青稞HbSnRK2.4的克隆及其序列特征与表达特性分析

Cloning and Characterization of HbSnRK2.4 in Tibetan Hulless Barley(Hordeum vulgare L. var. nudum HK.f.)

干旱胁迫是制约农作物生产的重要限制因素之一,研究并增强作物的抗旱性具有重要意义。Sn RK2(Sucrose nonfermenting 1-related protein kinase 2)基因编码一类蔗糖非酵解型蛋白激酶,该酶在ABA信号转录途径和抗渗透胁迫中起着重要作用。以青稞(Hordeum vulgare subsp.vulgare)抗旱品种喜马拉雅10号为材料,利用RT-PCR技术克隆获得了Sn RK2基因全长c DNA序列,命名为Hb Sn RK2.4(登录号:KJ699389)。生物信息学分析表明,该基因全长1 310 bp,编码362个氨基酸序列,蛋白分子量为41.94 k D,等电点(p I)为5.96。Prosite Scan分析结果表明,Hb Sn RK2.4含有多个干旱胁迫响应蛋白的作用位点,如酪蛋白激酶Ⅱ磷酸化位点、酪氨酸激酶磷酸化位点、蛋白激酶C磷酸化位点及N-豆蔻酰化位点等。利用实时定量PCR方法研究了Hb Sn RK2.4在干旱胁迫条件下及复水后不同时间点的表达情况,发现Hb Sn RK2.4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8 h时恢复正常表达水平。

Drought stress has become one of the important factors that hamper the production of agriculture. Varieties with enhanced drought resistance can be an effective way to solve this problem. SnRK2 gene encoded a kind of Sucrose non-fermenting 1-related protein kinase,which have been thought to play an important role in the ABA signaling pathways and resistance to osmotic stress. However,the exact mechanism underlining is still to be elusive. We presented the isolation of a new SnRK2 gene from Hordeum vulgare subsp. vulgare, nominated as HbSnRK2.4（Accession No. Kj699389）by RT-PCR. The plants were proved to be highly drought tolerance. The gene was 1 310 base pair in length,encoding a peptide of 362 amino acids,about 41.94 kD in molecular weight and with pI=5.96. Results from Prosite Scan indicated that HbSnRK2.4 contains multiple loci of drought stress response cis-element such as casein Kinase II phosphorylation sites,tyrosine kinase phosphorylation sites,protein kinase c-phosphorylation and n-cardamom acylation locus. Expression patterns of HbSnRK2.4 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%）,HbSnRK2.4 restored to a higher level. When plants were drought stressed（＆lt;15.5%of absolute moisture）,the gene were repressed again. After recovery from draught stress,the plants possessed a normal expression level of HbSnRK2.4.