百合LoSAUR10基因的表达特征及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0502

摘要/Abstract

摘要：

【目的】对百合LoSAUR10基因进行克隆和组织表达特异性分析，在拟南芥中对其功能进行探究，为百合鳞茎发育机理提供参考依据。【方法】利用外源处理比较生长素对百合鳞片扦插籽球发生的影响，在此基础上以‘西伯利亚’百合cDNA为模板克隆 LoSAUR10，并对其进行生物信息学分析，利用实时荧光定量PCR和过表达转基因拟南芥植株对LoSAUR10 基因的表达特征及其在叶片发育中的功能进行分析。【结果】生长素IAA处理能显著促进百合鳞片扦插过程中的籽球发生和膨大，0.05 g/L的生长素处理后百合籽球发生率较对照组增加了69.04%。在扦插产生的籽球发生早期，LoSAUR10 基因的表达受外源生长素诱导，在籽球进入膨大阶段时，LoSAUR10 基因的表达逐渐下降。测序结果表明，LoSAUR10基因编码区长297 bp，无内含子，编码98个氨基酸，含有Auxin-inducible结构域。进化树分析结果表明LoSAUR10蛋白序列与郁金香中TgSAUR10及模式植物拟南芥中AtSAUR9和AtSAUR10具有较高的序列相似性。荧光定量PCR结果表明，LoSAUR10基因在转录水平受生长素诱导，且在多个组织中均有表达，但在百合鳞片中表达量较高。与野生型植株相比，过表达LoSAUR10的转基因拟南芥植株表现出莲座叶增大，叶片数量增加，叶片表皮细胞面积增大，且抽薹时间提前的表型。【结论】百合LoSAUR10基因参与调控植物的叶片发育和开花过程，研究结果为揭示百合籽球发生机理奠定了基础。

关键词: 百合, LoSAUR10, 籽球, 组织表达特异性, 转基因拟南芥

Abstract:

【Objective】LoSAUR10 was cloned and its expression patterns were analyzed, and then the function of LoSAUR10 was identified using over-expressed transgenic plants in Arabidopsis thaliana, which may provide reference for the developmental mechanism of lily bulbs.【Method】According to the effect of exogenous auxin treatment on the occurrence of bulblets, lily cultivar ‘Siberian’ cDNA was used as template to clone LoSAUR10 for bioinformatics analysis. Real-time quantitative PCR and over-expressed transgenic Arabidopsis plants were used to analyze the expression characteristics of LoSAUR10 and its function in leaf development.【Result】The exogenous IAA treatment significantly promoted the occurrence and enlargement of bulblets. The occurrence rate of lily bulblets increased by 69.04% compared to the control treatment at a treatment with concentration of 0.05 g/L IAA. In the early stage of bulblets formation, the expression of LoSAUR10 gene was induced by exogenous auxin. As the bulblets enter the enlargement stage, the expressions of LoSAUR10 gene gradually decreased under IAA treatment. Results of gene sequencing indicated that the length pf coding region of the LoSAUR10 gene was 297 bp without introns, encoding 98 amino acids and containing an auxin-inducible domain. The phylogenetic tree analysis showed that the LoSAUR10 protein sequence had high similarity with Tulipa gesneriana TgSAUR10 and A. thaliana AtSAUR9 and AtSAUR10. Real-time RT-qPCR indicated that LoSAUR10 was induced by auxin treatment at the transcriptional level and expressed in most detected tissues with high expression in lily scales. Compared to wild-type plants, transgenic Arabidopsis plants overexpressing LoSAUR10 had the phenotypes of enlarged rosette leaves, increased leaf cell area, as well as early bolting.【Conclusion】LoSAUR10 is involved in the regulation of plant leaf growth and flowering processes, and the results lay a foundation for revealing the mechanism of lily bulbs development.

Key words: Lilium, LoSAUR10, bulblet, tissue-specific expression, transgenic Arabidopsis

田栩瑞, 霍信屹, 郭云涵, 向林, 产祝龙, 王艳平. 百合LoSAUR10基因的表达特征及功能分析[J]. 生物技术通报, 2025, 41(1): 221-229.

TIAN Xu-rui, HUO Xin-yi, GUO Yun-han, XIANG Lin, CHAN Zhu-long, WANG Yan-ping. Expression and Functional Analysis of Gene LoSAUR10 from Lilium Oriental Hybrids[J]. Biotechnology Bulletin, 2025, 41(1): 221-229.

图/表 7

表1 引物信息

Table 1 Information of primers

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
LoSAUR10-cloning-F	CTCCGTCTACTTCATTCTTTC
LoSAUR10-cloning-R	GTATCAGTGGTGGGTCTTCT
pCAMBIA1307-LoSAUR10-F	ACCGTCGACGAGCTCTCTAGAATGGCAGCAAGGAGGTTGAA
pCAMBIA1307-LoSAUR10-R	TTTGCGGAGTACCCGGGTACCGCAGAGACTGGAGGTGAGAGATCG
qRT-LoActin-F	TGCTGACCGTATGAGCAAGG
qRT-LoActin-R	GACGATGGCTGGACCAGATT
qRT-LoSAUR10-F	CATCGTCCCCATCTCTCTACTTG
qRT-LoSAUR10-R	AGGTGAGAGATCGGAAGACAGTA
qRT-AtActin-F	TGAGAGATTCAGATGCCCAGA
qRT-AtActin-R	TGGATTCCAGCAGCTTCCAT
qRT-At-LoSAUR10-F	GCTTCATCGTCCCCATCTC
qRT-At-LoSAUR10-R	GAAGACAGTATCCTCGCATGG

图1 生长素处理对百合扦插籽球发育的影响 A：IAA处理促进百合鳞片扦插籽球膨大；bar=0.5 cm；B：IAA处理促进籽球繁殖率；C：不同周径籽球占比分析；* P<0.05，*** P<0.001，下同

Fig. 1 Effects of auxin application on the development of lily bulblets A: IAA treatment enhanced bulblets enlargement in lily; bar=0.5 cm. B: Reproduction rates were induced by IAA treatment. C: Comparison of the proportion of bulblets. * P<0.05, *** P<0.001. The same below

图2 IAA处理对百合籽球发生和LoSAUR10基因表达的影响 A：IAA处理不同时间百合籽球发生比较；bar=0.5 cm；B：IAA处理不同时间LoSAUR10基因的表达分析

Fig. 2 Effects of IAA treatment on the bulblets occurrence and relative expressions of LoSAUR10 in lily A: Comparison of the bulblets at different time courses after IAA treatment; bar=0.5 cm. B: Relative expressions of LoSAUR1 gene after IAA treatment

图3 IAA处理不同时间（A）及不同组织（B）LoSAUR10基因表达水平分析不同字母表示P<0.05

Fig. 3 Relative expressions of LoSAUR10 in different treatment time (A) and different tissues with (B) IAA treatment Different letters indicate P<0.05

图4 百合LoSAUR10蛋白序列和进化树分析 A：LoSAUR10蛋白保守序列比对；红色框为SAUR结构域；B：LoSAUR10与拟南芥AtSAURs、郁金香TgSAUR10蛋白序列进化树分析

Fig. 4 Protein sequence and phylogenetic tree construction of LoSAUR10 in lily A: Alignment of conserved sequence of LoSAUR10 protein; the red box indicates SAUR domain. B: Phylogenetic tree of LoSAUR10 and other homologous proteins from Arabidopsis thaliana and Tulipa gesneriana

图5 LoSAUR10蛋白生物信息学分析 A：LoSAUR10跨膜结构域分析；B：LoSAUR10蛋白亲水性分析；C：LoSAUR10的结构域分析；D：LoSAUR10的蛋白二级结构域分析

Fig. 5 Bioinformatics analysis of LoSAUR10 protein A: Transmembrane domain analysis of LoSAUR10. B: Hydrophilicity analysis of LoSAUR10 protein. C: Conserved domain analysis of LoSAUR10. D: Secondary structure of LoSAUR10 protein

图6 LoSAUR10过表达转基因植株表型分析 A：生长3周的植物叶片大小比较，bar=1.0 cm；B：荧光定量PCR分析LoSAUR10基因的表达量；M：marker；WT：野生型；P：质粒；C：第6-7片莲座叶片长度比较；D：第6-7片莲座叶宽度比较；E：叶片表皮细胞大小比较；F：莲座叶数量比较；G：植株株幅比较；H：抽薹天数比较

Fig. 6 Phenotype analysis of transgenic plants with over-expressed LoSAUR10 gene A: Comparison of leaves sizes after 3 weeks growing; bar=1.0 cm. B: Relative expression of LoSAUR10 analyzed by RT-qPCR; M: marker; WT: wild-type; P: plasmid. C: Length comparison of the 6th-7th rosette leaves. D: Width comparison of the 6th-7th rosette leaves. E: Area comparison of epidermal cells. F: Comparison of numbers of rosette leaves.G: Plant width comparison. H: Bolting time comparison

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