不同桑树品种响应干旱胁迫的比较转录组学分析

Comparative transcriptome analysis of drought stress responses in mulberries of differing drought resistances

【目的】发掘响应干旱胁迫的关键抗旱基因,从转录水平上揭示桑树的抗旱分子机制,为后续开展桑树分子抗旱性育种工作提供科学依据。【方法】以干旱敏感性品种德果1号和耐旱性品种湖桑32号为研究对象,通过盆栽种植方式开展干旱胁迫及复水处理试验,采集24个桑叶样本提取总RNA后构建cDNA文库,在Illumina HiSeq^(TM) 4000测序平台上进行高通量测序,并结合生物信息学对相关基因进行注释分析。【结果】经转录组测序,各样本的Clean reads范围为45723096~67280168条,有效碱基数(Clean bases)集中在6.86~10.09 Gb,GC含量在44.84%~46.48%(平均为45.61%),Q30在90.36%~93.07%。差异表达基因(DEGs)筛选结果显示,6个差异分组(A2 vs A1,B2 vs B1,A3 vs A1,B3 vs B1,A4 vs A1,B4 vs B1)分别筛选出3510、3399、5677、5507、5124和2734个差异表达基因;经干旱胁迫处理后,桑叶功能组基因中呈下调表达的差异表达基因明显多于呈上调表达的差异表达基因,说明桑树在生长过程中存在不同的功能基因以控制桑叶生长发育。不同抗旱性桑叶转录组测序数据中以涉及生物学过程的差异表达基因最多,且主要集中在小分子代谢过程(Small molecule metabolic process)、跨膜运输(Transmembrane transport)及碳水化合物代谢过程(Carbohydrate metabolic process)等方面;与分子功能相关的差异表达基因次之,主要涉及转移酶活性(Transferase activity)和水解酶(Hydrolase activity)等。干旱胁迫下不同抗旱性桑叶差异表达基因主要富集到59条KEGG信号通路上,可划分为代谢、遗传信息处理、环境信息处理、细胞过程和生物系统五大信号通路;其中,抗旱性桑树品种通过提高能量代谢、碳水化合物代谢、增强光合作用及脂质代谢来更好地适应干旱胁迫。综合GO功能注释分析和KEGG信号通路富集分析,得到以下可能与干旱相关的基因:LOC21410404、LOC21404884、LOC21409623、LOC21401352、LOC21398977、LOC21388561、LOC21401447、LOC21398764、LOC21385254、LOC21384661、LOC-21410404及LOC21408971。【结论】不同桑树品种的耐旱性存在显著差异,其中抗旱性桑树品种是通过提高能量代谢、碳水化合物代谢、脂质代谢及光合作用共同应对干旱胁迫。

【Objective】To identify drought-resistance genes operating in mulberry under drought stress and reveal the molecular mechanism of drought resistance in mulberry at the transcriptional level,so as to provide a theoretical basis to support the breeding for and the selection of drought-resistance in mulberry.【Method】The drought-sensitive mulberry variety of Deguo 1 and drought-tolerant mulberry variety Husang 32 were used as the research materials.The drought stress and the control group of rehydration treatment were carried out through pot cultivation.Total RNA was extracted from 24 mulberry leaf samples,cDNA libraries was constructed,and then subject to high-throughput sequencing on the Illumina HiSeq^(TM) 4000 platform.Drought-related genes were annotated and analyzed in combination with bioinformatics.【Result】The results showed that clean reads ranged from 45723096 to 67280168,with clean bases concentrated in 6.86 Gb to 10.09 Gb,with a GC content of 44.84%-46.48%(average 45.61%)and Q30 scores of 90.36%-93.07%.The number of differentially expressed genes(DEGs)between samples(A2 vs A1,B2 vs B1,A3 vs A1,B3 vs B1,A4 vs A1,B4 vs B1)were:3510,3399,5677,5507,5124 and 2734,respectively.There were a smaller number of up-regulated genes than down-regulated genes after drought stress treatment of mulberry.DEGs between the mulberries of differing drought resistance were most enriched in biological processes,consisting mainly of small molecule metabolic process,transmembrane transport and carbohydrate metabolic process.DEGs involved in molecular function were the second most enriched,consisting mainly of transferase activity and hydrolase activity.DEGs were enriched in 59 KEGG signaling pathways,which can be divided into five categories:metabolism,genetic information processing,environmental information processing,cellular processes and biological systems.Drought-resistant mulberry varieties could better adapt to drought stress by their increased expression of genes involved in energy metabolism,carbohydrate metabolism,photosynthesis and lipid metabolism.GO functional annotation and KEGG signal pathway enrichment analyses were combined to obtain the droughtresistance genes:LOC21410404,LOC21404884,LOC21409623,LOC21401352,LOC21398977,LOC21388561,LOC21401447,LOC21398764,LOC21385254,LOC21384661,LOC21410404 and LOC21408971.【Conclusion】The higher drought tolerance of the mulberry variety Husang 32 relative to the variety Deguo 1 involved significant differential expression of genes under drought stress related with enhancements in energy metabolism,carbohydrate metabolism,photosynthesis and lipid metabolism.