Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (15): 3149-3157.doi: 10.3864/j.issn.0578-1752.2021.15.001


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;


【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

Table 1

Primers used in the study"

标记Marker 正向引物Forward primer sequence (5′-3′) 反向引物Reverse primer sequence (5′-3′)

Fig. 1

Regulation of rice leaf color formation by YGL3 A: Phenotype of the wild-type and ygl3 at seedling stage; B: Determination of photosynthetic pigment content of the wild-type and ygl3 at seedling stage; C: Identification of CRISPR-Cas9 knockout sites; D: Amino acid sequence alignment of wild type and the mutants; E: Expression analysis of LOC_Os01g73450"

Fig. 2

Chloroplast ultrastructure of the wild type and the ygl3 mutant A, B: Ultrastructure of the chloroplast of wild type; C, D: Ultrastructure of the chloroplast of the ygl3 mutant. Bar=5 μm (A, C), Bar=2 μm (B, D)"

Fig. 3

Analysis of tissue expression of YGL3"

Fig. 4

Sequence alignment of multiple amino acids from YGL3 and its homologs NP_188498.1: Arabidopsis; NP_001137013.1: Zea mays; OEL23965.1: Dichanthelium oligosanthes; XP_002456998.1: Sorghum bicolor; XP_015693434.1: Oryza brachyantha; YGL3: Oryza sativa. Amino acids that were fully or partially conserved are shaded blue and pink, respectively"

Fig. 5

Transcript levels of genes associated with chlorophyll biosynthesis in wide type and ygl3 at the seedling stage HEMA: Encoding glutamyl-tRNA synthetase; HEME: Encoding uroporphyrinogen decarboxylase; HEML: Encoding glutamyl-1-semialdehyde transaminase; HEMB: Encoding bilinogen synthase; HEMC: Encoding hydroxymethylated retrobilinogen synthase; HEMF: Encoding fecal porphyrin III oxidase; URO-D: Encoding uroporphyrinogen oxydecarboxylase. **: Significant difference at 0.01 level"

Fig. 6

The interaction between YGL3 and MORF8 in yeasts DDO: The basic nutrient deficient medium SD/-Leu/-Trp; QDO: The selective nutrient deficient medium SD/-Leu/-Trp/-His/-Ade"

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