野生大豆盐碱胁迫响应基因GsZFP1的克隆及序列分析

摘要

盐碱胁迫是影响植物生长、发育和产量的重要因素,野生大豆是优良的非生物胁迫抗性材料.为了获得在植物渗透胁迫反应中起关键作用的功能基因,研究以耐盐碱东北野生大豆(Glycine soja L.G07256)为试材,利用前期构建的野生大豆碱胁迫基因芯片表达谱,从中选出一个在盐碱胁迫早期上调表达,经Blast分析预测属于C2H2类型的锌指转录因子(探针号Gma.17534.1.S1 at),命名为GsZFP1.对GsZFP1基因进行芯片结果的sqRT-PCR验证,并通过同源克隆的方法克隆得到GsZFP1的cDNA序列.利用“ORF Finder”及ExPASy的ProtParam工具分析表明GsZFP1蛋白长度为325 aa,分子质量约35.14 ku,预测等电点为9.99.其锌指结构特征为Cys-X2-Cys-X3-Phe-X5-Phe-X2-His-X3-4-His,并且不含QALGGH保守结构域.该基因是野生大豆中首次被发现的不含QALGGH motif C2H2类型的锌指蛋白,并且参与到非生物胁迫反应.该结果将为研究GsZFP1基因在非生物胁迫中的功能,并为认识不含QALGGH保守结构域的C2H2类型的锌指蛋白在非生物胁迫中的作用奠定基础,为耐渗透胁迫基因工程研究提供潜在的基因资源,为植物耐渗透胁迫机理研究提供依据.%Salt and alkali stress seriously affects plant growth and development as well as reduces rnproductivity of crops. Wild soybean (Glydne soja) is an ideal material for isolation of abiotic stress related genes because of its stress-tolerance and adaptability. In order to gain some key functional genes in plant's response to osmotic stress, a global transcriptome profile of wild soybean {Glycine soja) roots under NaHCO3 treatment was constructed, other functionally relevant genes involved in the same process were identified using Affymetrix? Soybean Genome Array in our previous study. A zinc finger gene, namely GsZFPI was found. Its probe set is Gma.17534.1.S1_at. This gene was significantly induced by NaHCO3. Microarray expression level of GsZFPI gene was validated by semi-quantitative PCR (sqRT-PCR). The cDNA of GsZFPI was cloned from Glycine soja based on the method of homologous cloning. GsZFPI encodes a 35.14 ku protein which contains 325 aa and the predicted isoelectric point was 9.99. GsZFPI contains one C2H2-type zinc finger motif and the architectural feature is Cys-X2-Cys-X3-Phe-X5-Phe-X2-His-X3-4-His. The QALGGH domain, conserved in most plant C2H2-type ZFPs, is absent in the GsZFPI protein sequence. GsZFR1 was first discovered gene which did not contain the QALGGH motif in the ZFP domain. This study will lay a strong foundation for the functional analysis of GsZFPI and understanding the role of C2H2-type ZFP which did not contain the QALGGH motif in abiotic stress. The further functional analysis would facilitate to elucidate the mechanisms of osmotic stress tolerance. This gene could become a new gene source for genetic engineering of stress tolerance and provide the theoretical basis for elucidating plants osmotic stress tolerance mechanism.