中國農業科學 ?? 2019, Vol. 52 ?? Issue (8): 1308-1323.doi: 10.3864/j.issn.0578-1752.2019.08.002

? 作物雄性不育專題 ? 上一篇    下一篇

玉米C型細胞質雄性不育花藥不同發育時期的轉錄組分析

薛亞東1,楊露1,楊慧麗1,李冰1,林亞楠1,張懷勝1,郭戰勇1,湯繼華1,2()   

  1. 1 河南農業大學農學院/省部共建小麥玉米作物學國家重點實驗室,鄭州450002
    2 長江大學主要糧食作物產業化湖北省協同創新中心, 湖北荊州 434025
  • 收稿日期:2018-12-10 接受日期:2019-02-14 出版日期:2019-04-16 發布日期:2019-04-26
  • 通訊作者: 湯繼華 E-mail:[email protected]
  • 作者簡介:薛亞東,E-mail:[email protected]
  • 基金資助:
    國家自然科學基金(31471504)

Comparative Transcriptome Analysis Among the Three Line of Cytoplasmic Male Sterility in Maize

XUE YaDong1,YANG Lu1,YANG HuiLi1,LI Bing1,LIN YaNan1,ZHANG HuaiSheng1,GUO ZhanYong1,TANG JiHua1,2()   

  1. 1 College of Agronomy, Henan Agricultural University/Key Laboratory of Wheat and Maize Crops Science, Zhengzhou 450002
    2 Hubei Collaborative Innovation Center for Grain Industry, Yangtze University, Jingzhou 434025, Hubei
  • Received:2018-12-10 Accepted:2019-02-14 Online:2019-04-16 Published:2019-04-26
  • Contact: JiHua TANG E-mail:[email protected]

摘要:

【目的】通過分析玉米C型胞質雄性不育“三系”材料花藥不同發育時期的轉錄組數據,以期闡明玉米C型胞質的不育和恢復機制,并解析不育基因與恢復基因之間相互作用的調控網絡,為玉米C型細胞質雄性不育在不育化制種中的利用提供理論依據。【方法】以中國玉米生產上的骨干自交系豫自87-1為背景的C型胞質不育系、保持系、恢復系為材料,通過對3種材料減數分裂的前期Ⅰ、中期Ⅰ及末期Ⅱ(四分體)時期的花藥進行轉錄組測序并利用hisat2、ballgown及DESeq2等工具進行生物信息學分析,尋找三系花藥不同時期、相同時期不同材料間以及發育時序中差異表達的基因,預測C型胞質不育機制與育性恢復的調控網絡;同時通過實時定量PCR對測序分析結果進行驗證;通過酶活測定驗證推測的C型胞質不育及恢復假說。【結果】所有材料的轉錄組測序共產生156.59 Gb的序列數據,比對并組裝共得到53 035個基因;在恢復系與不育系、保持系與不育系以及“三系”花藥不同時期之間共篩選出非重復差異基因5 676個,其中發育階段差異基因4 705個,同時期材料間差異基因2 693個,發育時序差異基因135個。GO分子功能分析顯示ATP和DNA結合相關的基因和鋅離子結合基因得到高度富集;細胞組分中膜基本組分、核內及質膜內的基因得到富集;以DNA為模板的轉錄、轉錄調控、氧化還原及初級代謝等生物學過程中的基因得到富集。KEGG分析表明,差異基因主要富集于氧化磷酸化、碳代謝及糖酵解等能量代謝相關的途徑中。不育系相對保持系而言,多個與氧化磷酸化相關的基因下調表達,而恢復系中不但相應基因的表達水平得到恢復,而且同時協調調節了同一能量代謝途徑中的其他基因,定量分析顯示差異基因的表達差異及趨勢與轉錄組測序結果基本一致。ATP酶活結果表明不育系相比保持系,ATP酶活顯著降低,恢復系中由于恢復基因的作用其活性得到大幅恢復。【結論】玉米C型胞質不育基因引起基因表達變化可能發生在減數分裂中期Ⅰ之后,末期Ⅱ之前;玉米C型胞質不育的形成可能是由于不育基因引起的能量虧損所致,而恢復基因則通過能量補償促使育性得以恢復。

關鍵詞: 玉米, C型細胞質雄性不育, 轉錄組, 差異表達基因, 調控網絡

Abstract:

【Objective】It is one of the most efficient ways to utilize cytoplasmic male sterile (CMS) lines in hybrid seed production, which could improve the purity of seeds, reduce the cost in creating hybrid seeds and enhance the competitiveness of Chinese seed companies. The comparative transcriptome analysis of the anthers at different development stages from the CMS line, the maintainer line and the restorer line (the three lines) were performed in order to understand the mechanism of sterility and restoration of CMS-C in maize, and also to elucidate the regulation network between the restorer gene and the sterile gene, which will provide the fundamental basis for the employment of maize CMS in hybrid seed production.【Method】The transcriptome sequencing was carried out on the anthers at the prophaseⅠ, the metaphaseⅠand the tetrad stage from the three lines based on the elite inbred line Yu87-1. Method of comparative analysis was used to deal with all the transcripts by the tools such as hisat2, ballgown and DESeq2, and to predict genes involved in the regulation network between the sterile gene and the restorer gene, between the different development stages and through the development time series. qRT-PCR was used to verify the differentially expressed genes. The activity of ATPase was quantified with by the spectrophotometric method for the verification of the putative hypothesis.【Result】Transcriptome sequencing totally produced 156.59 Gb sequence data. After mapping and assembling, 53035 Unigenes were obtained. A total of 5676 differentially expressed (DE) genes were identified from the pairwise comparisons (except for comparisons between the restorer lines and the maintainer lines) in the anthers at the different stages from the three lines. Of those, 4705 DE genes between the comparisons of the development stages, 2693 DE genes between the comparisons of the different lines and 135 DE genes related to the time series. The GO molecular functional analysis showed that the genes related to ATP binding, DNA binding and zinc ion binding were highly enriched, in cell component analysis, genes located in integral component of membrane, nucleus and plasma membrane were enriched, and in biological process, genes involved in DNA-templated transcription, regulation of transcription, oxidation-reduction process and primary metabolic process were enriched. KEGG pathway analysis indicated that the oxidative phosphorylation pathways, the carbon metabolism pathways and glycolysis pathways were mostly enriched. Compared to the maintainer lines, several genes involving in the oxidative phosphorylation pathways were significantly down-regulated in the sterile lines, while those down-regulated genes were recovered, besides other genes in the same pathways were also coordinately regulated. The expression trend determined by qRT-PCR on the selected DE genes was in accordance with that in the transcriptome data. The enzyme activity results show that the activity of ATPase in the sterile line was greatly reduced compared to the maintainer line, while in the restorer line, the activities the ATPase were restored due to the existence of the restorer gene.【Conclusion】 The onset of the changes in the gene expression caused by the sterile gene in the anthers of CMS-C maize may happen after metaphaseⅠ and before telophase Ⅱ in meiosis before visible phenotype occurred. The energy deficiency model may account for the mechanism of the sterility in maize CMS-C, and the energy requirements were compensated by the restorer gene through direct or indirect manner.

Key words: Zea mays, cytoplasmic male sterility, transcriptome, differentially expressed gene, regulation network

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