Molecular cloning of rice cDNAs related to pollen development using the subtraction hybridization technique

The meiotic stage of pollen mother cell is a very important stage in controlling the development and formation of pollen. In order to clone the rice cDNA(s) of this stage, a normal rice, Annong N and its thermosensitive mutant, Annong S-1 were used as the plant material. The mRNA has been extracted from the young panicle at the meiotic stage. By using the cDNA subtraction hybridization technique, three cDNA fragments, RP-1, RP-2 and RP-3 have been successfully cloned from Annong N. Northern blot analysis reveals that the mRNA of these three clones are expressed only in anthers, and not leaves. The mRNA levels of these clones are lower in anthers of Annong S-1 than in Annong N. Furthermore, the amount of mRNA extracted from anthers of Annong S-1 growing under high temperature (28℃) is lower than plants growing at lower temperature (25℃). Sequence analysis and homology search indicate that these three clones display no similarity to the current database. It is concluded that the three novel cDNA cloned are related to pollen development in rice.     

Development and characterization of interspecific hybrids between Oryza sativa and O. latifolia by in situ hybridization

Oryza sativa and O. latifolia belong to the AA and CCDD genomes of Oryza, respectively. In this study, interspecific hybrids of these species were obtained using the embryo rescue technique. Hybrid panicle traits, such as long awns, small grain, exoteric large purple stigma, grain shattering and dispersed panicles, resemble that of the paternal parent, O. latifolia, whereas there is obvious heterosis in such respects as plant height, tillering ability and vegetative vigor. Chromosome pairing and the genomic components of the hybrid were subsequently investigated using genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) analysis. Based on the mitotic metaphase chromosome numbers of the root tips investigated, the hybrid is a triploid with 36 chromosomes. The genomic constitution of the hybrid is ACD. In the meiotic metaphase I of the hybrid pollen mother cell, poor chro- mosome pairing was identified and most of the chromosomes were univalent, which resulted in com- plete male sterility in the hybrid.     

Comparative physical mapping of rice BAC clones linked to resistance genes Glh,Bph-3 and xa-5 in Oryza sativa L. and O. granulata Nees et Arn. ex Watt.

Oryza granulata Nees et Arn. ex Watt. is one of the three wild relatives of rice,which are the most valuable for study and utilization in China.In this study,the homology and physical locations of three rice resistance genes,Glh,Bph-3 and xa-5 are comparatively analyzed between O.sativa and O. granulata by Southern blotting and fluorescence in situ hybridization (FISH).The results of Southern blotting indicate that there exist homologous sequences of the tested RFLP markers in O. granulata.By using three bacterial artificial chromosome (BAC) clones scanned by the tested RFLP as probes, FISH signals are detected on both mitotic and pachytene chromosomes in O.sativa and O.granulata.Dual-color FISH demonstrates that two of the three BAC clones (14E16 and 38J9) are located on the short arm of the same chromosome pair in O. granulata. Additionally, colinearity is shown for the two clones between O.sativa and O.granulata. Another BAC clone 44B4 is located on the end of the short arm of other chromosome pair in these two species.Although the phylogenetic relationship between O.sativa and O.granulata is the most distinct in Oryza and these two species have evidently different biological features and ecological habits, the relative lengths and arm ratios of the detected chromosomes and the relative positions of the tested clone signals on chromosomes in O. granulata are quite similar to those in O. sativa.     

Transfer of bacterial blight resistance from <Emphasis Type="Italic">Oryza meyeriana</Emphasis> to <Emphasis Type="Italic">O. sativa</Emphasis> L. by asymmetric somatic hybridization

Asymmetric somatic hybrid plants were produced between cultivated rice (Oryza sativa L.) and wild species [O. meyeriana (Zoll. etMor, exSteud.)] with high resistance to rice bacterial blight. X-ray-irradiated protoplasts of the wild species were used as donor and chemically fused with iodoacetamide-inactivated protoplasts of rice cv. 02428 to produce hybrids. Seventy-two plants were regenerated from 623 calli based on metabolic complementation. The morphological characters of the plants closely resembled that of the rice. Simple sequence repeats were employed to identify their hybridity. Cytological analysis of root-tips revealed that their chromosome number varied in the range of 27--38. The somatic hybrids were inoculated with strains of Xanthamonas oryzae pv. oryzae at adult growth stage and demonstrated the resistance to bacterial blight introgression from the O. meyeriana.     

不同品级拟黑多刺蚁(Polyrhachis vicina)脑部雌激素相关受体基因的表达

采用原位杂交方法对不同品级拟黑多刺蚁(Polyrhachis vicina)脑部雌激素相关受体pvERRmRNA的表达进行了检测.结果表明,pvERRmRNA在不同品级的蕈形体、附叶、视叶、中脑、食道下神经节和球状细胞中都有表达.蚁后蕈形体和球状细胞中pvERRmRNA的阳性表达最为明显;工蚁中脑pvERR mRNA的阳性反应强度明显高于蚁后和雄蚁;pvERR mRNA在雄蚁视叶中有较强的表达.pvERR mRNA在不同品级的拟黑多刺蚁脑部的表达差异可能与它们各自在蚁群中的功能和行为有关.     

Determination of copy number for 5S rDNA and centromeric sequence RCS2 in rice by Fiber-FISH

The copy number of 5S rDNA and centromeric sequence RCS2 was determined by extended DNA fiber based fluorescence in situ hybridization (Fiber-FISH) in rice (Oryza sativa ssp. indica cv. Guangluai No. 4) genome. In order to determine the copy number, it is necessary to know the basepair number that a given length DNA fiber contains under a microscope. Therefore, the length of two DNA fragments, in which the basepair number had been already known, was measured. The insert sequence of the tested BAC 38D17 was 136 kb and its extended DNA was 56.4 μm long, 2.41 kb/μm on average, while that of the tested BAC 44B4 was 144.5 kb in total and 55.7 μm long, 2.60 kb/μm on average under the microscope. They were very close to the theoretical value of B-DNA in the Watson-Crick DNA model, which is 2.97 kb/μm. According to the average value of basepair number per μm of the two samples mentioned above, that is, 2.51 kb/μm, it could be estimated that the copy number was about 686 for 5S rDNA and 286-1121 for the centromere sequence RCS2.     

Centromere inactivation in a dicentric rice chromosome during sexual reproduction

T0135 is a variant selected from the progeny of a rice line telotrisomic for the short arm of chromosome 11 （2n＋IIS＇）. Fluores- cent in situ hybridization （FISH） results indicated that T0135 contained two telocentric chromosomes, which have two centro- mere-specific molecular markers （5S rDNA） for chromosome 11; thus T0135 is a newly-described rice chromosome variant with two dicentric chromosomes, named 22＋11L-＋11L＇＋I IS.11S-＋I 1S-11S. （22 represents the 22 chromosomes excluding chromo- some 11 in the rice genome, ＂-＂ represents the centromere）. To investigate the genetic stability of the rice dicentric chromosomes during sexual reproduction, we observed the chromosome types in the progeny. Ninety-four percent of the progeny had the same chromosome type as the parental line. This result indicates that the dicentric chromosomes are mostly stable during mitosis and meiosis. Immunofluorescence analysis for centromere specific histone H3 （CENH3） revealed that only one centromere is active and the other centromere is inactivated in the rice dicentric chromosomes.     

Comparative analysis of A,B,C and D genomes in the genus Oryza with C_0t-1 DNA of C genome

The genus Oryza includes two cultivated species, O. sativa L. and O. glaberrima Steud. and comprises more than 20 species[1]. The genomes of Oryza are classified into 10 types: AA, BB, CC, BBCC, CCDD, EE, FF, GG, HHJJ and HHKK[2,3]. Morphological variatio…     

Comparative genome research between maize and rice using genomicin situ hybridization

Using the genomic DNAs of maize and rice as probes respectively, the homology of maize and rice genomes was assessed by genomic in situ hybridization. When rice genomic DNAs were hybridized to maize, all chromosomes displayed many multiple discrete regions, while each rice chromosome delineated a single consecutive chromosomal region after they were hybridized with maize genomic DNAs. The results indicate that the genomes of maize and rice share high homology, and confirm the proposal that maize and rice are diverged from a common ancestor.     

In situ localization of proteinase inhibitor mRNA in rice plant challenged by brown planthopper

Proteinase inhibitor (PI) mRNA was localized by in situ hybridization in tissue sections of root, stem and leaf of the resistant rice (B5) plant fed by brown planthopper nymphs. In the rice material without BPH feeding, PI gene was expressed in the root, stem and leaf, while the abundance of PI mRNA was low. In the rice material fed by BPH,PI gene was expressed substantially in the parenchyma of rice stem and leaf, but weakly in the root. The results indicated that the PI gene was up-regulated in the rice plant challenged by brown planthopper. For the first time, we reported the expression changes of proteinase inhibitor gene in plant which was infested by a piercing/sucking insect.     

Mapping of the rice （Oryza sativa L.） thermo-sensitive genic male sterile gene tms5 with EST and SSR markers

VELOPING“TWO-LINE”HYBRID RICE.THE POLLEN FERTILITY OF TGMS IS REGULATED BY THE TEMPERATURE OF ENVIRONMENT.THE POLLENS OF TGMS LINES ARE STERILE WHEN THE ENVI-RONMENT TEMPERATURE IS ABOVE A CRITICAL POINT,BUT FERTILE BELOW THIS POINT.SO FAR,A NUMBER OF T…     

乐安油田注汽后火烧油层开发参数敏感性研究

火烧油层开发效果的影响因素很多 ,但在油藏条件一定的情况下 ,可通过分析开发参数的敏感性并设计其合理值来提高火驱的经济效益。以乐安油田火烧试验区为例 ,在注蒸汽热采历史拟合及蒸汽驱预测的基础上 ,利用工程计算及数值模拟方法 ,研究了射孔层位、射开厚度、注气速度、燃烧方式、转湿烧时机、湿烧水气比等参数对火烧效果的影响 ,分析了火烧前缘推进速度 ,优选了开发参数。结果表明 ,底层射孔可减轻火线超覆 ,射开厚度大可提高地层吸气能力。由空气油比和采收率来确定分阶段变速率注气开采效果较好 ,最大注气速度为 80 0 0 0m3 /d。湿式燃烧能提高采收率 ,降低空气油比 ,其合理的转湿时机为燃烧距离达到 2 5 % ,水气比为 2 .0kg/m3 。预测结果与实际结果吻合较好 ,为火驱试验的现场实施提供了理论依据。     

Generation of selectable marker-free transgenic rice resistant to chewing insects using two co-transformation systems

To produce selectable marker-free (SMF) transgenic rice resistant to chewing insects, the Bacillus thuringiensis cryIA(c) gene (Bt) was introduced into two elite japonica rice varieties by using two Agrobacterium-mediated co-transformation systems. One system is with a single mini-twin T-DNA binary vector in one Agrobacterium strain, which consists of two separate T-DNA regions, one carrying the Bt while the other contains the selectable marker gene, hygromycin resistant gene (HPT). The other system uses two separate binary vectors in two separate Agrobacterium cultures, containing the Bt or HPT gene on individual plasmids. A lot of independent transgenic rice lines harboring both Bt and selectable marker genes were obtained. The results showed that the co-transformation frequency of the Bt gene and HPT gene was much higher by using the mini-twin T-DNA vector system (29.87%) than that by the two separate binary vector systems (4.52%). However, the frequency of the SMF transgenic rice plants obtained from the offspring of co-transgenic plants (21.74%) was lower for the mini-twin T-DNA vector system than that for the latter (50-60%). The data of ELISA implied that the expressed Bt proteins were quantitated as 0.025-0.103% of total leaf soluble proteins in the transgenic plant. Therefore, several elite transgenic rice lines, free of the selectable marker gene, were chosen. The results from both in vitro and in vivo insect bioassays indicated that the SMF transgenic rice was shown to be highly resistant to the striped stem borer and rice leaf folder. Moreover, in a natural field condition without any insecticide applied, all the transgenic rice plants were found to be not injured by the rice leaf folder, whereas the wild types were impaired seriously.     

Microscopic and ultramicroscopic localizations and quantitative analysis of 5-HT receptors in human placentas

The localizations of 5-hydroxytryptamine receptor (5-HTR) at light and electron microscopic levels and its quantitative analysis in human placentas were studied by using immunohistochemistry and in situ hybridization. Both syncytiotrophoblast and cytotrophoblast in placental villi and fetal white blood cells in villose capillary cavity showed 5-HT receptor immunoreactivity, with 5-HT 1A receptor mRNA hybridized signal detected in cytoplasm. But the stromal cells and capillary endothelium in placental villi showed 5-HT receptor immunoreactivity in cytoplasm, without 5_HT\-1A receptor mRNA hybridized signal detected. This suggested that two layers of trophoblast cells may produce 5-HT 1 and 5-HT 2 receptors, that the stromal cells and capillary endothelium in placental villi may only produce 5-HT 2 receptor. By immunohistochemistry at electron microscopic level, the small flattened vesicles and large dense cored vesicle within trophoblast cells showed 5-HT receptor immunoreactivity. This suggested that it may be the result of 5-HT receptors internalization and transportion. Using a quantitative immunohistochemical method, the contents of 5-HT receptor in placenta were higher during the 6th week of gestation, and decreased in 7th and 8th, reoccurred the second peak in the 9th, reduced gradually during the 10th, 20th and 40th of the gestation period. These changes paralleled the contents of 5-HT in the authors' studies, reflecting that 5-HT may be one of the most important bioactive substances in placental self-regulation.     

Breeding and molecular cytogenetic identification of wheat-Thinopyrum intermedium addition lines resistant to powdery mildew

Wheat-related species Th. intermedium was used to cross with common wheat Yannong 15. In the self progenies of the hybrid, two addition lines, Ⅱ-1-7-1 and Ⅱ-3-3-2, stable in cytology, were developed by cytology and powdery mildew resistance identification. Their chromosome number were 2n = 44 and formed 22 bivalents at PMC MI. In F1 of the two addition lines crossing with Yannong 15, there appeared about one univalent at PMC MI, respectively. Resistance identification in greenhouse and field using the No. 15 and mixed strains of E. gramnis f. sp. tritici showed that they were immune to powdery mildew. Chromosome number and resistance identification using the F2 single plants of the addition line crossing with Yannong 15 indicated that the resistant gene was located on the alien chromosomes. In situ hybridization using St and E genomic DNA as probe showed that the added chromosome in the two addition lines probably came from the E genome of Th. intermedium, which indicated that a pair of E genome chromosomes carried a new resistant gene to powdery mildew.