ZmpTAC2,RNA-seq,基因表達調控," /> 福彩3d玩法介绍及中奖规则

中國農業科學 ?? 2020, Vol. 53 ?? Issue (5): 874-889.doi: 10.3864/j.issn.0578-1752.2020.05.002

? 作物遺傳育種·種質資源·分子遺傳學 ? 上一篇    下一篇

玉米pTAC2影響苗期葉片葉綠素合成的轉錄組分析

張穩,孟淑君,王琪月,萬炯,馬拴紅,林源,丁冬(),湯繼華()   

  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
  • 收稿日期:2019-07-22 接受日期:2019-09-30 出版日期:2020-03-01 發布日期:2020-03-14
  • 通訊作者: 丁冬,湯繼華 E-mail:[email protected];[email protected]
  • 作者簡介:張穩,E-mail:[email protected]。|孟淑君,E-mail:[email protected]
  • 基金資助:
    國家自然科學基金(31871641);國家自然科學基金(U1604113);國家自然科學基金(31801379)

Transcriptome Analysis of Maize pTAC2 Effects on Chlorophyll Synthesis in Seedling Leaves

ZHANG Wen,MENG ShuJun,WANG QiYue,WAN Jiong,MA ShuanHong,LIN Yuan,DING Dong(),TANG JiHua()   

  1. College of Agronomy, Henan Agricultural University/National Key Laboratory of Wheat and Maize Crop Science, Zhengzhou 450002

摘要:

【目的】葉綠素是參與光合途徑最為重要的光合色素。葉綠體的發育及葉綠素的合成在很大程度上依賴于質體基因組與核基因組之間的雙向信號傳導來精確協調基因表達。通過對白化表型的CRISPR/Cas9-ZmpTAC2轉基因陽性純合突變材料進行RNA-seq研究,篩選和鑒定參與葉綠素合成的相關基因,為明確葉綠素的合成途徑奠定基礎。【方法】以CRISPR/Cas9-ZmpTAC2玉米轉基因編輯純合突變株系為研究材料,使用透射電鏡觀察葉綠體超微結構和分光光度法測定葉片葉綠素含量,確定葉綠體發育狀態及葉綠素合成情況。對轉基因陰性材料(CK)和CRISPR/Cas9-ZmpTAC2轉基因純合編輯材料(zmptac2)苗期葉片取樣進行轉錄組測序。通過生物信息學分析,尋找CK與zmptac2間差異表達的基因;qRT-PCR對差異表達基因進行驗證。通過酵母雙雜交篩選與玉米pTAC2互作的蛋白質。【結果】共獲得15株T0轉基因植株,包括綠色植株(7株)和白色植株(8株)。綠色幼苗中3株為轉基因陰性材料,4株為轉基因陽性(2株為未編輯,2株為雜合編輯突變),白色植株(8株)均為轉基因陽性純合編輯。與CK相比,突變體(zmptac2)葉綠體發異常,葉綠素含量顯著降低。RNA-seq的結果顯示,CK與zmptac2之間共檢測到1 367個基因差異表達,其中618個基因上調表達(zmptac2/CK),749個基因下調表達(zmptac2/CK)。GO富集分析顯示,下調基因顯著富集到葉綠體和質體中。KEGG分析表明下調表達基因顯著富集在苯丙氨酸代謝、酪氨酸代謝和異喹啉生物堿生物合成等途徑。選取的15個差異基因表達模式均與測序數據相一致,表明測序結果是可靠的。與CK相比,zmptac2中依賴PEP(plastid-encoded RNA polymerase)轉錄的基因表達量顯著降低,而依賴NEP(nuclear gene-encoded RNA polymerase)轉錄的基因表達量則顯著上升。通過對玉米cDNA文庫篩選和互作驗證,鑒定出ZmpTAC3與ZmpTAC2存在互作。【結論】ZmpTAC2突變會導致葉綠體早期生物合成受阻,該基因參與葉綠體發育及葉綠素合成,且該種作用是由ZmpTAC2調控PEP相關基因表達而實現的。

關鍵詞: 玉米, 葉綠素, ZmpTAC2, RNA-seq, 基因表達調控

Abstract:

【Objective】Chlorophyll is the most important photosynthetic pigment involved in plant photosynthesis. The development of chloroplasts and the synthesis of chlorophyll depend on the bi-directional signaling between the plastid and the nuclear genome. Plastid transcriptionally active chromosome proteins (pTACs) are essential for maintaining the transcriptional activity of PEP (plastid-encoded RNA polymerase) genes, whereas the function of pTACs in maize is still poorly understood. 【Method】The CRISPR/Cas9-ZmpTAC2 maize transgenic editing homozygous mutants were supplied. Transmission electron microscope were used to observe the ultrastructure of the chloroplast, with spectrophotometer to measure the chlorophyll content in maize leaves, respectively. Transcriptome sequencing of the negative material (CK) and CRISPR/Cas9-ZmpTAC2 transgenic homozygous (zmptac2) seedling leaves was performed. Bioinformatics toolbox was performed to identify the differently expressed genes between CK and zmptac2 leaves. Relative quantification of expression of selected differently expressed genes were verified using qRT-PCR. The interacting proteins of ZmpTAC2 were screened by yeast two-hybrid system. 【Result】 A total of 15 T0 transgenic seedlings were obtained, including 7 with green leaves and 8 with white ones. Among the seven green-leaf seedlings, 3 were transgenic negative together with 4 transgenic positive including 2 seedlings were edited. On the other hand, all 8 white-leaf seedlings were transgenic positive with homozygous editing. Compared with CK, zmptac2 chloroplast was abnormal with significantly reduced chlorophyll content. The results of RNA-seq showed that 1 367 genes were differentially expressed between CK and zmptac2, of which 618 genes were up-regulated (zmptac2/CK) and 749 genes down-regulated (zmptac2/CK). GO enrichment analysis revealed that the down-regulated genes were significantly enriched in chloroplasts and plastids. KEGG analysis indicated that down-regulated genes were abundant in the pathways of phenylalanine, tyrosine, and iso-quinoline alkaloid metabolism. Relative expression values of 15 selected differently expressed genes showed similar expression patterns and are consistent with sequencing data, which indicated that the sequencing results were reliable. The expression of PEP (plastid-encoded RNA polymerase) dependent genes in zmptac2 was significantly decreased, while the expression of NEP (nuclear gene-encoded RNA polymerase) genes increased. ZmpTAC3 was identified to be interacted with ZmpTAC2 by cDNA screening and verified by interaction assay. 【Conclusion】 This study first reported that mutations in the ZmpTAC2 gene cause early chloroplast biosynthesis to be hindered, indicating that this gene is involved in chloroplast development and chlorophyll synthesis, which is achieved by ZmpTAC2 regulated PEP-related gene expression.

Key words: maize (Zea mays L.), chlorophyll content, ZmpTAC2, RNA-seq, gene expression regulation

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