用RNAi沉默技术对水稻OsHAD1基因的功能分析

生物信息学分析推测了水稻OsHAD1基因属于HAD(haloacid dehalogenase)超级家族水解酶.为研究其功能,本实验设计构建了OsHAD1-RNAi载体,通过农杆菌介导的方法转化水稻,成功获得了转基因植株.结果表明,转基因植株与野生型(日本晴)相比,转基因植株从T0代到T2代结实率均有20%～50%的降低;实时荧光定量PCR分析显示,T2代独立转基因植株中OsHAD1基因的表达量均有50%以上的下调;花粉染色结果显示,转基因植株花粉染色异常,败育率高,平均败育率达71.19%;石蜡切片结果显示,转基因植株花粉形状不规则,在花粉囊中排列松散,并且植株表现出明显的雄性不育.因此,推测OsHAD1基因参与水稻的生殖发育.     

水稻OsMDH基因的克隆及其生物信息学分析

水稻(Oryza sativa L.)OsAAP6基因是控制水稻种子蛋白质含量的主效数量性状座位(quantitative trait locus, QTL)基因，对水稻品质性状产生重要影响，而OsMDH蛋白能够与OsAAP6基因发生相互作用。利用生物信息学策略对OsMDH蛋白的理化性质、蛋白结构及功能进行预测，探究OsMDH基因的生物学功能。结果表明：水稻OsMDH基因的编码区(coding sequence, CDS)区长999 bp,编码332个氨基酸；编码蛋白为疏水蛋白且不存在信号结构，包含两个跨膜结构。通过蛋白互作分析，发现OsMDH可能与柠檬酸合酶、延胡索酸酶等发生相互作用，参与三羧酸循环过程。     

转水稻osRACD反义基因拟南芥植株的育性分析

利用反义RNA技术,将水稻低分子量GTP结合蛋白基因osRACD反向置于CaMV 35 S启动子的调控下,构成反义基因表达载体pBID,并用真空渗透法转化拟南芥植株.转基因植株的荚果自花谢之后就停止生长,其后荚果便自顶端开始逐渐枯黄并死亡;而对照植株的荚果在花谢之后仍继续生长直到成熟.花粉离体萌发生长实验显示,转基因植株花粉萌发后的生长延伸过程受到抑制,形成短而略粗的花粉管;而对照植株花粉萌发后的生长状况正常,形成长圆柱形的花粉管.这些结果表明,osRACD基因的功能是参与控制花粉管的生长延伸过程,其编码的蛋白质可能是调控光敏核不育水稻58S育性的重要因子之一.     

小麦雌性不育基因的微卫星标记定位

以普通小麦新601×雌性不育小麦XND126的F2群体作为育性调查以及基因标记群体.通过对育性基因的分析,确定在此组合中雌性不育基因由1对主效基因控制;结合混合分组分析法(Bulk Segregant Analysis,BSA),首次对小麦雌性不育基因进行了SSR分子标记,通过对一千对微卫星引物的筛选,确定微卫星引物cfd36标记与主效基因连锁,遗传距离为20.2cM.     

水稻中ACOS5同源基因在小穗中的表达分析

ACOS5是控制拟南芥雄性育的关键基因之一,它也是编码花粉素合成必需的酶.利用氨基酸同源序列分析,在数据库中检索与拟南芥ACOS5同源性较高的植物同源蛋白.将检索出的水稻中同源氨基酸序列与ACOS5进行比对,两者一致性为64%.并且通过同源蛋白系统发生分析,两者在进化上关系也较为密切.根据水稻中同源蛋白基因组序列设计引物,进行低温处理下该基因在小花发育各时期的表达量分析.半定量RT-PCR显示,在水稻小孢子母细胞形成期其表达量较低,而当小孢子开始减数分裂时,表达量急剧增高.在这两个时期,低温(20℃)会诱导OsACOSmRNA积累增加;但当处于小孢子形成阶段时,低温下积累量却呈现相反的降低趋势.这说明环境温度显著影响水稻中ACOS5同源基因的转录表达,而这种影响与小花发育时期有关,提示水稻ACOS基因可能参与环境温度调控水稻雄性育性的过程.     

水稻蛋白质相互作用网络的预测与分析

通过同源映射的方法,利用6个模式物种的蛋白质相互作用数据预测水稻的蛋白质相互作用网络.预测到水稻中有4483个蛋白质参与了24942个蛋白质相互作用.通过GO注释,结构域相互作用,基因共表达等3个证据评估预测网络的质量,并对网络进行了拓扑属性分析.结果表明水稻的蛋白质相互作用网络符合scale-free属性.通过对网络中功能模块的分析,可以预测蛋白质的功能和亚细胞定位信息.     

温敏核不育水稻HD9802-9S育性相关基因的BSA测序初步分析

HD9802-9S是以HD9802S为母本、以固优12为父本杂交选育的一个温敏核不育系。人工气候箱鉴定该不育系转育起始温度高于HD9802S,表明HD9802-9S和HD9802S两个品系的育性调控基因有差异。构建HD9802-9S/R446 F_2群体和HD9802-9S/R144 F_2群体,每个群体中随机选择100个不育单株和100个高度可育单株分别构建可育混池和不育混池进行BSA重测序。统计并计算每个BSA重测序结果的ΔSNP-index值,并以95%和99%置信水平作为筛选的阈值。高于95%置信水平的ΔSNP-index定位于2号染色体上总长为3. 24 Mb的候选区域和7号染色体上0. 69 Mb长度的候选区域内;高于99%置信水平的ΔSNP-index定位于2号染色体上总长为1. 87 Mb的候选区域内,在7号染色体上没有候选SNP,两个群体的定位结果相同。因此,HD9802-9S的不育主效基因定位在2号染色体,7号染色体上可能存在其他控制育性的QTL位点。HD9802-9S的2号染色体上存在64个控制花粉育性表达的候选基因。这些结果为进一步研究它们与tms5之间的关系奠定基础。     

bar基因表达框和完整质粒转化对水稻农艺性状的变异效应比较

通过分析转基因植株的株高、分蘖数、结实率、千粒重四个主要农艺性状的变异,比较基因枪法转化的基因表达框(仅含启动子、基因开放阅读框和终止子序列)和完整质粒两种基因载体形式对水稻农艺性状的变异效应.结果表明,与非转基因亲本相比,转基因植株的千粒重和分蘖数与对照无显著差异;株高变异也不大,17个转基因株系中,仅基因表达框转化的2个株系XFb-36和XFb-63株高变矮;而结实率变异最大,有9个转基因株系结实率显著降低,变异率达50%以上.总体上看,基因表达框和完整质粒两种基因载体形式对水稻农艺性状的变异效应差异不明显,基因枪转化对水稻农艺性状的变异效应主要表现为降低植株的育性,转基因水稻植株的农艺性状变异主要来源于转基因过程中组织培养引起的无性系变异.     

Fertility analysis of the Arabidopsis transformed with antisense rice osRACD gene

The full length osRACD cDNA sequence was subcloned into the pBI121 plasmid in the antisense orientation under the control of the CaMV35S promoter to construct the expression vector pBID, and the constructs were introduced into Arabidopsis plants by using the vacuum infiltration method. The siliques of the transformants stopped growing after anthesis, and they turned yellow or died later; and the siliques from the control plants transformed by the pBI continued growing after anthesis and matured normally. In vitro pollen germination demonstrated that the growth and elongation process of the pollens of the transgenic plants was inhibited, the pollen tubes were shorter and slightly fatter than the tubes of the control plants, which grew normally with long cyclindrical tubes. The above results suggest the function of osRACD gene involved in regulation of the growth and elongation process of pollen tube, its encoding protein may be one of the important factors in regulation of fertility transition of the photoperiod-sensitive genic male-sterile rice Nongken 58S.     

Isolation of a strong matrix attachment region (MAR) and identification of its function in vitro and in vivo

Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. Four new MARs (TM2, TM3, AM1 and AM2) were isolated from tobacco and Arabidopsis by PCR method. The nuclei isolated from suspension- cultured cells of rice were used to prepare nuclear matrix. With a characterized MAR (TM1) as a positive control, the Matrix-MAR interactions were tested by an in vitro binding assay to identify the DNA sequences as MARs and their binding strength to nuclear matrix in vitro was compared. The results showed that TM2 and TM3 had stronger binding strength than TM1. To determine the functions of the four new MARs in vivo, binary vectors pBI121 carrying a uidA GUS reporter gene were modified with direct repeat MARs inserted on both sides of the reporter gene cassette and were transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assays of the transgenic tobaccos showed that when flanking a GUS reporter gene TM1, TM2, TM3 and AM1 increased uidA GUS gene expression level approximately 1.5-fold, 5-fold, 1.35-fold, 1.3-fold respectively and AM2 has no effect on gene expression. TM2 was found to be a strong MAR that could effectively increase gene expression level and could be used as an effective enhancing element to construct high efficient expression vectors. In this note the relations among the sequence features, binding strength in vitro and function in vivo of the five MARs were analyzed, and the potential significance of TM2 in plant genetic engineering was dis- cussed.     

Molecular tagging of a genic male-sterile gene in rice

The sterility of Pingxiang male-sterile rice (Pms), possibly derided from a spontaneous mutation in Pingxiang fertile rice (Pmf), was previously reported to be controlled by a single dominant nuclear gene. It can be restored to fertility either by a dominant epistatic gene or by higher temperature treatment at the early stage of inflorescence development. In order to tag the genic male-sterile gene, Pms, Pmf and Ce 64, a cytoplasmic male-sterile restoring line without the epistatic gene for Pms, were used to construct mapping populations. Two segregation populations, “(Pms/Ce 64) F1s (sterile plant)//Pmf ” F1 and “Pms//(Pmf/Ce 64) F1” F1, were simultaneously developed. Subsequently, the genic male- sterile gene was mapped between a simple sequence length polymorphism marker, RM228, and a restriction fragment length polymorphism marker, G2155, with distances of 14.9 and 2.6 cM, respectively. The tagged dominant genic male-sterile gene is temporarily designated Ms-p.     

Identifying neutral allele Sb at pollen-sterility loci in cultivated rice with Oryza rufipogon origin

Pollen sterility is commonly found in the intra-specific hybrids of indica and japonica rice, which is one of the main constrains for the utilization of heterosis between indica and japonica. Six loci controlling the pollen sterility of F1 between indica and japonica have been identified from previous studies. Neutral alleles at each locus are potential to overcome the F1 pollen sterility associated with the locus. Therefore, exploitation and utilization of neutral alleles are of significant importance. The present research was based on fine mapping of the F1 pollen-sterility gene Sb and the abundant genetic diversity of Oryza rufipogon Griff. indigenous to Gaozhou, Guangdong Province （referred to as Gaozhou wild rice）. Crosses were made using Taichung65 （with the genotype of Sb^jSb^j and referred to as El） and its near-isogenic line of F1 pollen sterility gene Sb（with the genotype of Sb^iSb^i, E2） as female parents, and 12 different accessions of Gaozhou wild rice as male parents. F1 pollen fertility was examined to identify the materials having the neutral alleles at the F1 pollen-sterility locus. Segregation of 4 molecular markers tightly linked with the Sb locus was analyzed in the F2 populations derived from the FlS carrying the neutral gene. The pollen fertility related to the 3 genotypes of the molecular markers was also checked by statistical test to determine whether it was consistent with the hypothesis. The results showed that the pollen fertility of two F1s from one accession of Gaozhou wild rice （GZW099） with E1 and E2 was （89.2±21.07）% and （85.65±1.05）%, respectively. Both of them were fertile and showed no significant difference by t-test. Segregation of the 3 genotypes of the 4 molecular markers followed the expected Mendelian ratio （1：2：1） in the F2 populations. There was no significant difference for the averaged pollen fertility of the plants related to the 3 genotypes, suggesting that no interaction exists between the alleles at the Sb locus in GZW099 and Taichung65 or E2. Evidentially, GZW099 carried the neutral gene （named Sb″Sb″） at the Sb locus, which provides valuable theoretical basis and resources for further studying and overcoming the sterility of indica-japonica hybrids.     

水稻基因组中U2snRNA基因连锁的分析

根据Ｕ２ｎＲＮＡ基因的保守性和结构特点设计引物,通过ＰＣＲ法扩增Ｕ２ｓｎＲＮＡ连锁基因间区的ＤＮＡ片段并进行顺序分析,证实在水稻中至少存在一组Ｕ２ｓｎＲＮＡ连锁基因。对水稻总ＤＮＡ的ＰＣＲ分析结果表明,水稻中可能还存在其他形式的连锁基因。ＰＣＲ方法可以有效地应用于结构保守基因的连锁分析中。     

Genetic analysis and fine mapping of an enclosed panicle mutant esp2 in rice （Oryza sativa L.）

The phenomenon of panicle enclosure in rice is mainly caused by the shortening of uppermost internode.Elucidating the molecular mechanism of panicle enclosure will be helpful for solving the problem of panicle enclosure in male sterile lines and creating new germplasms in rice.We acquired a monogenic recessive enclosed panicle mutant,named as esp2 (enclosed shorter panicle 2),from the tissue culture progeny of indica rice cultivar Minghui-86.In the mutant,panicles were entirely enclosed by flag leaf sheaths and the uppermost internode was almost completely degenerated,but the other internodes did not have obvious changes in length.Genetic analysis indicated that the mutant phenotype was controlled by a recessive gene,which could be steadily inherited and was not affected by genetic background.Apparently,ESP2 is a key gene for the development of uppermost internode in rice.Using an F 2 population of a cross between esp2 and a japonica rice cultivar Xiushui-13 as well as SSR and InDel markers,we fine mapped ESP2 to a 14-kb region on the end of the short arm of chromosome 1.According to the rice genome sequence annotation,only one intact gene exists in this region,namely,a putative phosphatidylserine synthase gene.Sequencing analysis on the mutant and the wild type indicated that this gene was inserted by a 5287-bp retrotransposon sequence.Hence,we took this gene as a candidate of ESP2.The results of this study will facilitate the cloning and functional analysis of ESP2 gene.