Functional Study of Rice Eukaryotic Translation Initiation Factor OseIF6.2 in Grain Size Regulation

doi:10.16819/j.1001-7216.2025.2401010

Abstract

Abstract:

【Objective】This study aims to investigate the functional role of the rice eukaryotic translation initiation factor OseIF6.2 in rice growth and development using a reverse genetics approach, thereby laying a foundation for further exploration of the functions of OseIF6.2.【Method】First, bioinformatics methods were employed to perform multiple sequence alignment and a phylogenetic tree was constructed to analyze the evolutionary relationship between OseIF6.2 and eIF6 proteins in other eukaryotes. CRISPR/Cas9 gene editing technology was used to precisely edit the OseIF6.2 gene in rice (Nipponbare).Next, the expression levels of OseIF6.2 in different tissues of rice were detected using quantitative real-time PCR (qRT-PCR) technology. The precise localization of OseIF6.2 within cells was observed using a confocal laser microscope.Finally, the interaction between OseIF6.2 and its potential interacting protein, OseIF6.1, was validated using yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. 【Result】Phylogenetic analysis revealed that OseIF6.2 is highly conserved among eukaryotic eIF6 proteins, underscoring its universal role in translation regulation.qRT-PCR results indicated that OseIF6.2 expression was relatively high in rice stems. Subcellular localization analysis using GFP-tagged OseIF6.2 in tobacco leaf cells showed fluorescence signals in both the nucleus and cytoplasm, confirming its dual localization.Additionally, compared to the wild type, the oseif6.2 mutant exhibited significant reductions in grain length, grain thickness, and 1000-grain weight. Both Y2H and BiFC experiments confirmed a direct interaction between OseIF6.2 and OseIF6.1【Conclusion】In summary, OseIF6.2, a eukaryotic translation initiation factor in rice, plays a crucial role in regulating grain morphology, further highlighting its importance in rice growth, development, and grain formation.

Key words: eukaryotic translation initiation factor, OseIF6.2, rice, grain shape, growth and development

摘要：

【目的】本研究旨在通过反向遗传学策略，深入探究水稻真核翻译起始因子OseIF6.2在水稻生长发育中的具体功能，以期为OseIF6.2功能的进一步解析奠定基础。【方法】首先，利用生物信息学方法进行多重序列比对及进化树的构建，以分析OseIF6.2与其他真核生物中eIF6蛋白的系统发育关系；运用CRISPR/Cas9基因编辑技术，对水稻(日本晴)中的OseIF6.2基因进行精准编辑。其次，通过qRT-PCR技术，检测OseIF6.2在水稻不同组织中的表达水平；借助激光共聚焦显微镜，观察OseIF6.2在细胞内的精确定位。然后对野生型与oseif6.2突变体的粒长、粒宽、粒厚及千粒重等关键农艺性状进行比较分析。最后，通过酵母双杂交(Y2H)及双分子荧光互补(BiFC)实验，验证OseIF6.2与其潜在互作蛋白OseIF6.1的相互作用。【结果】系统发育分析结果表明，OseIF6.2与其他真核生物中的eIF6蛋白具有高度保守性，凸显了其在真核生物翻译调控中的普遍重要性。qRT-PCR结果显示，OseIF6.2在水稻茎中的表达水平较高。通过将OseIF6.2蛋白与GFP融合后注入本氏烟叶片细胞，并在激光共聚焦显微镜下观察，发现OseIF6.2在细胞核及细胞质区域均有荧光信号分布，明确了其亚细胞定位。此外，与野生型相比，oseif6.2突变体在粒长、粒厚及千粒重等性状上均表现为显著降低。Y2H及BiFC实验均成功验证了OseIF6.2与OseIF6.1蛋白之间的直接相互作用。【结论】综上所述，OseIF6.2作为水稻真核翻译起始因子，其功能异常显著影响水稻的粒型特征，进一步揭示了该因子在水稻生长发育及粒型形成中的关键作用。

关键词: 真核翻译起始因子, OseIF6.2, 水稻, 粒型, 生长发育

MA Shunting, HU Yungao, GAO Fangyuan, LIU Liping, MOU Changling, LÜ Jianqun, SU Xiangwen, LIU Song, LIANG Yuyu, REN Guangjun, GUO Hongming. Functional Study of Rice Eukaryotic Translation Initiation Factor OseIF6.2 in Grain Size Regulation[J]. Chinese Journal OF Rice Science, 2025, 39(3): 331-342.

马顺婷, 胡运高, 高方远, 刘利平, 牟昌铃, 吕建群, 苏相文, 刘松, 梁毓玉, 任光俊, 郭鸿鸣. 水稻真核翻译起始因子OseIF6.2调控粒型的功能研究[J]. 中国水稻科学, 2025, 39(3): 331-342.

Figures/Tables 7

Table 1. Primers used in this study

引物名称 Primer name	引物序列 Sequence (5＇-3＇)	用途 Purpose
sgRNA序列	GCGAGATTGGAGTGTTCGCGAGG	OseIF6.2基因sgRNA序列
eIF6.2-CDS-F	ATGATTTTTTTTTTCAAAAATCGGTTCTGCATTGCA	CDS 扩增
eIF6.2-CDS-R	CTATAGGTAGCTGCTGACGACGAGCGA	CDS amplification
K21-F	AGTTTGTAACAGCTGCTGGGATTAC	亚细胞定位载体
K21-NOSR-R	CCATCTCATAAATAACGTCATGCATTACATGTT	Subcellular localization vector
eIF6.2-Cas9-F	AGGAGTTCTTGGTTGGCTGACTGCGA	基因编辑材料鉴定
eIF6.2-Cas9-R	ACGCACATCCTCCCGATGATCCT	Identification of gene editing materials
OsCas9-1000-F	GAGGAGACAATCACCCCCTGGAACT	Cas9蛋白鉴定
OsCas9-1000-R	TTATCGGACTTGCCCCTGTTCTTGT	Identification of Cas9 protein
eIF6.1-2YC-767-F	ACGATAGTTAATTAACATGGCGACCCGTATTCAGTTTG	BiFC载体
eIF6.1-2YC-767-R	GATCGACAGTTATGTGCACTAGTGGCGCGCCC	Bimolecular fluorescence complementation vector
eIF6.1-CDS-759-F	CCTTAATTAAGGATGGCGACCCGTATTCAGTTTGAGAAC	CDS 扩增
eIF6.1-CDS-759-R	TTGGCGCGCCAACACATAACTGTCGATCAAAG	CDS amplification
eIF6.2-BD-798-F	GAGGACCTGCATATGATGATTTTTTTTTTCAAAAATCG	酵母双杂载体
eIF6.2-BD-798-R	GATCCCCGGGAATTCCTATAGGTAGCTG	Construction of yeast two-hybrid vector
eIF6.2-2YN-797-F	AC GATAGTTAATTAACATGATTTTTTTTTTCAAAAATCGG	BiFC载体构建
eIF6.2-2YN-797-R	CACTAGTGGCGCGCCCTAGGTAGCTGC	Constructionof of bimolecular fluorescence complementation vector
Actin-qPCR-F	TGTATGCCAGTGGTCGTACCA	内参基因定量引物
Actin-qPCR-R	CCAGCAAGGTCGAGACGAA	Primers of internal reference gene
eIF6.2-qPCR-148-F	AGAGAACTTCTTCAGCGTGTTC	OseIF6.2 定量引物
eIF6.2-qPCR-148-R	CTCTTGGTCTGTGGTGGTATG	qRT-PCR primers of OseIF6.2
OsEXPA5-F	AAGGCTGTGGCTTGATTGACA	细胞膨大相关基因定量引物
OsEXPA5-R	TTAGGCCCAATTTTGCTATTTTG	qRT-PCR primers of genes related to cell expansion
OsEXPA8-F	GCGATGAGCCGCAACTG	细胞膨大相关基因定量引物
OsEXPA8-R	CTCTTCCATCCTATGCCACG	qRT-PCR primers of genes related to cell expansion
OsEXPA30-F	CCAAGTTCAGGGCGATGCAG	细胞膨大相关基因定量引物
OsEXPA30-R	ACGGTGATCGCCGGCGAGCC	qRT-PCR primers of genes related to cell expansion
OsEXPB3-F	CTTTGAGTGGTTGGAGTGGTGG	细胞膨大相关基因定量引物
OsEXPB3-R	GCAGCCTTCTTGGAGATGGAA	qRT-PCR primers of genes related to cell expansion

Table 1. Primers used in this study

引物名称 Primer name	引物序列 Sequence (5＇-3＇)	用途 Purpose
sgRNA序列	GCGAGATTGGAGTGTTCGCGAGG	OseIF6.2基因sgRNA序列
eIF6.2-CDS-F	ATGATTTTTTTTTTCAAAAATCGGTTCTGCATTGCA	CDS 扩增
eIF6.2-CDS-R	CTATAGGTAGCTGCTGACGACGAGCGA	CDS amplification
K21-F	AGTTTGTAACAGCTGCTGGGATTAC	亚细胞定位载体
K21-NOSR-R	CCATCTCATAAATAACGTCATGCATTACATGTT	Subcellular localization vector
eIF6.2-Cas9-F	AGGAGTTCTTGGTTGGCTGACTGCGA	基因编辑材料鉴定
eIF6.2-Cas9-R	ACGCACATCCTCCCGATGATCCT	Identification of gene editing materials
OsCas9-1000-F	GAGGAGACAATCACCCCCTGGAACT	Cas9蛋白鉴定
OsCas9-1000-R	TTATCGGACTTGCCCCTGTTCTTGT	Identification of Cas9 protein
eIF6.1-2YC-767-F	ACGATAGTTAATTAACATGGCGACCCGTATTCAGTTTG	BiFC载体
eIF6.1-2YC-767-R	GATCGACAGTTATGTGCACTAGTGGCGCGCCC	Bimolecular fluorescence complementation vector
eIF6.1-CDS-759-F	CCTTAATTAAGGATGGCGACCCGTATTCAGTTTGAGAAC	CDS 扩增
eIF6.1-CDS-759-R	TTGGCGCGCCAACACATAACTGTCGATCAAAG	CDS amplification
eIF6.2-BD-798-F	GAGGACCTGCATATGATGATTTTTTTTTTCAAAAATCG	酵母双杂载体
eIF6.2-BD-798-R	GATCCCCGGGAATTCCTATAGGTAGCTG	Construction of yeast two-hybrid vector
eIF6.2-2YN-797-F	AC GATAGTTAATTAACATGATTTTTTTTTTCAAAAATCGG	BiFC载体构建
eIF6.2-2YN-797-R	CACTAGTGGCGCGCCCTAGGTAGCTGC	Constructionof of bimolecular fluorescence complementation vector
Actin-qPCR-F	TGTATGCCAGTGGTCGTACCA	内参基因定量引物
Actin-qPCR-R	CCAGCAAGGTCGAGACGAA	Primers of internal reference gene
eIF6.2-qPCR-148-F	AGAGAACTTCTTCAGCGTGTTC	OseIF6.2 定量引物
eIF6.2-qPCR-148-R	CTCTTGGTCTGTGGTGGTATG	qRT-PCR primers of OseIF6.2
OsEXPA5-F	AAGGCTGTGGCTTGATTGACA	细胞膨大相关基因定量引物
OsEXPA5-R	TTAGGCCCAATTTTGCTATTTTG	qRT-PCR primers of genes related to cell expansion
OsEXPA8-F	GCGATGAGCCGCAACTG	细胞膨大相关基因定量引物
OsEXPA8-R	CTCTTCCATCCTATGCCACG	qRT-PCR primers of genes related to cell expansion
OsEXPA30-F	CCAAGTTCAGGGCGATGCAG	细胞膨大相关基因定量引物
OsEXPA30-R	ACGGTGATCGCCGGCGAGCC	qRT-PCR primers of genes related to cell expansion
OsEXPB3-F	CTTTGAGTGGTTGGAGTGGTGG	细胞膨大相关基因定量引物
OsEXPB3-R	GCAGCCTTCTTGGAGATGGAA	qRT-PCR primers of genes related to cell expansion

Fig. 1. Expression pattern and subcellular localization of OseIF6.2 A, Comparative phylogenetic analysis of eIF6 protein cross diverse eukaryotic species. B, Relative expression of OseIF6.2 in root, stem, leaf, and sheath of young seedlings and developing panicles at lengths of 4-5, 8-10, and 10-12 cm. OsActin served as the internal control. Values represent means ± SE from at least three independent experiments. C, Subcellular localization of OseIF6.2 in N. benthamiana leaves. YFP: Yellow fluorescent protein, RFP: Red fluorescent protein. Scale bars = 20 μm.

Fig. 2. Types and phenotypic analysis of OseIF6.2 mutants A, Mutation types of OseIF6.2. B, Plants and panicles of WT and oseif6.2 mutant at the mature stage. Scale bars = 10 cm for plant height and 1 cm for panicle length. C, Panicle length of WT and oseif6.2 mutant. D, Number of primary branches per panicle of WT and oseif6.2 mutant. E, Number of secondary branches of WT and oseif6.2 mutant. F, Seed setting rate of WT and oseif6.2 mutant. Values represent means ± SE from at least three independent experiments, Student's t-test: *P< 0.05, **P< 0.01, ***P< 0.001.

Fig. 3. Mutation of OseIF6.2 alters grain shape A, Mature grains of WT and oseif6.2 mutants. Scale bars =1 cm. B, Grain length of WT and oseif6.2 mutants; C, Grain width; D, Grain thickness; E, 1000-grain weight; F, Filled grains per panicle; G, Panicle number per plant; H, Grain yield per plant. Values represent means ± SE from at least three independent experiments, Student's t-test: *P < 0.05, **P < 0.01, ***P< 0.001.

Fig. 4. OseIF6.2 regulates grain size by affecting cell expansion A, Scanning electron microscopy images of the glume outer surfaces of WT and oseif6.2 mutants. Scale bars = 100 μm. Cell length (B) and width (C) of the outer epidermal of WT and oseif6.2 mutant lemmas. D, Cell area of the outer epidermal of WT and oseif6.2 mutant lemmas. Values represent means ± SE from at least three independent experiments. Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5. Expression levels of genes related to cell expansion in WT and oseif6.2 mutant lines The values represent means ± SE derived from at least three independent experiments. Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6. OseIF6.2 physically interacts with OseIF6.1 A, OseIF6.2 interacts with OseIF6.1 in yeast cells. Transformed cells were cultured on DDO or QDO/X/A media. B, Bimolecular fluorescence complementation assays verifies the interaction between OseIF6.2 and OseIF6.1 in N. benthamiana. OseIF6.2-nYFP was coexpressed with OseIF6.1-cYFP in cells of N. benthamiana. Scale bars = 10 μm.

References 51

[1]	Su S, Hong J, Chen X, Zhang C, Chen M, Luo Z, Chang S, Bai S, Liang W, Liu Q, Zhang D. Gibberellins orchestrate panicle architecture mediated by DELLA-KNOX signalling in rice[J]. Plant Biotechnology Journal, 2021, 19(11): 2304-2318.
[2]	Merrick W C, Pavitt G D. Protein synthesis initiation in eukaryotic cells[J]. Cold Spring Harbor Perspectives in Biology, 2018, 10(12): a033092.
[3]	Jackson R J, Hellen C U T, Pestova T V. The mechanism of eukaryotic translation initiation and principles of its regulation[J]. Nature Reviews Molecular Cell Biology, 2010, 11(2): 113-127.
[4]	Guo J, Jin Z, Yang X, Li J F, Chen J G. Eukaryotic initiation factor 6, an evolutionarily conserved regulator of ribosome biogenesis and protein translation[J]. Plant Signaling & Behavior, 2011, 6(5): 766-771.
[5]	Aitken C E, Lorsch J R. A mechanistic overview of translation initiation in eukaryotes[J]. Nature structural & Molecular Biology, 2012, 19(6): 568-576.
[6]	Raabe K, Honys D, Michailidis C. The role of eukaryotic initiation factor 3 in plant translation regulation[J]. Plant Physiology and Biochemistry, 2019, 145: 75-83.
[7]	Castellano M, Merchante C. Peculiarities of the regulation of translation initiation in plants[J]. Current Opinion in Plant Biology, 2021, 63: 102073.
[8]	Singha D L, Maharana J, Panda D, Dehury B, Modi M K, Singh S. Understanding the thermal response of rice eukaryotic transcription factor eIF4A1 towards dynamic temperature stress: insights from expression profiling and molecular dynamics simulation[J]. Journal of Biomolecular Structure and Dynamics, 2021, 39(7): 2575-2584.
[9]	Ma L, Yang Y, Wang Y, Cheng K, Zhou X, Li J, Zhang J, Li R, Zhang L, Wang K, Zeng N, Gong Y, Zhu D, Deng Z, Qu G, Zhu B, Fu D, Luo Y, Zhu H. SlRBP1 promotes translational efficiency via SleIF4A2 to maintain chloroplast function in tomato[J]. The Plant Cell, 2022, 34(7): 2747-2764.
[10]	Rausell A, Kanhonou R, Yenush L, Serrano R, Ros R. The translation initiation factor eIF1A is an important determinant in the tolerance to NaCl stress in yeast and plants[J]. Plant Journal, 2003, 34: 257-267.
[11]	Sahoo R K, Gill S S, Tuteja N. Pea DNA helicase 45 promotes salinity stress tolerance in IR64 rice with improved yield[J]. Plant Signaling＆Behavior, 2012, 7: 1042-104.
[12]	Zhang L, Liu X, Gaikwad K, Kou X, Wang F, Tian X, Xin M, Ni Z, Sun Q, Peng H, Vierling E. Mutations in eIF5B confer thermosensitive and pleiotropic phenotypes via translation defects in Arabidopsis thaliana[J]. The Plant Cell, 2017, 29(8): 1952-1969.
[13]	Zheng T C, Zang L N, Dai L J, Yang C P, Qu G Z. Two novel eukaryotic translation initiation factor 5A genes from Populus simonii×P. nigra confer tolerance to abiotic stresses in Saccharomyces cerevisiae[J]. Journal Frontiers Research, 2017, 28: 453-46.
[14]	Li L S, Luo C, Huang F, Liu Z L, An Z Y, Dong L, He X H. Identification and characterization of the mango eIF gene family reveals MieIF1A-a, which confers tolerance to salt stress in transgenic Arabidopsis[J]. Scientia Horticulturae, 2019, 248: 274-281.
[15]	Wang L, Xu C, Wang C, Wang Y. Characterization of a eukaryotic translation initiation factor 5A homolog from Tamarix androssowii involved in plant abiotic stress tolerance[J]. BMC Plant Biology, 2012, 12(1): 118.
[16]	王雷, 孙尧, 李瑶, 吴琼, 夏新莉. 罗布麻eIF-5A基因功能[J]. 北方园艺, 2017(23): 190-193.
	Wang L, Sun Y, Li Y, Wu Q, XIia X L. Function of eIF-5A gene in Apocynum venetum L[J]. Norther Horticulture, 2017(23):190-193.
[17]	Kenia S D, Mayra A L, Eloísa H L, Brenda N R, Marlon A P T, Jesús H J P, Marina G R, Tzvetanka D D. Arabidopsis thaliana eIF4E1 and eIF(iso)4E participate in cold response and promote translation of some stress-related mRNAs[J]. Frontiers in Plant Science, 2021, 12(12): 698585.
[18]	Wang W, Xu M, Liu X, Tu J. The rice eukaryotic translation initiation factor 3 subunit e (OseIF3e) influences organ size and pollen maturation[J]. Frontiers in Plant Science, 2016, 7: 1399.
[19]	Huang Y, Zheng P, Liu X, Chen H, Tu J. OseIF3h regulates plant growth and pollen development at translational level presumably through interaction with OsMTA2[J]. Plants, 2021, 10(6): 1101.
[20]	Kim T H, Kim B H, Yahalom A, Chamovitz D A, von Arnim A G. Translational regulation via 5' mRNA leader sequences revealed by mutational analysis of the Arabidopsis translation initiation factor subunit eIF3h[J]. The Plant Cell, 2004, 16(12): 3341-3356.
[21]	Kim B H, Cai X, Vaughn J N, vonArnim A G. On the functions of the h subunit of eukaryotic initiation factor 3 in late stages of translation initiation[J]. Genome Biology. 2007, 8: R60.
[22]	Li N, Li Y. Signaling pathways of seed size control in plants[J]. Current opinion in Plant Bitology, 2016, 33: 23-32.
[23]	Xia C, Wang Y J, Li W Q, Chen Y R, Deng Y, Zhang X Q, Chen L Q, Ye D. The Arabidopsis eukaryotic translation initiation factor 3, subunit F (AteIF3f), is required for pollen germination and embryogenesis[J]. The Plant Journal, 2010, 63(2): 189-202.
[24]	Feng H, Chen Q, Feng J, Zhang J, Yang X, Zuo J. Functional characterization of the Arabidopsis eukaryotic translation initiation factor 5A-2 that plays a crucial role in plant growth and development by regulating cell division, cell growth, and cell death[J]. Plant Physiology, 2007, 144: 1531-1545.
[25]	Hopkins M T, Lampi Y, Wang T W, Liu Z, Thompson J E. Eukaryotic translation initiation factor 5A is involved in pathogen-induced cell death and development of disease symptoms in Arabidopsis[J]. Plant Physiology, 2008, 148: 479-489.
[26]	Wang T W, Lu L, Wang D, Thompson J E. Isolation and characterization of senescence-induced cDNAs encoding deoxyhypusine synthase and eucaryotic translation initiation factor 5A from tomato[J]. Journal of Biological Chemistry, 2001, 276(20): 17541.
[27]	Lin L, Cao J, Du A, An Q, Chen X, Yuan S, Batool W, Shabbir A, Zhang D, Wang Z, Norvienyeku J. EIF3k domain-containing protein regulates conidiogenesis, appressorium turgor, virulence, stress tolerance, and physiological and pathogenic development of Magnaporthe oryzae Oryzae[J]. Frontiers in Plant Science, 2021, 12: 748120.
[28]	Zhang X, Yin Y, Su Y, Jia Z, Jiang L, Lu Y, Zheng H, Peng J, Rao S, Wu G, Chen J, Yan F. eIF4A, a target of siRNA derived from rice stripe virus, negatively regulates antiviral autophagy by interacting with ATG5 in Nicotiana benthamiana[J]. PLoS Pathogens, 2021, 17(9): e1009963.
[29]	Roy B, von Arnim A G. Translational regulation of cytoplasmic mRNAs[J]. The Arabidopsis book/American Society of Plant Biologists, 2013, 11: e0165.
[30]	Si K, Maitra U. The Saccharomyces cerevisiae homologue of mammalian translation initiation factor 6 does not function as a translation initiation factor[J]. Molecular and Cellular Biology, 1999, 19(2): 1416-1426.
[31]	Basu U, Si K, Warner J R, Maitra U. The Saccharomyces cerevisiae TIF6 gene encoding translation initiation factor 6 is required for 60S ribosomal subunit biogenesis[J]. Molecular and Cellular Biology, 2001, 21(5): 1453-1462.
[32]	Ceci M, Gaviraghi C, Gorrini C, Sala L A, Offenhäuser N, Marchisio P C, Biffo S. Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly[J]. Nature, 2003, 426(6966): 579-584.
[33]	Miluzio A, Beugnet A, Volta V, Biffo S. Eukaryotic initiation factor 6 mediates a continuum between 60S ribosome biogenesis and translation[J]. EMBO Reports, 2009, 10(5): 459-465.
[34]	Gandin V, Miluzio A, Barbieri A M, Beugnet A, Kiyokawa H, Marchisio P C, Biffo S. Eukaryotic initiation factor 6 is rate-limiting in translation, growth and transformation[J]. Nature, 2008, 455(7213): 684-688.
[35]	Kausik S, Jayanta C, Jorge C H. Molecular cloning and functional expression of a human cDNA encoding translation initiation factor 6[J]. Proceedings of the National Academy of Sciences, 1997, 94(26): 14285-14290.
[36]	Sanvito F, Piatti S, Villa A, Bossi M, Lucchini G, Marchisio PC, Biffo S. The beta4 integrin interactor p27(BBP/eIF6) is an essential nuclear matrix protein involved in 60S ribosomal subunit assembly.[J]. The Journal of Cell Biology, 1999, 144(5): 823-837.
[37]	Kapp L D, Lorsch J R. The molecular mechanics of eukaryotic translation[J]. Annual Review of Biochemistry, 2004, 73(1): 657-704.
[38]	Sonenberg N, Hinnebusch A G. Regulation of translation initiation in eukaryotes: Mechanisms and biological targets[J]. Cell, 2009, 136(4): 731-745.
[39]	Russell D W, Spremulli L L. Mechanism of action of the wheat germ ribosome dissociation factor: Interaction with the 60S subunit[J]. Archives of Biochemistry and Biophysics, 1980, 201(2): 518-526.
[40]	Kato Y, Konishi M, Shigyo M, Yoneyama T, Yanagisawa S. Characterization of plant eukaryotic translation initiation factor 6 (eIF6) genes: The essential role in embryogenesis and their differential expression in Arabidopsis and rice[J]. Biochemical and Biophysical Research Communications, 2010, 397(4): 673-678.
[41]	Guo H M, Lv J Q, Su X W, Chen L, Ren J S, Liu L P, Ren M X, Liu S, Dai M L, Ren G J, Gao F Y. Rice OseIF6. 1 encodes a eukaryotic translation initiation factor and is essential for the development of grain and anther[J]. Frontiers in Plant Science, 2024, 15: 1366986.
[42]	Sparkes I A, Runions J, Kearns A, Hawes C. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants[J]. Nature Protocols, 2006, 1(4): 2019-2025.
[43]	Aslanidis C, Jong P J, Schmitz G. Minimal length requirement of the single-stranded tails for ligation-independent cloning (LIC) of PCR products[J]. Genome Research, 1994, 4(3): 172-177.
[44]	Raychaudhuri P, Stringer E A, Valenzuela D M, Maitra U. Ribosomal subunit antiassociation activity in rabbit reticulocyte lysates. Evidence for a low molecular weight ribosomal subunit antiassociation protein factor (Mr= 25,000)[J]. Journal of Biological Chemistry, 1984, 259(19): 11930-11935.
[45]	Xing Y, Zhang Q. Genetic and molecular bases of rice yield[J]. Annual Review of Plant Biology, 2010, 61:421-442.
[46]	何秀英, 伍时照, 宋美芳, 林贤琛. 水稻籽粒发育和胚乳淀粉粒形成的研究[J]. 广东农业科学, 2000(2): 8-10.
	He X Y, Wu S Z, Song M F, Lin X C. Research on rice grain development and endosperm starch grain formation[J]. Guangdong Agricultural Science, 2000, (2): 8-10. (in Chinese with English abstract)
[47]	黄海祥, 钱前. 水稻粒形遗传与长粒型优质粳稻育种进展[J]. 中国水稻科学, 2017, 31(6): 665-672.
	Huang H X, Qian Q. Progress in Rice Grain Shape Genetics and Breeding of Long Grain High Quality Japonica Rice[J]. Chinese Journal of Rice Science, 2017, 31(6): 665-672. (in Chinese with English abstract)
[48]	况慧云, 张慧, 许寿增, 黄英金. 水稻产量基因设计育种及其发展前景[J]. 安徽农学通报, 2014, 20(8):42-45.
	Kuang H Y, Zhang H, Xu S Z, Huang Y J. Design and breeding of rice yield genes and their development prospects[J]. Anhui Agricultural Bulletin, 2014, 20(8): 42-45. (in Chinese with English abstract)
[49]	李孝琼, 韦宇, 高国庆, 邓国富, 郭嗣斌. 水稻遗传图谱构建及粒形相关性状的QTL定位[J]. 南方农业学报, 2014, 45(7): 1154-1159.
	Li X Q, Wei Y, Gao G Q, Deng G F, Guo S B. Construction of rice genetic map and QTL mapping of grain shape related traits[J]. Journal of Southern Agriculture, 2014, 45(7): 1154-1159. (in Chinese with English abstract)
[50]	Wood L C, Ashby M N, Grunfeld C, Feingold K R. Cloning of murine translation initiation factor 6 and functional analysis of the homologous sequence YPR016c in Saccharomyces cerevisiae[J]. Journal of Biological Chemistry, 1999, 274(17): 11653-11659.
[51]	Guo H M, Cui Y C, Huang L J, Ge L, Xu X R, Xue D Y, Tang M, Zheng J S, Yi Y, Chen L. The RNA binding protein OsLa influences grain and anther development in rice[J]. The Plant Journal, 2022, 110(5): 1397-1414.