Van De Loo F. J., Turner S., Somerville C.
Carnegie Institution of Washington, Department of Plant Biology, 290 Panama Street, Stanford, California 94305.
Plant Physiol. 1995 Jul;108(3):1141-1150. doi: 10.1104/pp.108.3.1141.
To expand the availability of genes encoding enzymes and structural proteins associated with storage lipid synthesis and deposition, partial nucleotide sequences, or expressed sequence tags (ESTs), were obtained for 743 cDNA clones derived from developing seeds of castor (Ricinus communis L.). Enrichment for seed-specific cDNA clones was obtained by selecting clones that did not detectably hybridize to first-strand cDNA from leaf mRNA. Similarly, clones that hybridized to storage proteins or other highly abundant mRNA species from developing seeds were selected against. To enrich for endomembrane-associated proteins, some clones were selected for sequencing by immunological screening with antibodies prepared against partially purified endoplasmic reticulum membranes. Comparison of the deduced amino acid sequences of the ESTs with the public data bases resulted in the assignment of putative identities of 49% of the clones selected by differential hybridization and 71% of the clones selected by immunological screening. Open reading frames in 100 of the ESTs exhibited higher homology to 78 different nonplant gene products than to any previously known plant gene product.
为了扩大与储存脂质合成和沉积相关的酶及结构蛋白编码基因的可利用性,从蓖麻(Ricinus communis L.)发育中的种子中获得了743个cDNA克隆的部分核苷酸序列,即表达序列标签(ESTs)。通过选择与来自叶片mRNA的第一链cDNA无明显杂交信号的克隆,实现了种子特异性cDNA克隆的富集。同样,淘汰了与发育中种子的储存蛋白或其他高丰度mRNA种类杂交的克隆。为了富集内膜相关蛋白,通过用针对部分纯化的内质网膜制备的抗体进行免疫筛选,选择了一些克隆进行测序。将ESTs推导的氨基酸序列与公共数据库进行比较,结果显示,通过差异杂交选择的克隆中有49%以及通过免疫筛选选择的克隆中有71%被赋予了推定的身份。100个ESTs中的开放阅读框与78种不同的非植物基因产物的同源性高于任何先前已知的植物基因产物。