中国农业科学 ›› 2019, Vol. 52 ›› Issue (22): 3987-4001.doi: 10.3864/j.issn.0578-1752.2019.22.005

• 分子遗传 • 上一篇    下一篇

高粱苗期耐盐性转录组分析和基因挖掘

董明1,再吐尼古丽·库尔班2,吕芃1,杜瑞恒1,叶凯2,侯升林1(),刘国庆1()   

  1. 1 河北省农林科学院谷子研究所/河北省杂粮重点实验室,石家庄050035
    2 新疆农业科学院生物质能源研究所,乌鲁木齐 830091
  • 收稿日期:2019-06-17 接受日期:2019-09-24 出版日期:2019-11-16 发布日期:2019-11-16
  • 通讯作者: 侯升林,刘国庆
  • 作者简介:董明,E-mail:dddongming@126.com|再吐尼古丽·库尔班,E-mail:zaytungul@sohu.com
  • 基金资助:
    国家自然科学基金(31660435);国家现代农业产业技术体系(CARS-06-13.5-A10)

Transcriptome Analysis and Gene Mining of Salt Tolerance in Sorghum Seedlings (Sorghum bicolor L. Moench)

DONG Ming1,KUERBAN Zaituniguli2,Lü Peng1,DU RuiHeng1,YE Kai2,HOU ShengLin1(),LIU GuoQing1()   

  1. 1 Institute of Millet Crops, Hebei Academy of Agriculture & Forestry Sciences/The Key Minor Cereal Crops Laboratory of Hebei Province, Shijiazhuang 050035;
    2 Institute of Bioenergy, Xinjiang Academy of Agriculture Sciences, Urumqi 830091
  • Received:2019-06-17 Accepted:2019-09-24 Online:2019-11-16 Published:2019-11-16
  • Contact: ShengLin HOU,GuoQing LIU

摘要:

【目的】探究高粱耐盐胁迫响应机制,挖掘高粱耐盐胁迫基因,为高粱耐盐育种提供理论基础。【方法】 以高粱感盐品种L甜和耐盐品种石红137为供试材料,采用水培试验。待高粱植株长至三叶一心期,使用2%NaCl溶液对幼苗进行盐胁迫,分别设置0(对照)、1和24 h处理,每个处理3次重复。测定不同处理样品株高、根长、干物重、Na +含量和叶绿素相对含量(SPAD值),并依托Illumina HiSeq 2000平台进行转录组测序分析。利用FPKM方法计算基因表达量,在差异表达基因检测过程中,将差异表达倍数(fold change)≥2且FDR<0.001作为筛选标准。通过Gene Ontology和KEGG Pathway数据库对参与高粱不同时间盐胁迫差异表达基因进行分析注释。 【结果】 盐胁迫处理对高粱株高、根长、干物重等性状无显著影响,对钠离子含量和SPAD值影响显著。石红137株高、根长、钠离子含量和SPAD值均高于L甜。转录组测序结果鉴定得到已知基因26 628个,新基因866个。石红137中的差异基因数目高于L甜。石红137中,0 h VS 1 h、0 h VS 24 h、1 h VS 24 h三组的差异基因数目分别为375、4 206和3 750个。感盐品种L甜中,0 h VS 1 h、0 h VS 24 h、1 h VS 24 h三组的差异基因数目分别为167、2 534和1 612个。GO分析共获得25个功能注释,分别为光合作用、细胞物质代谢、翻译过程以及激素合成等与盐胁迫相关的差异表达基因。KEGG分析发现盐胁迫1 h表达差异基因富集在植物激素信号转导途径,涉及脱落酸(abscisic acid,ABA)、生长素(auxin,AUX)、细胞分裂素(cytokinin,CTK)、赤霉素(gibberellins,GS)、乙烯(ethylene,ETH)过程等共71个基因。盐胁迫24 h表达差异基因富集于光合作用相关途径,涉及Lhca、Lhcb、磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase,PPC)、磷酸核酮糖激酶(phosphorylribonucleic kinase,PRK)等20个基因。类黄酮生物合成代谢途径差异可能是引起石红137和L甜的耐盐能力差异的原因之一,花青素还原酶(anthocyanidin reductase,ANR)和黄酮醇合成酶(flavonol synthase,FLS)参与类黄酮生物合成途径。【结论】 高粱的盐胁迫过程是一个复杂的生物过程,依赖于多个基因在复杂网络中的平衡表达。盐胁迫条件下,高粱应对环境刺激受到激素信号转导和光合作用的控制。类黄酮生物合成途径在耐盐品种中起到了重要作用。

关键词: 高粱, 盐胁迫, 转录组, 基因挖掘

Abstract:

【Object】The primary aim of this study was to identify salt tolerance genes and explore the tolerance response mechanism under salt stress in sorghum, which may provide a theoretical basis for sorghum salt tolerance breeding. 【Method】Two sorghum varieties, L-Tian, salt-sensitive and Shihong 137, salt-tolerant were employed as plant materials. The sorghum seedlings were treated with 2% NaCl solution at three-leaf and one heart stage. Three treatments including 0 h (CK), 1 h and 24 h were conducted. The plant height, root length, dry matter weight, Na + content and relative content of chlorophyll (SPAD value) were determined, and the transcriptome sequencing was performed on Illumina HiSeq 2000 platform. The FPKM method was employed to calculate the gene expression level. Both the differential expression fold (Fold Change) ≥ 2 and FDR<0.001 were used as screening criteria to detect the differentially expressed genes. The Gene Ontology and KEGG Pathway databases were used to analyze the differentially expressed genes involved in salt stress at different time points. 【Result】Salt stress had no significant effect on plant height, root length and dry matter weight of sorghum, but had significant effect on Na + content and SPAD value. The plant height, root length, Na + content and SPAD value of Shihong 137 were higher than those of L-tian. Totally 26628 known genes and 866 new genes have been identified from RNA-seq, of which, the number of differentially expressed genes from Shihong 137 is higher than that from L-tian. 375, 4206 and 3750 differentially expressed genes in 0 h VS 1 h, 0 h VS 24 h and 1 h VS 24 h groups had been identified from Shihong 137 respectively. The number of differentially expressed genes of 0 h VS 1 h, 0 h VS 24 h, and 1 h VS 24 h of L-tian was 167, 2534 and 1612, respectively. GO and KEGG analysis revealed that plant hormones such as abscisic acid, auxin, cytokinin, gibberellin and ethylene were involved in the salt tolerance of sorghum at an early stage of salt stress (1h), while Lhca, Lhcb, phosphoenolpyruvate carboxylase, and ribulose phosphate kinase were involved in the salt tolerance at a late stage of salt stress (24 h). The difference in salt tolerance between Shihong 137 and L-tian was mainly caused by the flavonoid biosynthetic metabolic pathway. 【Conclusion】The sorghum response to salt stress is a complex biological process that relies on the balanced expression of multiple genes in complex networks. Under salt stress, sorghum response to environmental stimuli was controlled by both hormonal signal transduction and photosynthesis. The flavonoid biosynthesis pathway played an important role in salt-tolerant varieties.

Key words: sorghum (Sorghum bicolor L.), salt stress, transcriptome, gene mining