中国农业科学 ›› 2021, Vol. 54 ›› Issue (15): 3149-3157.doi: 10.3864/j.issn.0578-1752.2021.15.001

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻黄绿叶突变体ygl3的鉴定与基因功能分析

许子怡(),程行,沈奇,赵亚男,汤佳玉,刘喜()   

  1. 淮阴师范学院/江苏省环洪泽湖生态农业生物技术重点实验室/江苏省区域现代农业与环境保护协同创新中心,江苏淮安 223300
  • 收稿日期:2020-12-14 接受日期:2021-02-10 出版日期:2021-08-01 发布日期:2021-08-10
  • 通讯作者: 刘喜
  • 作者简介:许子怡,E-mail: 2631140968@qq.com
  • 基金资助:
    江苏省高等学校自然科学研究面上项目(19KJB180009);淮阴师范学院大学生创新创业计划(202019009XJ)

Identification and Gene Functional Analysis of Yellow Green Leaf Mutant ygl3 in Rice

XU ZiYi(),CHENG Xing,SHEN Qi,ZHAO YaNan,TANG JiaYu,LIU Xi()   

  1. Huaiyin Normal University/Jiangsu Key Laboratory for Eco-agriculture Biotechnology Around Hongze Lake/Jiangsu Collaborative Innovation Center of Regional Modern Agriculture and Environment Protection, Huaian 223300, Jiangsu
  • Received:2020-12-14 Accepted:2021-02-10 Online:2021-08-01 Published:2021-08-10
  • Contact: Xi LIU

摘要:

【目的】 为了丰富和加深人们对植物叶色形成的分子机理认识,对水稻黄绿叶突变体ygl3yellow green leaf 3)进行表型鉴定和基因克隆,阐明YGL3的分子功能,为解析YGL3调控水稻叶色形成的分子机理奠定基础。【方法】 从水稻中花11 CRISPR-Cas9敲除突变体库中鉴定出2份稳定遗传的等位黄绿叶突变体,命名为ygl3-1ygl3-2,对突变体的表型进行鉴定,测定野生型和突变体苗期的叶绿素含量,运用透射电镜观察野生型和突变体ygl3的叶绿体结构。利用实时荧光定量PCR分析YGL3的组织表达模式,并使用BioXM 2.6软件对YGL3及其同源蛋白序列进行比对,采用酵母双杂交方法筛选YGL3的互作蛋白。【结果】 在苗期,与野生型相比,突变体ygl3叶片黄化,叶绿素、类胡萝卜素和总光合色素含量显著降低。透射电镜结果表明,突变体ygl3叶绿体形态异常,类囊体片层结构较少,而野生型叶绿体形态正常,类囊体片层结构排列有序。CRISPR-Cas9敲除位点鉴定结果表明,LOC_Os01g73450发生单碱基插入,导致蛋白翻译提前终止,该基因编码351个氨基酸的蛋白突变为55个氨基酸的截短蛋白。与野生型相比,LOC_Os01g73450的表达水平在突变体中显著下调。qRT-PCR结果表明YGL3在水稻根、穗、种子、叶鞘以及叶片中均有表达,且叶片中表达水平最高。YGL3编码一个质体定位的尿嘧啶核苷酸激酶。蛋白氨基酸序列比对表明YGL3蛋白在玉米、高粱、拟南芥等物种中均较为保守,与拟南芥同源蛋白氨基酸的同源性为59.4%。qRT-PCR结果表明,叶绿素合成基因(如HEMCHEMEURO-D)在突变体ygl3中显著下调,而HEMBHEMFHEML等叶绿素合成基因在野生型与突变体之间无显著差异。酵母双杂交系统筛选水稻叶片酵母cDNA文库,发现YGL3与RNA编辑因子MORF8存在互作。【结论】 水稻黄绿叶突变体ygl3的表型是由LOC_Os01g73450突变导致,该基因与已报道的水稻黄绿叶基因YL2/YGL8等位。YGL3在叶片中高度表达,同时YGL3与MORF8在酵母中互作。

关键词: 水稻(Oryza sativa L.), 黄绿叶, YGL3, CRISPR-Cas9, 叶绿体发育

Abstract:

【Objective】 To enrich and deepen people’s understanding of the molecular mechanism of plant leaf color, the phenotype identification and gene cloning of the yellow green leaf mutant ygl3 (yellow green leaf 3) were carried out to clarify the molecular function of YGL3 and lay the foundation for elucidating the molecular mechanism of YGL3 regulating rice leaf color.【Method】 Two stable genetic allelic yellow green leaf mutants, ygl3-1 and ygl3-2, were isolated from the CRISPR-Cas9 knockout mutant library of Zhonghua 11. The phenotype of the mutant was identified, and the chlorophyll contents of the wild-type and ygl3 were determined. The chloroplast structure of the wild-type and ygl3 was observed by transmission electron microscope. qRT-PCR was used to analyze the tissue expression of YGL3, and BioXM2.6 software was used for sequence alignment of YGL3 and its homologs. Yeast two hybrid was used to screen the interacting proteins of YGL3.【Result】 Compared with the wild type, the leaves of ygl3 were yellowing, and the contents of chlorophyll, carotenoid and total photosynthetic pigment at seedling stage in ygl3 were significantly decreased. Transmission electron microscopy showed that the chloroplast morphology of ygl3 was abnormal, and the thylakoid lamellar structure was less, whereas the chloroplast morphology of the wild type was normal and the thylakoid lamellar structure was orderly arranged. CRISPR-Cas9 knock-out site identification showed that the LOC_Os01g73450 gene had a single base insertion, which resulted in the early termination of protein translation. The gene encoding 351 amino acids was mutated into a truncated protein with 55 amino acids. Compared with the wild type, the expression level of LOC_Os01g73450 was significantly down-regulated in the mutants. qRT-PCR showed that YGL3 was expressed in roots, panicles, seeds, leaf sheaths and leaves. YGL3 was highly expressed in leaves. YGL3 encodes a plastid localized UMP kinase. The YGL3 protein was conserved in Zea mays, Sorghum bicolor and Arabidopsis thaliana. YGL3 shared the high sequence homology (59.4% amino acid identity) to Arabidopsis. qRT-PCR showed that chlorophyll synthesis genes, including HEMC, HEMC and URO-D, were significantly down-regulated in ygl3, whereas the expression levels of HEMB, HEMF and HEML were no significant difference between the wild type and ygl3. Yeast two hybrid screen showed that YGL3 interacted with RNA editing factor MORF8.【Conclusion】 The phenotype of the yellow leaf mutant ygl3 resulted from the LOC_Os01g73450 mutation. YGL3 was an allele of the yellow green leave gene YL2/YGL8. YGL3 was highly expressed in leaves, and YGL3 interacted with MORF8 in yeasts.

Key words: rice (Oryza sativa L.), yellow green leaf, YGL3, CRISPR-Cas9, chloroplast development