青稞HbTsi1的克隆及其序列特征与表达特性分析

Cloning and Characterization of HbTsi1 in Tibetan Hulless Barley

为了探究DREB类基因在青稞干旱胁迫下的表达模式,通过RT-PCR技术从抗旱品种‘喜马拉雅10号’中克隆其中的一个基因全长cDNA,并利用Real Time PCR方法研究其在干旱胁迫、复水条件下的表达情况。结果表明:从抗旱材料中克隆一个1 237bp全长cDNA序列,命名为HbTsi1(登录号:KJ699390)。生物信息学分析表明,该序列开放阅读框长为837bp,编码278个氨基酸序列,由HbTsi1的ORF推测所编码的蛋白,预测分子质量为30.33ku,等电点(pI)为6.11。Prosite Scan分析结果表明,该基因含有AP2/ERF domain profile家族特征基序、1个Bipartite nuclear localization signal profile、6个蛋白激酶C磷酸化位点、6个酪蛋白激酶Ⅱ磷酸化位点、5个N-豆蔻酰化位点、2个cAMP和cGMP依赖性的蛋白激酶磷酸化位点、2个氮糖基化位点。TMHMM预测该蛋白不含跨膜转移功能区。Signal IP3.0预测该蛋白没有信号肽,不属于分泌蛋白。PSORT亚细胞定位预测该基因所编码蛋白位于细胞质。推导的氨基酸序列同源比对分析结果表明,HbTsi1蛋白与短芒大麦的DREB蛋白具有较高的相似性。利用实时定量PCR方法研究HbTsi1在干旱胁迫条件下及复水后不同时间点的表达情况,发现HbTsi1在土壤绝对含水量为33.4%时表达量最高,随着土壤绝对含水量的下降而下调表达;当达到作物正常生长所需的土壤绝对含水量时又开始上调表达;进行干旱胁迫后(<15.5%)基因表达量下降;复水后8h时恢复至最高表达水平。说明,HbTsi1基因可能是青稞抗旱节水的关键基因。

In order to explore the expression patterns of DREB genes under drought stress in Tibetan Hulless barley, we cloned a full-length DREB gene cDNA by RT-PCR from the material of the drought barley variety ‘Himalaya 10 ’, and its expression under conditions of rehydration situation was studied through the use of Real Time PCR. The results showed that：a 1 237 bp cDNA sequence was cloned from a drought-resistant material, named HbTsil （GenBank accession No：K J699390）. The sequence of the open reading frame was 837 bp, encoding a peptide of 278 amino acids, about 30.33 ku in molecular weight and with pI=6.11. Results from Prosite Scan indicated thatHbTsil contains mul-tiple loci of drought stress response cis-element such as AP2/ERF domain profile family signature motif site, one Bipartite nuclear localization signal profile site, six protein kinase C phosphorylation sites, six casein kinase Ⅱ phosphorylation sites, five N-myristoylation sites, two cAMP and cGMP dependent protein kinase phosphorylation sites, and two N-glycosylation sites. TMHMM predicted the protein without transmembrane transfer zone, and Signal IP 3.0 showed that the protein was not secreted proteins with no signal peptide. PSORT subcellular localization predicted the HbTsil protein was in the cytoplasm. Amino acid sequence homology to guide comparative analysis of the results showed that, HbTsil protein has a high similarity with Hordeum Breyisubulatum. Expression patterns of HbTsil were also investigated by RT-qPCR at serious waterlogging or drought and various time points after recovery. The highest expression level was observed in plants growing in soil absolute moisture 33. 4%, which may brought waterlogging to Tibet barley. As soil absolute moisture declined, transcripts decreased sharply. But when the soil water content turned to be normal for Hordeum vulgare （15. 5%）, HbTsil restored to a higher level. When plants were drought stressed （〈15.5 % of absolute moisture）, the gene were repressed again. After recovery from drought stress, the plants possessed the highest expression level ofHbTsil , which indicated theHbTsil gene may be the key gene of the Tibetan Hulless barley during the drought and water conservation.