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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (3): 543-555.doi: 10.3724/SP.J.1006.2024.32023


Functional analysis of OsFLZ13, the gene encoding a small peptide zinc finger protein in rice

ZHANG Li-Jie1,2(), ZHOU Hai-Yu1,2, MUHAMMAD Zeshan1,2, MUNSIF Ali Shad1,2, YANG Ming-Chong1,2, LI Bo1,2, HAN Shi-Jian1, ZHANG Cui-Cui1,3, HU Li-Hua1,3,*(), WANG Ling-Qiang1,2,*()   

  1. 1State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004, Guangxi, China
    2College of Agriculture, Guangxi University, Nanning 530004, Guangxi, China
    3College of Life Science and Technology, Guangxi University, Nanning 530004, Guangxi, China
  • Received:2023-06-20 Accepted:2023-09-13 Online:2024-03-12 Published:2023-10-09
  • Contact: *-mail: hulihua@gxu.edu.cn; E-mail: lqwang@gxu.edu.cn
  • Supported by:
    Regional Science Foundation Project, the National Natural Science Foundation of China(31860296);Key Projects of Guangxi Natural Science Foundation(2020GXNSFDA238027)


FCS-like zinc finger (FLZ) is a protein associated with plant growth and stress. At present, there are few reports on FLZ gene family analysis and functional studies in rice. In this study, TBtools were used to blast rice genome, and a total of 29 OsFLZ genes were identified. Their gene location, gene structure, motif and promoter sequences were analyzed. The relative expression level of FLZ genes in rice from CREP database showed that, OsFLZ13, a member of this family, was predominantly expressed in anthers before flowering. β-D-glucuronidase (GUS) staining assays exhibited that OsFLZ13 began to express at stage 9 and gradually peaked at stage 14 of stamen development before flowering. Furthermore, two independent mutant lines, namely Osflz13-1 and Osflz13-2, were obtained with CRISPR/Cas9 gene editing system. Compared with the 94% seed-setting rate of wild type Zhonghua 11, the setting rates of Osflz13-1 and Osflz13-2 were reduced to 44% and 36%, respectively. This study throws light on the evolution of FLZ in planta and indicates the roles of OsFLZ13 in anthers development and pollen fertility, which will be beneficial further studies of its functions. Additionally, it provides a reference for exploring the function of the FLZ family and highlights its potential value for the utilization in male sterility systems in rice.

Key words: rice, pollen, seed-setting rate, FLZ gene family

Table 1

Primers and sequences in this study"

Primer name
Forward sequence (5'-3')
Reverse sequence (5'-3')

Table 2

Physicochemical properties of proteins encoded by OsFLZ family genes"

Gene LOC
Gene name
染色体Chr. 核酸长度Nucleotide length
Number of amino acids (aa)
Molecular weight
Isoelectric point
系数Instability index
指数Aliphatic index
average of hydropathicity
LOC_Os01g08520 OsFLZ1 1 1167 389 39.42 5.44 59.23 66.83 -0.13
LOC_Os01g41010 OsFLZ2 1 783 261 28.59 4.33 51.77 72.31 -0.31
LOC_Os01g52100 OsFLZ3 1 789 263 28.34 4.68 54.71 79.69 -0.25
LOC_Os02g07820 OsFLZ4 2 657 219 23.02 5.40 63.96 69.13 -0.13
LOC_Os02g37970 OsFLZ5 2 381 127 13.72 8.86 57.81 57.54 -0.39
LOC_Os02g46180 OsFLZ6 2 441 147 16.09 8.71 61.70 42.95 -0.79
LOC_Os02g46190 OsFLZ7 2 378 126 14.29 6.90 81.23 51.60 -0.83
LOC_Os02g46210 OsFLZ8 2 321 107 11.49 5.26 55.94 60.00 -0.36
LOC_Os02g51550 OsFLZ9 2 444 148 15.16 8.98 50.35 57.28 -0.42
LOC_Os03g08520 OsFLZ10 3 492 164 17.87 9.98 92.52 72.02 -0.34
LOC_Os03g46260 OsFLZ11 3 909 303 31.79 6.39 80.69 65.26 -0.25
LOC_Os04g49620 OsFLZ12 4 453 151 15.70 5.92 50.62 63.80 -0.49
LOC_Os04g49650 OsFLZ13 4 360 120 13.63 9.50 56.63 51.76 -0.93
LOC_Os04g49660 OsFLZ14 4 384 128 14.69 6.64 77.52 49.29 -1.02
LOC_Os04g49670 OsFLZ15 4 423 141 15.88 6.27 67.54 82.36 0.05
LOC_Os04g49680 OsFLZ16 4 315 105 11.45 8.56 94.87 50.96 -0.52
LOC_Os05g08800 OsFLZ17 5 984 328 34.01 5.68 67.42 75.35 -0.14
LOC_Os06g03520 OsFLZ18 6 435 145 15.47 6.88 79.40 57.78 -0.31
LOC_Os06g05970 OsFLZ19 6 612 204 21.97 5.94 63.27 73.60 -0.33
LOC_Os06g11980 OsFLZ20 6 429 143 15.03 9.22 66.34 40.07 -0.73
LOC_Os06g14070 OsFLZ21 6 309 103 10.75 4.65 76.24 44.22 -0.53
LOC_Os06g50080 OsFLZ22 6 411 137 14.63 6.83 81.82 54.85 -0.41
LOC_Os07g42390 OsFLZ23 7 594 198 20.59 8.67 59.04 74.62 0.03
LOC_Os08g31510 OsFLZ24 8 600 200 20.86 4.84 50.77 58.69 -0.38
LOC_Os08g34984 OsFLZ25 8 576 192 20.60 11.05 57.41 68.59 -0.47
LOC_Os09g20240 OsFLZ26 9 576 192 20.10 9.02 66.86 42.04 -0.66
LOC_Os09g26370 OsFLZ27 9 525 175 19.61 9.84 66.76 71.26 -0.73
LOC_Os10g28680 OsFLZ28 10 903 301 31.79 5.32 67.17 53.77 -0.60
LOC_Os11g43790 OsFLZ29 11 453 151 15.88 8.88 67.63 65.93 -0.15

Fig. 1

Analysis of conserved motifs, domains, and gene structure of OsFLZ family genes A: OsFLZ proteins conserved motif analysis, differently colored boxes indicate differently conserved motif, black lines indicate protein length. B: OsFLZ proteins domain analysis, differently colored boxes indicate different types of domains. C: OsFLZ family genes structure analysis, green boxes indicate 5' and 3' untranslated regions (UTR); yellow depicts the coding regions (CDS); Black lines indicate introns."

Fig. 2

Cis-regulatory elements analysis of OsFLZ family genes in rice"

Fig. 3

Relative expression pattern of FLZ family genes in rice A: the relative expression pattern of OsFLZ family genes in different tissues and panicle development gradient of rice. SD: seedling stage; BH: before heading stage; HT: heading stage; S2, S3, S4, and S5 refer to the second, third, fourth, and fifth stages of panicle development, respectively; BF: before flowering period; AF: after flowering and pollination. B: the relative expression level of OsFLZ13 in different tissues and stages of panicle development in Zhenshan 97 (ZS97) and Minghui 63 (MH63). Abbreviations are the same as those given in Fig. 3-A. C: heat map of OsFLZ family gene expression in seedlings after gibberellin 3 (GA3), kinetin (KT), naphthaleneacetic acid (NAA) treatments and in plumules and radicles under 48 hours light or 48 hours dark treatments."

Fig. 4

β-glucuronidase (GUS) staining of the tissues and organs across vegetative and reproductive stages from the pCAMBIA1381Z-OsFLZ13 transgenic rice plants A: germinated seeds GUS activity. Bar: 5 mm; B: GUS activity of roots and leaves at the seedling and booting stages. GUS activity of roots, internodes, leaves, and nodes at heading stage. Bar: 1 cm; C: GUS activity of florets at different stages of stamen development. St designates each stage of stamen development while letters a and b after stage number represent early and late phases, respectively. St9, st10, st11, st12, st13, and st14 refer to the 9th, 10th, 11th, 12th, 13th, and 14th stages of stamen development, respectively. Bar: 2 mm; D: GUS activity of pistils with stamen being removed. Abbreviations are the same as those given in Fig. 4-C. Bar: 500 μm; E: anthers GUS activity. Abbreviations are the same as those given in Fig. 4-C. Bar: 100 μm; F: pollens GUS activity. Abbreviations are the same as those given in Fig. 4-C. Bar: 100 μm."

Fig. 5

Subcellular localization of OsFLZ13 protein in Nicotona benthamiana 35S::eGFP: empty vector pD1301S; 35S::OSFLZ13::GFP: the recombinant vector pD1301S-OsFLZ13. Bar: 50 μm."

Fig. 6

Construction of CRISPR/Cas9 editing lines (Osflz13-1 and Osflz13-2) and Osflz13 gene sequences A: 2 gRNA target positions and base sequences of OsFLZ13; B: schematic diagram of the recombination of 2 target gRNA expression cassettes and PHK1-Cas-U3; C: sequence alignment analysis of mutant Osflz13 and OsFLZ13 gene in wild-type plants. Blue letters indicate target sequences, yellow highlights the PAM, strike through represents missing bases, red lowercase letters represent inserted bases, - indicates number of deletions, + indicates number of insertions, and WT is the wild-type plant Zhonghua 11."

Fig. 7

Stamen, pistil, pollen fertility, and seed-setting rates of Osflz13 mutant plants A: florets of wild-type (WT) and mutants (Osflz13-1, Osflz13-2) after removing glumes. Bar: 500 μm; B: pistils of wild-type (WT) and mutants (Osflz13-1, Osflz13-2). Bar: 500 μm; C: potassium iodide staining of pollens of wild-type (WT) and mutants (Osflz13-1, Osflz13-2). Bar: 100 μm; D: plants of wild-type (WT) and mutants (Osflz13-1, Osflz13-2) at the maturity. Bar: 10 cm; E: spikelet of wild-type (WT) and mutants (Osflz13-1, Osflz13-2) after seed formation. Bar: 2 cm; F: statistical analysis of seed-setting rate of wild type (ZH11) and mutant (Osflz13-1, Osflz13-2). Values represent the means, error bars depict SE (Student’s t-test, n=10, P<0.01)."

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