授粉后黄瓜果实膨大相关基因的鉴别

利用cDNA—AFLP技术分析了授粉前后黄瓜幼果的基因表达差异。有64条转录本片段（TDF）出现在授粉后6-72h的黄瓜幼果中,其中5条经反向Northern斑点杂交证实主要在授粉后的幼果组织中表达。序列分析确定,有一条TDF属于编码黄瓜扩张蛋白的新基因,命名为Cs．Expansin10（CsExplO）,可能与授粉后黄瓜果实膨大生长相关;另一条与谷胱甘肽还原酶基因高度同源;其余3务在基因库中未找到相似序列。     

Improving quality of expressed sequence tag (EST) databases: recovery of reversed, antisense cDNA sequences

Expressed sequence tag (EST) databases contain a significant number (5-20%) of reversed, antisense, cDNA sequences that can be recognized by the label "reversed clone: similarity on wrong strand" in the annotations to the sequence. Despite this high number of altered sequences, no attempt has been made to explain the alteration in molecular terms, or to evaluate their effect on the quality of the information curated in EST databases. In this paper we try to explain the way these altered sequences are originated, and propose a plausible mechanism: a "double priming" of the first strand oligo-dT primer at both ends of nascent cDNAs. In this way, a symmetrical cDNA intermediate is generated, an intermediate that can be cloned after partial digestion with the restriction enzyme used for the directional cloning. Furthermore, when "secondary" priming takes place inside the cDNA, the chain synthesized is prone to be truncated prematurely, with the subsequent loss of upstream information. One of the most subtle effects of this cloning alteration is the generation of virtual open reading frames (ORFs) in sequences with no homologues available for comparison. Nevertheless, and according to our model and our data, the "double priming mechanism" does not shift the ORF effected, so antisense sequences should be considered as normal ones after a simple transformation in their inverse-complementary forms.     

Analysis of expressed sequence tags of flower buds in Lotus japonicus.

In order to study gene expression in a reproductive organ, we constructed a cDNA library of mature flower buds in Lotus japonicus, and characterized expressed sequence tags (ESTs) of 842 clones randomly selected. The EST sequences were clustered into 718 non-redundant groups. From BLAST and FASTA search analyses of both protein and DNA databases, 58.5% of the EST groups showed significant sequence similarities to known genes. Several genes encoding these EST clones were identified as pollen-specific genes, such as pectin methylesterase, ascorbate oxidase, and polygalacturonase, and as homologous genes involved in pollen-pistil interaction. Comparison of these EST sequences with those derived from the whole plant of L. japonicus, revealed that 64.8% of EST sequences from the flower buds were not found in EST sequences of the whole plant. Taken together, the EST data from flower buds generated in this study is useful in dissecting gene expression in floral organ of L. japonicus.