利用抑制差减杂交技术分离受水稻抗性调控的褐飞虱基因

为分离受水稻抗性调控的褐飞虱Nilaparvata lugens基因, 以取食感虫水稻台中1号和高抗水稻B5的2叶1芯秧苗24 h的褐飞虱4龄若虫为起始材料, 采用抑制差减杂交技术构建了两个群体间的正反向差减cDNA文库。通过斑点杂交从差减文库中筛选代表受水稻抗性调控的基因的cDNA克隆, 进行测序和功能分析, 挑选具功能的基因进行Northern杂交验证。结果表明, 通过斑点杂交筛选到的98个阳性克隆代表92个互不重复的单基因, 其中25个与动物的已知蛋白基因存在较高的同源性。Northern杂交表明, 这25个基因有11个表达上调, 8个表达下调, 提示它们可能在褐飞虱适应抗性水稻过程中发挥了重要作用。本研究结果为克隆上述新基因的全长cDNA序列及进一步研究其在褐飞虱与水稻互作中的功能奠定了基础。     

Analysis of ESTs from multiple Gossypium hirsutum tissues and identification of SSRs.

In an effort to expand the Gossypium hirsutum L. (cotton) expressed sequence tag (EST) database, ESTs representing a variety of tissues and treatments were sequenced. Assembly of these sequences with ESTs already in the EST database (dbEST, GenBank) identified 9675 cotton sequences not present in GenBank. Statistical analysis of a subset of these ESTs identified genes likely differentially expressed in stems, cotyledons, and drought-stressed tissues. Annotation of the differentially expressed cDNAs tentatively identified genes involved in lignin metabolism, starch biosynthesis and stress response, consistent with pathways likely to be active in the tissues under investigation. Simple sequence repeats (SSRs) were identified among these ESTs, and an inexpensive method was developed to screen genomic DNA for the presence of these SSRs. At least 69 SSRs potentially useful in mapping were identified. Selected amplified SSRs were isolated and sequenced. The sequences corresponded to the EST containing the SSRs, confirming that these SSRs will potentially map the gene represented by the EST. The ESTs containing SSRs were annotated to help identify the genes that may be mapped using these markers.     

人参植物与皂苷生物合成相关的差减cDNA文库构建及基因差异表达分析

应用抑制差减杂交技术,分别以源于4年和1年生人参根组织cDNA群体作为检测子(tester)与驱赶子(driver),成功构建了与人参植物皂苷生物合成相关的差减cDNA文库,并时从中筛选的阳性cDNA克隆进行DNA测序及其序列分析、PCR及Northern印迹杂交鉴定．结果显示,获得的13个克隆为新基因序列．其中6个差减克隆系人参植物根生长发育阶段差异表达基因．目前,6个差异表达新基因的结构与功能仍在进一步研究中．     

Analysis of Medicago truncatula nodule expressed sequence tags

Systematic sequencing of expressed sequence tags (ESTs) can give a global picture of the assembly of genes involved in the development and function of organs. Indeterminate nodules representing different stages of the developmental program are especially suited to the study of organogenesis. With the vector lambdaHybriZAP, a cDNA library was constructed from emerging nodules of Medicago truncatula induced by Sinorhizobium meliloti. The 5' ends of 389 cDNA clones were sequenced, then these ESTs were analyzed both by sequence homology search and by studying their expression in roots and nodules. Two hundred fifty-six ESTs exhibited significant similarities to characterized data base entries and 40 of them represented 26 nodulin genes, while 133 had no similarity to sequences with known function. Only 60 out of the 389 cDNA clones corresponded to previously submitted M. truncatula EST sequences. For 117 cDNAs, reverse Northern (RNA) hybridization with root and nodule RNA probes revealed enhanced expression in the nodule, 48 clones are likely to code for novel nodulins, 33 cDNAs are clones of already known nodulin genes, and 36 clones exhibit similarity to other characterized genes. Thus, systematic analysis of the EST sequences and their expression patterns is a powerful way to identify nodule-specific and nodulation-related genes.     

人参植物皂苷生物合成相关新基因的筛选与鉴定

人参植物根进行的特定发育过程在药用次生物———人参皂苷生物合成和累积中发挥重要作用。为从人参根中分离出人参皂苷生物合成相关基因 ,采用抑制差减杂交技术 ,构建四年和一年生人参根组织mRNA群体间正向差减cDNA文库。对从差减文库中筛选的 4 0个阳性cDNA克隆进行酶切、PCR与逆向Northern斑点杂交鉴定、DNA测序以及核苷酸序列同源性比较。结果表明 ,获得的 6个差减克隆在GenBank/DDBJ/BMBL无对应的同源基因 ,代表新基因序列。与此同时 ,使用Northern印迹杂交验证及半定量RT PCR进一步确认 ,6个转录本为根发育阶段差异性表达基因。因而提示 ,它们可能在人参皂苷生物合成中发挥了重要作用。此外 ,在人参茎、叶与种子中亦能检测到上述基因转录本的表达。目前 ,6个新基因已被命名 ,在GenBank注册并获登录号 ,为克隆上述新基因cDNA全长序列及深入鉴定其在人参皂苷生物合成中的功能提供了重要实验依据。     

Screening and analysis of differentially expressed genes from an alien addition line of wheat Thinopyrum intermedium induced by barley yellow dwarf virus infection.

The alien addition line TAI-27 contains a pair of chromosomes of Thinopyrum intermedium that carry resistance against barley yellow dwarf virus (BYDV). A subtractive library was constructed using the leaves of TAI-27, which were infected by Schizaphis graminum carrying the GAV strain of BYDV, and the control at the three-leaf stage. Nine differentially expressed genes were identified from 100 randomly picked clones and sequenced. Two of the nine clones were highly homologous with known genes. Of the remaining seven cDNA clones, five clones matched with known expressed sequence tag (EST) sequences from wheat and (or) barley whereas the other two clones were unknown. Five of the nine differentially expressed sequences (WTJ9, WTJ11, WTJ15, WTJ19, and WTJ32) were highly homologous (identities >94%) with ESTs from wheat or barley challenged with pathogens. These five sequences and another one (WTJ18) were also highly homologous (identities >86%) with abiotic stress induced ESTs in wheat or barley. Reverse Northern hybridization showed that seven of the nine differentially expressed cDNA sequences hybridized with cDNA of T. intermedium infected by BYDV. Three of these also hybridized with cDNA of line 3B-2 (a parent of TAI-27) infected by BYDV. The alien chromosome in TAI-27 was microdissected. The second round linker adaptor mediated PCR products of the alien chromosomal DNA were labeled with digoxygenin and used as the probe to hybridize with the nine differentially expressed genes. The analysis showed that seven differentially expressed genes were homologous with the alien chromosome of TAI-27. These seven differentially expressed sequences could be used as ESTs of the alien chromosome of TAI-27. This research laid the foundation for screening and cloning of new specific functional genes conferring resistance to BYDV and probably other pathogens.     

日本血吸虫期别差异表达基因文库的构建及分析

为从期别差异表达基因分析入手研究血吸虫的生长发育机制,应用抑制性消减杂交 (suppressed subtractive hybridization , SSH) 技术首次构建了日本血吸虫尾蚴、虫卵和成虫的期别差异表达基因文库 . 经消减效率分析和三种文库克隆的 EST 的期别差异性鉴定,表明所建文库质量较高,为在整个基因组水平分离血吸虫的差异表达基因提供了重要材料 . 由三个文库选择 257 个插入片段大于 500 bp 的克隆测定了 EST 序列 . 同源性分析结果表明 257 个 EST 代表 182 种血吸虫基因,其中有 22 种为血吸虫已知基因,有 128 种为血吸虫已知 EST ,有 32 种为新发现的血吸虫基因 . 对 EST 编码蛋白的功能预测结果显示：尾蚴消减文库的基因多与运动、能量代谢、转录调节及致病性相关;虫卵消减文库的基因可能参与信号转导、细胞粘附、蛋白质和碳水化合物的代谢以及抗氧化反应;成虫消减文库的基因多参与蛋白质的合成、转运及分解代谢,参与虫体的运动等 . 大规模分离、分析血吸虫期别差异表达基因将对从分子水平去解读血吸虫的生长发育机制,筛选高效疫苗候选抗原、药物靶标及诊断制剂有重要意义 .     

Screening and comparison of differentially expressed genes between one MDR-TB strain and the virulent M. tuberculosis H37Rv

To screen and compare the differentially expressed genes between one MDR-TB strain separated from one child patient and the virulent Mycobacterium tuberculosis H37Rv, suppression subtractive hybridization (SSH) technology was used to build a library of cDNAs that were differentially expressed in the MDR and H37Rv. From this cDNA library, genes that were expressed in the MDR-TB but not in the H37Rv were selected for gene sequencing and homology analysis; 113 positive clones were obtained, their cDNA fragments were sequenced, and homology analysis was performed. Four novel sequences were identified. The results provide a partial list of genes differentially expressed in MDR-TB and four novel genes were found. Identification of these genes may contribute to our understanding of MDR-TB development.     

An in situ hybridization screen for the rapid isolation of differentially expressed genes

To rapidly isolate genes specifically expressed during medaka development we generated a cDNA library enriched for genes expressed in the head region of the developing embryo. Clones were spotted on filters automatically and preselected for abundantly expressed genes by hybridizing them with a probe derived from RNA of undifferentiated totipotent cells. Of the nonhybridizing clones 153 were chosen randomly and further analyzed by whole-mount in situ hybridization. There were 67 selected clones differentially expressed in the developing embryos, and 48 of these were expressed in the developing head. Differentially expressed genes were either of novel type or showed homology to known genes containing DNA binding motifs or to putative housekeeping genes. Received: 1 December 1998 / Accepted: 17 May 1999     

Assignment of 118 novel cDNAs of cynomolgus monkey brain to human chromosomes

In order to isolate genes that may not be represented in current human brain cDNA libraries, we have sequenced about 20,000 sequence tags of cDNA clones derived from cerebellum and parietal lobe of cynomolgus monkeys (Macaca fascicularis). We determined the entire cDNA sequence of approximately 700 clones whose 5'-terminal sequences showed no homology to annotated putative genes or expressed sequence tags in current databases of genetic information. From this, 118 clones with sequences encoding novel open reading frames of more than 100 amino acid residues were selected for further analysis. To localize the genes corresponding to these 118 newly identified cDNA clones on human chromosomes, we performed a homology search using the human genome sequence and fluorescent in situ hybridization. In total, 108 of 118 clones were successfully assigned to specific regions of human chromosomes. This result demonstrates that genes expressed in cynomolgus monkey are highly conserved throughout primate evolution, and that virtually all had human homologs. Furthermore, we will be able to discover novel human genes in the human genome using monkey homologs as probes.     

Levels of RNA from a family of putative serine protease genes are reduced in Drosophila melanogaster dunce mutants and are regulated by cyclic AMP.

We have isolated several genes expressed at abnormal levels in the memory mutant, dunce (dnc), of Drosophila melanogaster. These mutants have an elevated cyclic AMP (cAMP) content due to a mutation in the structural gene for cAMP phosphodiesterase, so the isolated genes are potentially ones regulated by cAMP. Here, we describe the characterization of a genomic clone and corresponding cDNA clones which contain sequences that are underexpressed in dnc mutants. Sequence analysis of portions of the genomic clone and representative cDNAs revealed the presence of two uninterrupted and complete open reading frames (SER1 and SER2) and part of a third (SER3). The predicted amino acid sequences of all of these were found to be homologous to the serine protease family of enzymes. The genomic clone was localized to the polytene chromosome region 99C-D, although genome-blotting experiments indicated the existence of several other genes related to the cloned serine protease-like genes. Hybridization experiments with probes representing each of the three sequenced genes showed that only the SER1-related genes were differentially expressed in dnc mutants. The putative serine protease genes were abundantly expressed in the larval gut, suggesting a major function in digestion. Feeding normal flies cAMP, isobutylmethylxanthine, or forskolin resulted in a decreased RNA level of the SER1-related genes. Thus, RNA levels of this serine protease gene family are negatively regulated by cAMP.     

Identification of differentially expressed genes in a porcine in vivo model of adipogenesis using suppression subtractive hybridization

Although they provide valuable information, in vitro models of adipocyte development often require high doses of hormones and growth factors, which may influence gene expression and adipocyte differentiation patterns. To overcome these problems, a novel in vivo model of adipose tissue development was used to characterize genes involved in adipogenesis. The suppression subtractive hybridization technique was used to identify genes showing differential expression between the adipose tissue of a day 90 gestating sow, which is enriched in adipocytes, and day 90 fetal adipose tissue, which is enriched in preadipocytes. A total of 149 expressed sequence tags corresponding to identified genes and tentative consensus sequences emerged. Thirty-seven clones matched expressed sequence tags or genomic DNA sequences and six novel sequences were also identified. Adipogenesis-related genes were identified, many of which have never been reported to be expressed in mammalian adipose tissue, and may play a role in regulation of adipose tissue differentiation. Validation of differentially expressed genes was confirmed for perilipin, monocyte to macrophage differentiation-associated, myocilin, paraoxonase 3, stearoyl-CoA desaturase, angiotensinogen and adiponectin genes using real-time RT-PCR.