Identification of Conserved and Novel MicroRNAs in the Pacific Oyster Crassostrea gigas by Deep Sequencing

MicroRNAs (miRNAs) play important roles in regulatory processes in various organisms. To date many studies have been performed in the investigation of miRNAs of numerous bilaterians, but limited numbers of miRNAs have been identified in the few species belonging to the clade Lophotrochozoa. In the current study, deep sequencing was conducted to identify the miRNAs of Crassostrea gigas (Lophotrochozoa) at a genomic scale, using 21 libraries that included different developmental stages and adult organs. A total of 100 hairpin precursor loci were predicted to encode miRNAs. Of these, 19 precursors (pre-miRNA) were novel in the oyster. As many as 53 (53%) miRNAs were distributed in clusters and 49 (49%) precursors were intragenic, which suggests two important biogenetic sources of miRNAs. Different developmental stages were characterized with specific miRNA expression patterns that highlighted regulatory variation along a temporal axis. Conserved miRNAs were expressed universally throughout different stages and organs, whereas novel miRNAs tended to be more specific and may be related to the determination of the novel body plan. Furthermore, we developed an index named the miRNA profile age index (miRPAI) to integrate the evolutionary age and expression levels of miRNAs during a particular developmental stage. We found that the swimming stages were characterized by the youngest miRPAIs. Indeed, the large-scale expression of novel miRNAs indicated the importance of these stages during development, particularly from organogenetic and evolutionary perspectives. Some potentially important miRNAs were identified for further study through significant changes between expression patterns in different developmental events, such as metamorphosis. This study broadened the knowledge of miRNAs in animals and indicated the presence of sophisticated miRNA regulatory networks related to the biological processes in lophotrochozoans.     

胡杨无性系幼苗响应盐胁迫的miRNA表达差异研究

植物miRNA在调控基因表达、细胞周期、生物体发育、抗逆等方面起重要作用。为研究胡杨(Populus euphratica Oliv.)的耐盐机制,以1年生胡杨无性系幼苗为材料,构建具有空间代表性的盐胁迫胡杨cDNA文库,利用二代测序技术测定NaCl胁迫下和正常培养条件下胡杨叶和根miRNA表达情况。结果表明,不同的miRNA之间表达量存在明显差异,表达丰度最高的miRNA有miR156、miR157、miR165、miR166和miR167等,合计占总表达量的90%以上。胡杨根部存在特异表达的miRNA,在整个耐盐调控机制中发挥着生理调节、分子调控和信号传导等极为重要的作用。盐处理样品中发现大量响应盐胁迫的miRNA,对这些转录因子进行靶基因预测和注释后,发现很多盐胁迫响应的miRNA与NAC和SPL等重要转录因子家族相关,与前人的结论一致,另外还发现许多miRNA的调控对象是ATP酶和激素响应因子。     

Identification of MicroRNAs in Meloidogyne incognita Using Deep Sequencing

MicroRNAs play important regulatory roles in eukaryotic lineages. In this paper, we employed deep sequencing technology to sequence and identify microRNAs in M. incognita genome, which is one of the important plant parasitic nematodes. We identified 102 M. incognita microRNA genes, which can be grouped into 71 nonredundant miRNAs based on mature sequences. Among the 71 miRANs, 27 are known miRNAs and 44 are novel miRNAs. We identified seven miRNA clusters in M. incognita genome. Four of the seven clusters, miR-100/let-7, miR-71-1/miR-2a-1, miR-71-2/miR-2a-2 and miR-279/miR-2b are conserved in other species. We validated the expressions of 5 M. incognita microRNAs, including 3 known microRNAs (miR-71, miR-100b and let-7) and 2 novel microRNAs (NOVEL-1 and NOVEL-2), using RT-PCR. We can detect all 5 microRNAs. The expression levels of four microRNAs obtained using RT-PCR were consistent with those obtained by high-throughput sequencing except for those of let-7. We also examined how M. incognita miRNAs are conserved in four other nematodes species: C. elegans, A. suum, B. malayi and P. pacificus. We found that four microRNAs, miR-100, miR-92, miR-279 and miR-137, exist only in genomes of parasitic nematodes, but do not exist in the genomes of the free living nematode C. elegans. Our research created a unique resource for the research of plant parasitic nematodes. The candidate microRNAs could help elucidate the genomic structure, gene regulation, evolutionary processes, and developmental features of plant parasitic nematodes and nematode-plant interaction.     

党参的体细胞胚发生及不同发育阶段几种同工酶的分析

以党参为材料,在附加0.1 mg·L-1 2,4-D、0.3 mg·L-1 6-BA和3%蔗糖的MS培养基上获得大量发育良好的体细胞胚. 附加6-BA可以使胚性愈伤组织进行芽的分化,而添加0.1%活性炭后仅有根的分化.利用梯度凝胶电泳进行同工酶的研究表明在胚性愈伤组织和体细胞胚之间,酶谱差异比较大;而在不同发育时期的体细胞胚之间,差异较小.并讨论了同工酶酶谱和酶活性变化与体细胞胚发生、发育的关系.     

Ultrastructural Changes in Leaves of Cichorium during Somatic Embryogenesis

A detailed electron microscopy study of early cellular eventsduring somatic embryogenesis in leaves of Cichorium is described.Leaves on in-vitro grown plantlets were sectioned and put at35°C, in darkness, in an agitated liquid induction medium.No sign of embryogenic predetermination, such as thick cellwall, dense cytoplasm and enlarged nucleus, could be seen inany cell before treatment. Perivascular cells were the firstto react. Addition of glycerol (330 mM) allowed the arrest ofembryogenic cells at an activated stage. The main events werea thickening of the wall, with extracellular secretion and anaccumulation of Ca²⁺ in the vacuole, demonstrated by an antimonateprocedure. After 5 d, leaves were transferred to glycerol-freemedium where multicellular proembryos could be observed. Theyshowed reduced vacuoles, cortical microtubules, numerous multivesicularbodies and lipid globules. The embryoid cells were lined alongthe mesophyll lacunae by an extracellular secretion with a tubularstructure; histochemical tests proved its complex lipo-glyco-proteicnature.Copyright 1993, 1999 Academic Press Cichorium, extracellular tubular protein, somatic embryogenesis, vacuolar calcium     

植物体细胞胚发生中ATP酶活性时空分布动态与内源激素的变化

本文以我们的研究结果为基础,并结合国内外近几年有关研究报道,对植物体细胞胚发生中的超策结构和ATP酶活性时空分布动脉及内源激素的变化和作用进行专题评述。（1）超微结构的变化：当植物体细胞一量转化为胚性细胞后,各种细胞器相继增加,不仅丰富而且活跃,特别是线粒体内发达,有的正处于分裂状态;核糖体聚集成多聚核糖体;质体中含大量淀粉粒,接着出现高尔基体等。早期胚性细胞与周围细胞还存在胞间连丝,随着胚性细胞壁的加厚,胞间连丝也随之消失。（2）ATP酶时空分布动态：早期的胚性细胞中ATP酶反应产物主要沉积于质和液泡上,后期ATP酶活性转入细胞内,液泡和细胞核中,而且在胚性细胞壁加厚处有活跃的A5P酶活性反应,并证明ATP酶活性是在胚性细胞发生过程中形成的。（3）内源激素的变化与作用：在体细胞胚诱导过程中内源激素起着关键性作用,内源生长素含量的提高为胚性细胞的诱导奠定了基础,细胞分裂素含量的增加可促进胚性细胞的分裂和增殖,ABA不仅提高了体细胞胚的诱导频率,而且促进了体细胞胚的正常发育。     

植物体细胞胚发生中ATP酶活性时空分布动态与内源激素的变化

本文以我们的研究结果为基础,并结合国内外近几年有关研究报道,对植物体细胞胚发生中的超微结构和ATP酶活性时空分布动态及内源激素的变化和作用进行专题评述。⑴ 超微结构的变化：当植物体细胞一旦转化为胚性细胞后,各种细胞器相继增加,不仅丰富而且活跃,特别是线粒体内嵴发达,有的正处于分裂状态;核糖体聚集成多聚核糖体;质体中含大量淀粉粒,接着出现高尔基体等。早期胚性细胞与周围细胞还存在胞间连丝,随着胚性细胞壁的加厚,胞间连丝也随之消失。⑵ ATP酶时空分布动态：早期的胚性细胞中ATP酶反应产物主要沉积于质膜和液泡膜上,后期ATP酶活性转入细胞内,液泡和细胞核中,而且在胚性细胞壁加厚处有活跃的ATP酶活性反应,并证明ATP酶活性是在胚性细胞发生过程中形成的。⑶ 内源激素的变化与作用：在体细胞胚诱导过程中内源激素起着关键性作用,内源生长素含量的提高为胚性细胞的诱导奠定了基础,细胞分裂素含量的增加可促进胚性细胞的分裂和增殖,ABA不仅提高了体细胞胚的诱导频率,而且促进了体细胞胚的正常发育。     

Single Circulating-Tumor-Cell-Targeted Sequencing to Identify Somatic Variants in Liquid Biopsies in Non-Small-Cell Lung Cancer Patients

Non-small-cell lung cancer (NSCLC) accounts for most cancer-related deaths worldwide. Liquid biopsy by a blood draw to detect circulating tumor cells (CTCs) is a tool for molecular profiling of cancer using single-cell and next-generation sequencing (NGS) technologies. The aim of the study was to identify somatic variants in single CTCs isolated from NSCLC patients by targeted NGS. Thirty-one subjects (20 NSCLC patients, 11 smokers without cancer) were enrolled for blood draws (7.5 mL). CTCs were identified by immunofluorescence, individually retrieved, and DNA-extracted. Targeted NGS was performed to detect somatic variants (single-nucleotide variants (SNVs) and insertions/deletions (Indels)) across 65 oncogenes and tumor suppressor genes. Cancer-associated variants were classified using OncoKB database. NSCLC patients had significantly higher CTC counts than control smokers (p = 0.0132; Mann–Whitney test). Analyzing 23 CTCs and 13 white blood cells across seven patients revealed a total of 644 somatic variants that occurred in all CTCs within the same subject, ranging from 1 to 137 per patient. The highest number of variants detected in ≥1 CTC within a patient was 441. A total of 18/65 (27.7%) genes were highly mutated. Mutations with oncogenic impact were identified in functional domains of seven oncogenes/tumor suppressor genes (NF1, PTCH1, TP53, SMARCB1, SMAD4, KRAS, and ERBB2). Single CTC-targeted NGS detects heterogeneous and shared mutational signatures within and between NSCLC patients. CTC single-cell genomics have potential for integration in NSCLC precision oncology.     

Somatic Embryogenesis and Plant Regeneration in Pigeonpea

Somatic embryogenesis in pigeonpea [Cajanus cajan (L.) Millsp.] has been achieved using cotyledon segments of mature seeds as explants. A large number of globular somatic embryos were induced directly from cotyledons of genotypes T-15-15, GAUT-82-90 and GAUT-82-99 when cultured on EC6 basal medium supplemented with 2.22, 4.44, 13.32 or 22.2 M N⁶-benzylaminopurine (BAP) and 0.45, 1.36, 2.27, 4.54 and 13.62 M thidiazuron. Somatic embryos developed into cotyledonary stage when the globular embryos were transferred to Murashige and Skoog's (MS) basal medium containing 2.89 – 14.43 M gibberellic acid. Maturation of somatic embryos was achieved on half strength MS medium with 0.38 M abscisic acid. The mature somatic embryos were germinated on MS medium supplemented with 0.44 M BAP and the plantlets were hardened and transferred to soil.     

Identification and Characterization of MicroRNAs in Pearl Oyster Pinctada martensii by Solexa Deep Sequencing

MicroRNAs (miRNAs) are short-nucleotide RNA molecules that function as negative regulators of gene expression in various organisms. However, miRNAs of Pinctada martensii have not been reported yet. P. martensii is one of the main species cultured for marine pearl production in China and Japan. In order to obtain the repertoire of miRNAs in P. martensii, we constructed and sequenced small RNA libraries prepared from P. martensii by Solexa deep sequencing technology and got a total of 27,479,838 reads representing 3,176,630 distinct sequences. After removing tRNAs, rRNAs, snRNAs, and snoRNAs, 10,596,306 miRNA reads representing 18,050 distinct miRNA reads were obtained. Based on sequence similarity and hairpin structure prediction, 258 P. martensii miRNAs (pm-miRNA) were identified. Among these pm-miRNAs, 205 were conserved across the species, whereas 53 were specific for P. martensii. The 3′ end sequence of U6 snRNA was obtained from P. martensii by 3′ rapid amplification of cDNA end PCR reaction and sequence-directed cloning. Eight conserved pm-miRNAs and two novel pm-miRNAs were validated by stem-loop quantitative real-time PCR with U6 snRNA as an internal reference gene. pm-miRNAs and the reported biomineralization-related genes were subjected to target analysis by using target prediction tools. Some of the pm-miRNAs, such as miR-2305 and miR-0046, were predicted to participate in biomineralization by regulating the biomineralization-related genes. Thus, this study demonstrated a large-scale characterization of pm-miRNAs and their potential function in biomineralization, providing a foundation to understand shell formation.     

Identification of MicroRNAs and Transcript Targets in Camelina sativa by Deep Sequencing and Computational Methods

Camelina sativa is an annual oilseed crop that is under intensive development for renewable resources of biofuels and industrial oils. MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play key roles in diverse plant biological processes. Here, we conducted deep sequencing on small RNA libraries prepared from camelina leaves, flower buds and two stages of developing seeds corresponding to initial and peak storage products accumulation. Computational analyses identified 207 known miRNAs belonging to 63 families, as well as 5 novel miRNAs. These miRNAs, especially members of the miRNA families, varied greatly in different tissues and developmental stages. The predicted miRNA target genes are involved in a broad range of physiological functions including lipid metabolism. This report is the first step toward elucidating roles of miRNAs in C. sativa and will provide additional tools to improve this oilseed crop for biofuels and biomaterials.