Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (20): 3875-3884.doi: 10.3864/j.issn.0578-1752.2022.20.001


Function of FCS-Like Zinc-Finger Protein OsFLZ18 in Regulating Rice Flowering Time

MA YaMei(),ZHANG ShaoHong,ZHAO JunLiang(),LIU Bin()   

  1. Rice Research Institute, Guangdong Academy of Agricultural Sciences/Guangdong Key Laboratory of New Technology in Rice Breeding/Guangdong Rice Engineering Laboratory, Guangzhou 510640
  • Received:2022-05-20 Accepted:2022-06-23 Online:2022-10-16 Published:2022-10-24
  • Contact: JunLiang ZHAO,Bin LIU;;


【Objective】Flowering time is an important agronomic trait which determines the yield and regional adaptability of rice, but the underlining molecular regulatory mechanism need further study. FCS-like Zinc finger proteins (FLZs) are a class of plant specific regulatory proteins which play essential roles in plant growth and stress response, but their functions in regulating flowering time have not been reported. This study aims to investigate the potential function of FLZ proteins in rice flowering time control. The finding will broaden our understanding on the molecular regulatory mechanism of rice flowering time.and provide new theoretical basis and gene resource for rice breeding. 【Method】Based on the target sequences published in RGAP database, OsFLZ18 overexpression vector and CRISPR-Cas9 vector were generated and introduced into Japonica variety Nipponbare by Agrobacterium tumefaciens-mediated genetic transformation assay. Homozygous CRISPR knockout mutants were screened by PCR and sequencing analyses. The quantitative real-time PCR (qRT-PCR) assay was used to examine the spatial-temporal expression and diurnal rhythmic expression of OsFLZ18, as well as the effects of OsFLZ18 on the transcription of several known flowering time-related genes. Yeast two-hybrid assay (Y2H) was used to test the interaction between OsFLZ18 and the flowering time-related regulatory proteins.【Result】OsFLZ18 was ubiquitously expressed in various rice tissues, with the highest expression level in 14 day-old seedling, followed by leaf sheaths and leaf blades at the tillering stage, and stem and young panicles at reproductive stages. The OsFLZ18-CRISPR vector was constructed and transformed into Nipponbare. Two independent homozygous OE lines (OE-2, OE-3) with higher OsFLZ18 expression level and two homozygous mutants (CRISPR-21, CRISPR-25) were selected for further study. Phenotypic observation showed that the OE lines flowered later than the wild-type plants under both natural long-day and short-day conditions in Guangzhou, while the CRISPR lines had no obvious differences in heading date when compared to the wild-type plants. The expression levels of Ehd1, Hd3a and RFT1 were significantly decreased in OE-2 plants compared with those in the wild-type plants under artificial short-day conditions, but no significant difference in the expression level of Hd1 was observed between them. The results of Y2H experiment showed that OsFLZ18 interacted with OsMADS51, a positive regulator of rice flowering time. Furthermore, OsFLZ18 exhibits a diurnal rhythmic expression profile, showing lower expression levels in the daytime and higher expression levels at night with a peak at midnight. 【Conclusion】Overexpression of OsFLZ18 delays rice flowering time.

Key words: rice, flowering time, FLZ, OsMADS51

Table 1

Primers used in this study"

引物名称 Primer name 引物序列 Primer sequence (5′-3′) 用途 Purpose
FLZ18-1-F CGCCTGCTTCCTCTGCAAGCgttttagagctagaaat 基因敲除载体
Gene editing
Target detection primer
Primers used for qRT-PCR

Fig. 1

Spatiotemporal expression pattern of OsFLZ18 by qRT-PCR assay"

Fig. 2

Diagram of the OE vector and molecular characterization of OsFLZ18-CRISPR knockout mutants A: Diagram of pUBQ10:OsFLZ18:GFP vector; B: Mutation sites in OsFLZ18 by the CRISPR-Cas9 gene editing system. The protospacer adjacent motif (PAM) site is depicted as underline. Inserted nucleotides are shown in red, and deleted nucleotides are depicted as dashes"

Fig. 3

Heading dates of OsFLZ18 transgenic rice plants A: Phenotypic comparison of OsFLZ18 transgenic and WT plants under natural SD conditions (GuangZhou, Aug-Nov); B: Flowering times of OsFLZ18 transgenic plants and Nip. **: P<0.01"

Fig. 4

Expression pattern of key regulatory genes involved in flowering by qRT-PCR assay The open and filled bars at the bottom represent light and dark periods, respectively; ZT: Zeitgeber time; Nip: Nippobare; OE-2: OsFLZ18-OE line"

Fig. 5

Verification the interaction between OsFLZ18 and OsMADS51 by yeast two-hybrid assay"

Fig. 6

Diurnal rhythmic expression of OsFLZ18 A: Diurnal rhythmic expression of OsFLZ18 under ASDs; B: Diurnal rhythmic expression of OsFLZ18 under LDs. Data is downloaded from the Rice Expression Profile Database ( D: Daytime; N: Night"

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