中国农业科学 ›› 2020, Vol. 53 ›› Issue (5): 874-889.doi: 10.3864/j.issn.0578-1752.2020.05.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米pTAC2影响苗期叶片叶绿素合成的转录组分析

张稳,孟淑君,王琪月,万炯,马拴红,林源,丁冬(),汤继华()   

  1. 河南农业大学农学院/省部共建小麦玉米作物学国家重点实验室,郑州 450002
  • 收稿日期:2019-07-22 接受日期:2019-09-30 出版日期:2020-03-01 发布日期:2020-03-14
  • 通讯作者: 丁冬,汤继华
  • 作者简介:张稳,E-mail:18339922359@163.com。|孟淑君,E-mail:18237116524@163.com
  • 基金资助:
    国家自然科学基金(31871641);国家自然科学基金(U1604113);国家自然科学基金(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
  • Received:2019-07-22 Accepted:2019-09-30 Online:2020-03-01 Published:2020-03-14
  • Contact: Dong DING,JiHua TANG

摘要:

【目的】叶绿素是参与光合途径最为重要的光合色素。叶绿体的发育及叶绿素的合成在很大程度上依赖于质体基因组与核基因组之间的双向信号传导来精确协调基因表达。通过对白化表型的CRISPR/Cas9-ZmpTAC2转基因阳性纯合突变材料进行RNA-seq研究,筛选和鉴定参与叶绿素合成的相关基因,为明确叶绿素的合成途径奠定基础。【方法】以CRISPR/Cas9-ZmpTAC2玉米转基因编辑纯合突变株系为研究材料,使用透射电镜观察叶绿体超微结构和分光光度法测定叶片叶绿素含量,确定叶绿体发育状态及叶绿素合成情况。对转基因阴性材料(CK)和CRISPR/Cas9-ZmpTAC2转基因纯合编辑材料(zmptac2)苗期叶片取样进行转录组测序。通过生物信息学分析,寻找CK与zmptac2间差异表达的基因;qRT-PCR对差异表达基因进行验证。通过酵母双杂交筛选与玉米pTAC2互作的蛋白质。【结果】共获得15株T0转基因植株,包括绿色植株(7株)和白色植株(8株)。绿色幼苗中3株为转基因阴性材料,4株为转基因阳性(2株为未编辑,2株为杂合编辑突变),白色植株(8株)均为转基因阳性纯合编辑。与CK相比,突变体(zmptac2)叶绿体发异常,叶绿素含量显著降低。RNA-seq的结果显示,CK与zmptac2之间共检测到1 367个基因差异表达,其中618个基因上调表达(zmptac2/CK),749个基因下调表达(zmptac2/CK)。GO富集分析显示,下调基因显著富集到叶绿体和质体中。KEGG分析表明下调表达基因显著富集在苯丙氨酸代谢、酪氨酸代谢和异喹啉生物碱生物合成等途径。选取的15个差异基因表达模式均与测序数据相一致,表明测序结果是可靠的。与CK相比,zmptac2中依赖PEP(plastid-encoded RNA polymerase)转录的基因表达量显著降低,而依赖NEP(nuclear gene-encoded RNA polymerase)转录的基因表达量则显著上升。通过对玉米cDNA文库筛选和互作验证,鉴定出ZmpTAC3与ZmpTAC2存在互作。【结论】ZmpTAC2突变会导致叶绿体早期生物合成受阻,该基因参与叶绿体发育及叶绿素合成,且该种作用是由ZmpTAC2调控PEP相关基因表达而实现的。

关键词: 玉米, 叶绿素, ZmpTAC2, RNA-seq, 基因表达调控

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