玉米EIN3/EIL家族基因ZmEIL9调控籽粒发育

doi:10.3864/j.issn.0578-1752.2024.18.002

摘要/Abstract

摘要：

【目的】玉米籽粒大小和粒重是影响其产量的重要因素。EIN3/EIL基因家族是乙烯信号转导途径中的关键转录因子，解析EIN3/EIL家族基因ZmEIL9在玉米籽粒发育中的生物学功能。【方法】利用生物信息学和RT-qPCR分析ZmEIL9在玉米籽粒发育中的表达特征，对ZmEIL9及其同源基因进行多序列比对，利用邻接法构建其系统进化树，分析ZmEIL9编码蛋白序列特征，对目的蛋白进行亚细胞定位。筛选ZmEIL9的玉米Mu转座子插入突变体和CRISPR/Cas9编辑突变体，鉴定突变体和对照的农艺性状表型，分析籽粒灌浆速率，测量淀粉粒、蛋白含量等籽粒储藏物质。【结果】根据玉米中EIN3/EIL家族成员，系统进化树分析显示，ZmEIL9编码蛋白与ZmEIL1、SbEIL1亲缘关系较近。玉米自交系B73籽粒转录组数据库中，ZmEIL9在籽粒发育早期和晚期表达量较高，但在自交系N04籽粒发育中期和晚期表达量较高。在自交系丹232和N04中ZmEIL9编码644个氨基酸，自交系B73中编码642个氨基酸；亚细胞定位表明，ZmEIL9编码蛋白定位于细胞核中。获得不同Mu转座子插入位点的ZmEIL9突变体，以及发生氨基酸移码突变的CRISPR/Cas9编辑突变体，表型分析表明，ZmEIL9的Mu转座子突变体和编辑突变体的株高、籽粒粒长和百粒重均比对照显著降低。对不同发育时期玉米籽粒干重进行分析，显示Zmeil9突变体的籽粒灌浆速率小于野生型。扫描电镜观察显示，Zmeil9突变体比野生型成熟籽粒中的淀粉粒明显变小，并表现不规则形态；进一步分析表明，Zmeil9编辑突变体籽粒中总淀粉含量和醇溶蛋白浓度均显著低于野生型。【结论】ZmEIL9在玉米籽粒发育过程中起重要调控作用。

关键词: 玉米, EIN3/EIL家族, ZmEIL9, 籽粒发育, 生物学功能

Abstract:

【Objective】 Grain size and weight are the important factors affecting the yield of maize. The EIN3/EIL gene family is a sort of key transcription factors in the ethylene signaling transduction pathway, and the functions and regulatory mechanisms of the EIN3/EIL gene ZmEIL9 were analyzed in maize kernel development to elucidate its molecular mechanisms.【Method】 The expression patterns of ZmEIL9 in maize kernel at different developmental stages were analyzed by bioinformatics and RT-qPCR. The multiple sequence alignment of ZmEIL9 and its homologs from different species was performed, and the phylogenetic trees was constructed based on the neighbor-joining method. The sequence characteristics of ZmEIL9 protein were analyzed, and subcellular localization of ZmEIL9 was performed. The insertion mutants of Mu transposon and CRISPR/Cas9 knockout mutants of ZmEIL9 were screened, and the agronomic traits including grain filling rate, storage substances such as starch granule and protein content were analyzed. 【Result】 According to the members of EIN3/EIL family in maize, phylogenetic trees showed that ZmEIL9 was closely related to ZmEIL1 and SbEIL1. In the transcriptomic database of maize inbred line B73, the expression levels of ZmEIL9 were higher in the grain at early and late developmental stages. However, the expression levels were higher in inbred line N04 at the middle and late developmental stages. ZmEIL9 encoded 644 amino acids in the inbred lines Dan232 and N04, while its homolog in inbred line B73 has 642 amino acids. Subcellular localization analysis indicated that ZmEIL9 was localized in nucleus. The ZmEIL9 mutants with different Mu transposon insertion sites and CRISPR/Cas9 knockout mutants with amino acid frameshift mutations were obtained, respectively. The plant height, grain length, and 100-grain weight of Mu mutants and knockout mutants were significantly lower than those of its wild counterpart. The grain dry weights at different developmental stages were also analyzed, and the grain filling rates of Zmeil9 mutant were lower than those of the wild type. The starch granules of Zmeil9 mutant were significantly smaller and had an irregular shape based on scanning electron microscopy (SEM) observations. The contents of total starch and the concentration of zein protein in the Zmeil9 mutant were significantly lower than those in the control. 【Conclusion】ZmEIL9 plays an important regulatory role in the kernel development of maize.

Key words: maize (Zea mays L.), EIN3/EIL family, ZmEIL9, grain development, biology function

朱俊杰, 张鑫悦, 潘梦影, 张静雯, 郑琦, 李玉玲, 董永彬. 玉米EIN3/EIL家族基因ZmEIL9调控籽粒发育[J]. 中国农业科学, 2024, 57(18): 3522-3532.

ZHU JunJie, ZHANG XinYue, PAN MengYing, ZHANG JingWen, ZHENG Qi, LI YuLing, DONG YongBin. An EIN3/EIL Family Gene, ZmEIL9 Regulates Grain Development in Maize[J]. Scientia Agricultura Sinica, 2024, 57(18): 3522-3532.

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