小G蛋白Rab在真核细胞内的小泡运转过程中起重要作用。本文通过反向Northern筛选，从小麦抗旱品种旱选10 号水分胁迫诱导表达的cDNA文库中分离到与小G蛋白Rab2基因高度同源的EST片段。利用电子克隆和RT-PCR方法，在小麦中克隆了该基因的全长cDNA，命名为TaRab2（GenBank编号为AY851657）。测序结果表明，TaRab2的cDNA长度为824 bp，包含一个完整的633 bp的ORF，推测编码一个210个氨基酸的蛋白质。氨基酸多重比对分析表明，TaRab2编码的蛋白质与玉米、水稻、拟南芥及Sporobolus stapfianus等植物小G蛋白Rab2的同源性均大于90%。Northern杂交分析结果表明，TaRab2为水分胁迫诱导上调表达的基因，在水分胁迫6 h的表达量最高，随着胁迫时间的推移表达量下降。
Wheat (Triticum aestivum L.) is an important crop in the world. However, drought greatly affects its yield in many areas. Discovering and cloning genes related to drought tolerance are the foundation for improving the drought tolerance in crops with genetic modification, which also plays a crucial role in understanding the genetic mechanism of drought tolerance in crops. The small GTP binding protein Rab plays an important role in vesicular transport in eukaryotic cells. The transportation of newly synthesized proteins from the endoplasmic reticulum to the Golgi complex and to secretory vesicles involves the movement of carrier vesicles. By the reverse Northern screening, some candidate ESTs were isolated from the cDNA libraries which were constructed from the 2-leaf seedlings of Hanxuan 10，a wheat cultivar with strong drought tolerance，treated with 24 h and 48 h water stress. One of them was highly identified with small GTP binding protein Rab2. The EST was extended using CAP3 software in the lab local net. Based on the extended sequence, a pair of primer was designed in the two side regions of its predicted ORF. Using RT-PCR method, an 824 bp cDNA was isolated from Hanxuan 10, which contains a complete 633 bp ORF and encodes a putative protein composed of 210 amino acids. The full length cDNA was named as TaRab2 (Accession No. AY851657). TaRab2 contains conserved structure of small GTPase including four conserved domains for guanine nucleotide binding and an effector domain. Multiple alignment analysis on the putative protein indicated that TaRab2 was more than 90% homologous to the small GTP-binding proteins Rab2 in maize, rice, Arabidopsis and Sporobolus stapfianus. Northern blots showed that TaRab2 was an up-regulated gene induced by water stress in wheat, but the expression levels were different in the different time points. The highest expression level was induced by the 6 h water stress. The longer the stress duration, the lower the expression level. The expression level of TaRab2 returned to original quantity by the 72 h water stress. Therefore, TaRab2 was inferred to possibly play a certain role in drought tolerance of plants.
全 文 ：