摘要
通过反向Northern筛查小麦旱选10号幼苗水分胁迫诱导表达的cDNA文库,选择一个在水分胁迫1、6和12 h均上调表达的cDNA克隆作为＂种子＂序列,利用电子延伸和RT-PCR方法,获得一个开放阅读框为1 434 bp,编码477个氨基酸的cDNA序列。由该序列推测编码的蛋白含有1个典型的ABC1保守域（123-243氨基酸）和1个AARF域（42~369氨基酸）,但是没有发现ABC1向线粒体转移的信号肽前体序列（PD017350）,因此,将该基因命名为TaABC1L。同源比对结果表明,TaABC1L只与水稻、拟南芥中4个尚未研究功能的基因编码的氨基酸序列高度同源。实时定量RT-PCR结果显示,TaABC1L对渗透、高盐、低温等逆境胁迫和ABA处理均表现出应答反应,但是在不同胁迫条件下表达高峰出现的时间和表达强度上存在差异。其中受NaCl胁迫1h,基因的表达量已达到对照的30倍,之后其表达量迅速回落,但仍高于对照;受ABA诱导时,其表达量略有增加,在12h的最高表达量也仅为对照的2倍。
Abiotic stresses such as drought, salinity and low temperature extremely limit crop productivity. However, abiotic stress tolerance is controlled by complex multigene, and many key genes in the transduction networks are still not investigated. Thus, it is important to discover new genes involved in abiotic stress response and/or tolerance. ABC1 subfamily is a member of the Protein kinase family. The exact functions of ABC1 are not clear; however results in yeast showed that ABC1 could reduce the mis-translation of a cytochrome b and stable bcl complex of the electron transport chain in the mitochondria. The abcl/coq8 null mutants led to defective CoQ biosynthesis and respiratory deficiency in yeast. CoQ analogues have been shown to be capable of regulating the mitochondfial permeability transition pore; therefore it was supposed that ABC1/COQ8 might be involved in the regulation of CoQ biosynthesis. Evidence from E. coli indicated that ABC1 acted as a lipid soluble anti-oxidant and required for the regulation of aerobic and anaerobic metabolisms through the ArcAArcB signal transduction system.
We cloned TaABC1L, an abiotic stress-responsive gene, from common wheat with a combination of reverse Northern blot screening, bioinformatics and RT-PCR strategies. A candidate EST, which was all up-regulated at 1, 6, and 12 h water stress, was selected from the cDNA library constructed with mRNA isolated from the 2-leaf seedling of Hanxuan 10 treated with 1 h water stress, by reverse Northern blotting. The candidate EST was extended by in silico cloning, and the full-length cDNA sequence, containing a 1 434 bp open reading frame, was obtained. The cDNA encodes a polypeptide of 477 amino acids with a calculated molecular mass of 54.55 kD and isoelectric point （pl） of 8.34. Sequence analysis indicated this putative protein includes ABC1 characteristic domain architecture （ 123-243 aa） and AARF domain （42-369 aa）, but ABC1 precursor mitochondrion transit peptide （PD017350） was not observed. Therefore the gene was designated TaABC1L （TaABCl-like）. Multiple alignment results showed that TaABCIL shared a homology of 88%, 81% with ABCIL and ABC transporter-like protein from Oryza sativa, and 62%, 62% with two unknown protein from Arabidopsis thaliana, respectively. The expression pattern of TaABC1L at transcription level was investigated by real-time quantitative RT-PCR, which revealed that TaABCIL was distinctly responsive to hyperosmolality （ - 0.5 MPa, PEG-6000）, high salinity （250 mmol·L^-1 NaC1）, low temperature （ 4℃ ） stresses and abscisic acid （ 50 μmol·L^-1 ABA） treatment. Expressional peaks of TaABC1L responsed to PEG, NaCI and 4℃ stresses appeared at 24, 1, and 48 h, with 13.9-, 30-, and 9.8-fold as high as that of the control, respectively. However, the expressional intensity of the peak with 12 h ABA treatment was only 2-fold as high as that of the control. Our studies strongly suggested that TaABC1L could respond to environmental stresses including salinity, low temperature, osmotic stresses and exogenous ABA treatment.
出处
《作物学报》
CAS
CSCD
北大核心
2007年第6期878-884,共7页
Acta Agronomica Sinica
基金
国家转基因植物研究与产业化开发专项项目(JY03-A-14)
