两种完熟相关基因反义RNA在转因番茄中的表达及其对果实完熟的仰制作用

植物激素乙烯作为一种信使分子调节控制果实完熟。ACC合成酶是植物体内乙烯生物合成途径的限速酶，其反义RNA的表达将能有效地抑制乙烯的生物合成而延缓果实完熟，利用反转录PCR技术克隆获得了ACC合成酶多基因家族成员之一LE-ACC2阅读框架约1.7kb的cDNA，经酶切图谱和序列分析鉴定无误后，反向连入植物表达载体pBin437中构建成组成型表达ACC合成酶反义RNA的双元载体。经农杆菌途径转化番茄“丽春”品种，获得了60株抗卡那再生杭株，PCR检测证明有6株为转基因植株，Southern杂交和Northern杂交分析进一步确证了外源基因的插入及其转录活性。反义番茄果实的乙烯释放受到明显抑制，表现出更好的耐储保鲜特性，并且与对照相比，在果实品质上没有明显差别。大田培育Fl和F2代转化番茄植株，反义番茄纯合品系的筛选工作正在进行之中。 同时，本研究利用已经获得的ACC合成酶和PG的cDNA克隆，构建了两个嵌合转化基因载体pPGACC1、pPGACC10，它包括1300bp的ACC合成酶cDNA编码序列，并分别含有反向与正向的250bp的5’端PG基因片断。酶切图谱和序列分析鉴定无误后，以pBin437为植物表达载体构建了双元载体pBPGACC1和pBPGACC10，分别表达PG正义RNA和反义RNA，并均表达ACC合成酶反义RNA。经农杆菌转化番茄子叶，植株的再生培育有待进行。通过对转基因植物的分析，我们期望阐明用单一嵌合基因表达载体通过反义抑制与抑制作用实现对内源两同源基因——PG和ACC合成酶下降调节的可能性，并可望得到具有更好耐储效果且品质优良的番茄品系。

金丝桃素在制备抗RNA病毒药物中的应用

本发明公开了金丝桃素的新用途。本发明发明人的实验证实，金丝桃素对ＲＮＡ病毒，特别是禽流感病毒，口蹄疫病毒和犬瘟热病毒具有较好的抑制和灭活效果，可以该化合物为活性成分，制备成抗ＲＮＡ病毒药物。该抗病毒药物可用于临床防治和治疗禽流感、犬瘟热、口蹄疫等由ＲＮＡ病毒引发的疾病，对禽业、犬业及畜牧养殖业等意义重大。此外，我国草药资源丰富，该药物具有工业化生产的可行性。综上所述，金丝桃素将在医学和生物制药领域，尤其是抗ＲＮＡ病毒药物的制备领域具有较大的实际意义和广阔的应用前景。

眼镜王蛇泛素融合蛋白基因和核糖体蛋白L30 基因的克隆及分析

从广西产眼镜王蛇( Ophiophagus hannah) 毒腺中抽提总RNA , 经mRNA 纯化后构建眼镜王蛇毒腺 cDNA 文库。从所构建的cDNA 文库中, 随机筛选200 个克隆测序, 得到两个在进化上高度保守的基因: 泛素融 合蛋白基因(GenBank 登录号为AF297036) 和核糖体蛋白L30 基因(GenBank 登录号是AF297033) 。前者cDNA 的开放阅读框为387 bp , 后者为348 bp 。前者编码128 个氨基酸残基组成的泛素融合蛋白前体; 后者编码115 个氨基酸残基组成的核糖体蛋白L30 前体。由cDNA 序列推导出的氨基酸序列分析表明, 泛素融合蛋白前体包 括N - 末端的泛素结构域(76 个氨基酸残基) 和C - 末端的核糖体蛋白L40 结构域(52 个氨基酸残基) 。该蛋 白为一高碱性蛋白, C 末端含有一个“锌指”模式结构。与16 个物种比较的结果表明, 眼镜王蛇与脊椎动物的 泛素融合蛋白氨基酸序列相似度较高, 具有高度的保守性。

Estimation of RNA/DNA ratio in two north-Indian natural populations of mahseer (Tor tor) and its relationship with growth and hydrobiology

Samples of Tor tor were collected from Bari Reservoir of Udaipur and Narmada River at Hoshangabad (India), in the months of July and November 2005, respectively. Twenty-five samples were collected from each location. Bari Reservoir samples ranged from 17.0 to 24.5 cm in total length and from 75 to 155 g in weight, while Narmada samples ranged from 20.0 to 42.0 cm in length and 90 to 425 g in weight. The nucleic acid content in body muscle of Tor tor and the RNA/DNA ratio were estimated. The age of fishes was estimated by the scale study method and specimens were classified into four age groups. RNA/DNA ratio showed significant linear increase with increase in weight and age till the age of three years after which, the growth rate reduced. The 1-2 year group was the only one common between the two water bodies and a comparison of RNA/DNA ratios showed higher growth rate in Bari Reservoir. The gross primary productivity was also higher in Bari Reservoir being 551 mg cmˉ³ dˉ¹ compared to 404 mg cmˉ³ dˉ¹ observed for Narmada River. The condition factor (K) was found to be higher (1.21) in the fish from the Bari Reservoir compared to those of Narmada River (1.14). The growth rate was higher in females compared to males in >100 g specimens.

RNA processing and Arabidopsis flowering time control.

Plants control their flowering time in order to ensure that they reproduce under favourable conditions. The components involved in this complex process have been identified using a molecular genetic approach in Arabidopsis and classified into genetically separable pathways. The autonomous pathway controls the level of mRNA encoding a floral repressor, FLC, and comprises three RNA-binding proteins, FCA, FPA and FLK. FCA interacts with the 3'-end RNA-processing factor FY to autoregulate its own expression post-transcriptionally and to control FLC. Other components of the autonomous pathway, FVE and FLD, regulate FLC epigenetically. This combination of epigenetic and post-transcriptional control gives precision to the control of FLC expression and flowering time.

Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning.

Small RNAs have several important biological functions. MicroRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) regulate mRNA stability and translation, and siRNAs cause post-transcriptional gene silencing of transposons, viruses and transgenes and are important in both the establishment and maintenance of cytosine DNA methylation. Here, we study the role of the four Arabidopsis thaliana DICER-LIKE genes (DCL1-DCL4) in these processes. Sequencing of small RNAs from a dcl2 dcl3 dcl4 triple mutant showed markedly reduced tasiRNA and siRNA production and indicated that DCL1, in addition to its role as the major enzyme for processing miRNAs, has a previously unknown role in the production of small RNAs from endogenous inverted repeats. DCL2, DCL3 and DCL4 showed functional redundancy in siRNA and tasiRNA production and in the establishment and maintenance of DNA methylation. Our studies also suggest that asymmetric DNA methylation can be maintained by pathways that do not require siRNAs.

An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana.

ARGONAUTE4 (AGO4) and RNA polymerase IV (Pol IV) are required for DNA methylation guided by 24 nucleotide small interfering RNAs (siRNAs) in Arabidopsis thaliana. Here we show that AGO4 localizes to nucleolus-associated bodies along with the Pol IV subunit NRPD1b; the small nuclear RNA (snRNA) binding protein SmD3; and two markers of Cajal bodies, trimethylguanosine-capped snRNAs and the U2 snRNA binding protein U2B''. AGO4 interacts with the C-terminal domain of NRPD1b, and AGO4 protein stability depends on upstream factors that synthesize siRNAs. AGO4 is also found, along with the DNA methyltransferase DRM2, throughout the nucleus at presumed DNA methylation target sites. Cajal bodies are conserved sites for the maturation of ribonucleoprotein complexes. Our results suggest a function for Cajal bodies as a center for the assembly of an AGO4/NRPD1b/siRNA complex, facilitating its function in RNA-directed gene silencing at target loci.

MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant.

The Arabidopsis genome contains a highly complex and abundant population of small RNAs, and many of the endogenous siRNAs are dependent on RNA-Dependent RNA Polymerase 2 (RDR2) for their biogenesis. By analyzing an rdr2 loss-of-function mutant using two different parallel sequencing technologies, MPSS and 454, we characterized the complement of miRNAs expressed in Arabidopsis inflorescence to considerable depth. Nearly all known miRNAs were enriched in this mutant and we identified 13 new miRNAs, all of which were relatively low abundance and constitute new families. Trans-acting siRNAs (ta-siRNAs) were even more highly enriched. Computational and gel blot analyses suggested that the minimal number of miRNAs in Arabidopsis is approximately 155. The size profile of small RNAs in rdr2 reflected enrichment of 21-nt miRNAs and other classes of siRNAs like ta-siRNAs, and a significant reduction in 24-nt heterochromatic siRNAs. Other classes of small RNAs were found to be RDR2-independent, particularly those derived from long inverted repeats and a subset of tandem repeats. The small RNA populations in other Arabidopsis small RNA biogenesis mutants were also examined; a dcl2/3/4 triple mutant showed a similar pattern to rdr2, whereas dcl1-7 and rdr6 showed reductions in miRNAs and ta-siRNAs consistent with their activities in the biogenesis of these types of small RNAs. Deep sequencing of mutants provides a genetic approach for the dissection and characterization of diverse small RNA populations and the identification of low abundance miRNAs.

Role of RNA polymerase IV in plant small RNA metabolism.

In addition to the three RNA polymerases (RNAP I-III) shared by all eukaryotic organisms, plant genomes encode a fourth RNAP (RNAP IV) that appears to be specialized in the production of siRNAs. Available data support a model in which dsRNAs are generated by RNAP IV and RNA-dependent RNAP 2 (RDR2) and processed by DICER (DCL) enzymes into 21- to 24-nt siRNAs, which are associated with different ARGONAUTE (AGO) proteins for transcriptional or posttranscriptional gene silencing. However, it is not yet clear what fraction of genomic siRNA production is RNAP IV-dependent, and to what extent these siRNAs are preferentially processed by certain DCL(s) or associated with specific AGOs for distinct downstream functions. To address these questions on a genome-wide scale, we sequenced approximately 335,000 siRNAs from wild-type and RNAP IV mutant Arabidopsis plants by using 454 technology. The results show that RNAP IV is required for the production of >90% of all siRNAs, which are faithfully produced from a discrete set of genomic loci. Comparisons of these siRNAs with those accumulated in rdr2 and dcl2 dcl3 dcl4 and those associated with AGO1 and AGO4 provide important information regarding the processing, channeling, and functions of plant siRNAs. We also describe a class of RNAP IV-independent siRNAs produced from endogenous single-stranded hairpin RNA precursors.

FY is an RNA 3' end-processing factor that interacts with FCA to control the Arabidopsis floral transition

The nuclear RNA binding protein, FCA, promotes Arabidopsis reproductive development. FCA contains a WW protein interaction domain that is essential for FCA function. We have identified FY as a protein partner for this domain. FY belongs to a highly conserved group of eukaryotic proteins represented in Saccharomyces cerevisiae by the RNA 3' end-processing factor, Pfs2p. FY regulates RNA 3' end processing in Arabidopsis as evidenced through its role in FCA regulation. FCA expression is autoregulated through the use of different polyadenylation sites within the FCA pre-mRNA, and the FCA/FY interaction is required for efficient selection of the promoter-proximal polyadenylation site. The FCA/FY interaction is also required for the downregulation of the floral repressor FLC. We propose that FCA controls 3' end formation of specific transcripts and that in higher eukaryotes, proteins homologous to FY may have evolved as sites of association for regulators of RNA 3' end processing.

从核糖体RNA基因序列探讨双尾虫的系统进化

双尾虫是否单系，以及双尾虫与其他六足动物系统关系是多年来动物分类学家争议的一个关键问题。测定了双尾虫的两大类群：康 类和铗 类，以及原尾虫、跳虫和蝗虫等核糖体RNA基因18SrDNA全序列和28SrDNA部分序列（D3－D5区），并选用甲壳动物卤虫为外群，采用最大筒约（MP）法构建分子系统树。结果表明：(i）18SrDNA和28SrDNA数据整合分析含有较强的系统发育信息，支持双尾虫单系性观点；（ii）双尾虫与原尾虫在系统中构成姊姝群，且支持率很高。

Ribosomal DNA gene and phylogenetic relationships of Diplura and lower Hexapods

The monophyly of Diplura and its phylogenetic relationship with other hexapods are important for understanding the phylogeny of Hexapoda. The complete 18SrRNA gene and partial 28SrRNA gene (D3-D5 region) from 2 dipluran species (Campodeidae and Japygidae), 2 proturan species, 3 collembolan species, and 1 locust species were sequenced. Combining related sequences in GenBank, phylogenetic trees of Hexapoda were constructed by MP method using a crustacean Artemia salina as an outgroup. The results indicated that: (i) the integrated data of 18SrDNA and 28SrDNA could provide better phylogenetic information, which well supported the monophyly of Diplura; (ii) Diplura had a close phylogenetic relationship to Protura with high bootstrap support.

Deep RNA sequencing at single base-pair resolution reveals high complexity of the rice transcriptome

Understanding the dynamics of eukaryotic transcriptome is essential for studying the complexity of transcriptional regulation and its impact on phenotype. However, comprehensive studies of transcriptomes at single base resolution are rare, even for modern organisms, and lacking for rice. Here, we present the first transcriptome atlas for eight organs of cultivated rice. Using high-throughput paired-end RNA-seq, we unambiguously detected transcripts expressing at an extremely low level, as well as a substantial number of novel transcripts, exons, and untranslated regions. An analysis of alternative splicing in the rice transcriptome revealed that alternative cis-splicing occurred in similar to 33% of all rice genes. This is far more than previously reported. In addition, we also identified 234 putative chimeric transcripts that seem to be produced by trans-splicing, indicating that transcript fusion events are more common than expected. In-depth analysis revealed a multitude of fusion transcripts that might be by-products of alternative splicing. Validation and chimeric transcript structural analysis provided evidence that some of these transcripts are likely to be functional in the cell. Taken together, our data provide extensive evidence that transcriptional regulation in rice is vastly more complex than previously believed.

Phylogeny of fireflies (Coleoptera : Lampyridae) inferred from mitochondrial 16S ribosomal DNA, with references to morphological and ethological traits

We sequenced partial mitochondrial 16S ribosomal DNA (16S rDNA) of 18 firefly species from Southwest of China. Combined with homologous sequences previously reported, phylogenetic trees including Japanese, Korean and Chinese species were reconstructed by