水稻基因整合平台系统供体质粒pURKMT的构建

提高水稻产量,改良稻米品质是育种学家广泛研究的课题．随着现代生物技术的发展,水稻已成为植物基因工程的重要研究对象．许多实验室已成功地建立了一系列供外源基因转化水稻的系统．但是这些转化系统主要应用Ｔｉ质粒衍生的载体,通过Ｔ－ＤＮＡ左右两端的序列将目的基...     

Ethylene signaling in rice

Ethylene regulates many aspects of growth, development and responses to environmental stresses in plants. Its signaling pathway has been established in model dicotyledonous plant Arabidopsis. However, its roles and signal transduction in monocotyledous rice plant remain largely unknown. In this review, we summarize the current advances in rice ethylene signaling studies and compare these with the results from Arabidopsis and other plants. Most of the components homologous to those in Arabidopsis ethylene signaling pathway have been found in rice, including five ethylene receptors, OsEIN2, OsEIL1, and OsERFs. Rice ethylene receptors are functionally more divergent than that of Arabidopsis. OsEIN2 and OsEIL1 display limited roles in regulation of rice ethylene responses compared with their Arabidopsis orthologs. ERF-like proteins OsERF1 and OsEBP-89 appear to be involved in rice ethylene signaling. However, whether they are activated through OsEIN2 and OsEIL1-mediated pathway needs further studies. Given that rice uses ethylene to control many processes that do not exist in Arabidopsis, it seems that new components or new mechanisms may exist in rice ethylene signaling pathway.     

Fine mapping of <Emphasis Type="Italic">qHUS6.1</Emphasis>, a quantitative trait locus for silicon content in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Silicon is essential for optimal growth of rice (Oryza sativa L.). This study was conducted to fine map qHUS6.1, a quantitative trait locus (QTL) for rice hull silicon content previously located in the interval RM510–RM19417 on the short arm of chromosome 6, and to analyze the effect of this QTL on the silicon content in different organs of rice. Selfed progenies of a residual heterozygous line of rice were detected using 13 microsatellite markers in the vicinity of qHUS6.1. Three plants with overlapping heterozygous segments were selected. Three sets of near isogenic lines (NILs) were developed from the selfed progenies of the 3 plants. They were grown in a paddy field and the silicon contents of the hull, flag leaf, and stem were measured at maturity. Based on analyses of the phenotypic distribution and variance among different genotypic groups in the same NIL set, a significant genotypic effect was shown in the NIL set that was heterogenous in the interval RM19410–RM5815, whereas a significant effect was not found in the remaining 2 NIL sets that were heterogenous in either of the intervals RM4923–RM19410 or RM19417–RM204. On comparison among the physical positions of the 3 heterogenous segments, qHUS6.1 was delimited to a 64.2-kb region flanked by RM19410 and RM19417 that contains nine annotated genes according to the genome sequence of Nipponbare. This QTL showed strong effects on all of the three traits tested, and the enhancing alleles were always derived from the paternal line Milyang 46. The present study will facilitate the cloning of qHUS6.1 and the exploration of new genetic resources for QTL fine mapping.     

Vesicle-related OsSEC27P enhances H<Superscript>+</Superscript> secretion in the iron deficient transgenic tobacco root

Iron is an essential micronutritional element for plants. In addition to the iron uptake mechanism Strategy I and Strategy II, the vesicle transport process was also found to participate in iron uptake and homeostasis. Herein, a new iron deficiency induced OsSEC27P gene was isolated and investigated in both its localization and its function in transgenic plants. The vesicle-related protein OsSEC27P may play a potential role in enhancing H⁺ secretion in roots under the iron deficiency conditions.     

含编码类铁氧还双亲性蛋白ap1基因的转基因水稻植株的获得

利用基因枪法将含有潮霉素抗性基因（hpt),gusA报告基因和ap1基因的2个质粒（pJIMB15和pBiSAP1)共同转化同转化由粳稻品种鄂宜105号种 子在胚诱导的愈伤组织（2－3周龄）。ap1基因编码一种双亲性的蛋白。该蛋白能延缓因假单孢菌感染所引起的非寄主植物中的过敏反应。经过2轮潮霉素（30mg/L)筛选,抗性愈伤组织被转入含30mg/L潮霉素的再生培养基中再生植株。从轰击的186块愈伤组织中共再生出32株独立的转基因水稻植株（转化率为17．2％）,PCR／Southern blot分析显示84％的转基因植株含有所有3个基因。     

通过遗传转化获得含多基因的水稻转基因纯合植株

利用基因枪法将含有4个不同基因的3个质粒共转化由粳稻品种鄂宜105号和鄂晚5号种子胚诱导的愈伤组织（5－10d龄）。从轰击的986块愈合组织中共再生出169株独立的转基因水稻植株（转化率为17％）。PCR／Southern blot分析显示70％以上的转基因植株含有所有4个基因。GUS组织化学分析、Western blot和或RT－PCR分析表明所有4个基因的共表达率为70％。未观察到任何质粒在整合中存在优势,转基因拷贝数也与基因表达量无关。遗传分析证实外源基因在后代植株中大多以孟德尔方式遗传。从其R1代为3：1孟德尔方式遗传的后代R2代植株中,鉴定含有3个或4个不同基因的转基因纯合植株系。PCR／Southern blot分析证实了这些转基因纯合植株系。这些系的植株具有相似的外源基因表达量。我们证实通过基因枪介导的共转化,结合常规育种方法筛选可以获得含多基因的转基因水稻纯合植株。这项技术为利用基因同时改良作用多个性状提供了一种途径。     

Expression analysis ofgdcsP promoter from C3-C4 intermediate plantFlaveria anomala in transgenic rice

ThegdcsP promoter isolated from C₃-C₄ intermediate plantFlaveria anomala was fused to the β-glucuronidase (GUS) gene. The chimeric gene was inserted into the binary vector pBin19 and introduced into the rice (Oryza sativa L.) cv. 8706 byAgrobacteriummediated gene transfer. GUS activity can be detected in leaf, leaf sheath, stem and root tissues via fluorometric GUS assay. However, no GUS activity was found in mature endosperm. Histochemical localization revealed that GUS expression was exclusively restricted to vascular tissues in transgenic plants. This promoter also showed spatial-temporal expression patterns that GUS expression declined significantly with the maturity of plants. These expression patterns make thegdcsP promoter extremely valuable in the applied biotechnology that needs target gene expression restricted to vascular tissues.     

Mapping of a new gene for brown planthopper resistance in cultivated rice introgressed fromOryza eichingeri

Wild rice species is an important source of useful genes for cultivated rice improvement. Some accessions of Oryza eichingeri (2n = 24, CC) from Africa confer strong resistance to brown planthopper (BPH), whitebacked planthopper (WBPH) and bacterial blight (BB). In the present study, restriction fragments length polymorphism (RFLP) and simple sequence repeats (SSR) analysis were performed on disomic backcross plants between Oryza sativa (2n = 24, AA) and O. eichingeri in order to identify the presence of O. eichingeri segments and further to localize BPH-resistant gene. In the introgression lines, 1—6 O. eichingeri segments were detected on rice chromosomes 1, 2, 6, or/and 10. The dominant BPH resistant gene, tentatively named Bph13(t), was mapped to chromosome 2, being 6.1 and 5.5 cM away from two microsatellite markers RM240 and RM250, respectively. The transfer and localization of this gene from O. eichingeri will contribute to the improvement of BPH resistance in cultivated rice.     

Salt tolerance of transgenic rice (Oryza sativa L.) with mtlD gene and gutD gene

Southern blot analysis indicated that mtlD gene (encoding mannitol-1-phosphate dehydrogenase) and gutD gene (encoding glucitol-6-phosphate dehydrogenase) had been integrated into the rice genome mediated by Agrobacterium tumefaciens LBA4404(pBIGM). The expression of the above two genes in transgenic rice plants was demonstrated by Northern blot analysis and enzymatic activity assay. Analysis of sugar alcohol showed that transgenic rice plants could produce and accumulate mannitol and sorbitol. The salt tolerance of transgenic plants was much higher than that of their controls.     

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。