Isolation by suppression-subtractive hybridization of genes preferentially expressed during early and late fiber development stages in cotton
Isolation by suppression-subtractive hybridization of genes preferentially expressed during early and late fiber development stages in cotton
复制标题
DOI：
10.1134/s0026893306050086
复制
发表时间：
2006-09-01
影响因子：
1.2
通讯作者：
Guo, X.
中科院分区：
文献类型：
--
作者：
Liu, D.;Zhang, X.;Guo, X.
棉花作为主要的天然纤维来源，在人类生活中扮演着重要的角色。为了筛选在棉纤维发育早期和后期优先表达的基因，我们在PCR选择消减的基础上构建了两个纤维消减文库，分别以开花后10天(DPA)和20天(DPA)的纤维细胞为测试者。通过差异筛选，两个文库中都有292个克隆在纤维发育过程中优先表达。序列分析表明，在10个DPA纤维文库中发现了31个唯一序列，在20个DPA纤维文库中获得了48个唯一序列。此外，在两个文库中都有13个独特的克隆。许多先前报道的棉纤维相关基因都包含在这两个文库中。Northern杂交进一步证实了差异表达，并确定了所选克隆的mRNA积累模式。结果表明，所有筛选的基因在棉纤维发育过程中都表现出高度的优势表达。在10DPA纤维消减文库中鉴定出4个基因--赤霉素调控蛋白、液泡膜固有蛋白、质膜固有蛋白和陆地棉膜蛋白，它们在纤维发育早期大量表达。另一方面，从20DPA纤维消减文库中筛选出的阿拉伯半乳糖蛋白和纤维糖基水解酶家族19蛋白等基因，在纤维从伸长到纤维沉积的发育过程中高表达，转录水平最高。通过纤维和5个非纤维组织的差减，鉴定出两组基因，它们的纤维特异或纤维偏好表达表明它们参与了控制棉花纤维发育的网络。
As a main natural fiber source, cotton plays an important role in human life. To identify genes preferentially expressed during early and late cotton fiber development, we constructed two fiber subtracted libraries on the basis of PCR-selected subtraction using a pool of nonfiber tissues as the same driver and 10 days postanthesis (DPA) and 20 DPA fiber cells as testers, respectively. Through differential screening, 292 clones in both libraries were identified as being preferentially expressed during fiber development. Sequence analysis showed that 31 unique sequences were found in the library of 10 DPA fibers and 48 unique sequences were obtained in the library of 20 DPA fibers. In addition, there were 13 unique clones in common in both libraries. Many previously reported cotton-fiber-related genes were included in both libraries. Northern hybridization was performed to further confirm the differential expression and to determine the pattern of mRNA accumulation of selected clones. As a result, all selected cDNAs showed highly preferential expression in developing cotton fibers. Four genes-putative gibberellin-regulated protein, putative tonoplast intrinsic protein, putative plasma membrane intrinsic protein, and Gossypium hirsutum putative membrane protein-were identified in the 10 DPA fiber subtracted library, and they were found to be expressed a lot during early fiber development. On the other hand, those genes screened out of the 20 DPA fiber subtracted library, like arabinogalactan protein and fiber glycosyl hydrolase family 19 protein, were found to be highly expressed in fibers with the maximal transcription level during the developmental switch from elongation to cellulose deposition. By subtraction between fibers and five nonfiber tissues, two sets of genes were identified, and their fiber-specific or fiber-preferential expression indicated that they are involved in the network that controls cotton fiber development.