兰州百合三个LdBBXs基因的克隆与表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-1174

摘要/Abstract

摘要：

目的 B-box（BBX）第Ⅳ亚组的许多成员在光调控花青素苷合成中扮演着重要角色，克隆兰州百合BBX基因并分析其对不同光照时间的应答及对鳞片变红的影响，为培育见光不变色兰州百合品种提供重要的候选基因。方法克隆从兰州百合转录组鉴定获得的3个BBX第Ⅳ亚组成员（LdBBX21、LdBBX22、LdBBX24），并对其编码的蛋白进行生物信息学分析和亚细胞定位，基于实时荧光定量PCR研究其在兰州百合不同组织及鳞片光处理后的表达模式。结果 LdBBX21、LdBBX22、LdBBX24均含有2个B-box保守结构域，属于第Ⅳ亚组的BBX蛋白。亚细胞定位结果显示，LdBBX21、LdBBX22、LdBBX24均定位于细胞核。组织特异性表达分析表明，LdBBX22和LdBBX24在花中的表达量最高，在鳞片中的表达量最低，LdBBX21与之相反。RT-qPCR分析表明，在不同光照时长处理的鳞片中，LdBBX21的基因表达量随光照时长的增加总体呈下降趋势；LdBBX22的基因表达量随光照时长的增加而上升，与花青素苷含量的变化趋势基本相同；LdBBX24随光照时长的增加，表达量总体呈先上升后下降趋势。结论 LdBBX21、LdBBX22、LdBBX24均可能在光调控兰州百合鳞片花青素苷合成中起着重要功能，本研究为后续研究兰州百合BBX基因功能验证奠定基础。

关键词: 兰州百合, BBX, 基因克隆, 表达分析

Abstract:

Objective Members of the BBX subgroup Ⅳ play an important role in regulating anthocyanins synthesis in response to light. The BBX gene of lily was cloned and its response to different light times and its effect on the reddening of scales were analyzed, which may provide important candidate genes for breeding Lilium davidii var. willmottiae varieties with scales keeping white upon light exposure. Method Three members of BBX subgroup Ⅳ (LdBBX21, LdBBX22,and LdBBX24) were cloned from L. davidii var. willmottia according to its transcriptome data, and the bioinformatics analysis and subcellular localization analysis of the proteins encoded by them were performed, and their expression patterns in different tissues and scales after light treatment were studied by quantitative real-time polymerase chain reaction (RT-qPCR). Result LdBBX21, LdBBX22, and LdBBX24 all contained two B-box conserved domains, and belonged to BBX subgroup Ⅳ. The results of subcellular localization showed that LdBBX21, LdBBX22 and LdBBX24 were all localized in the nucleus. Tissue-specific expression analysis showed that LdBBX22 and LdBBX24 had the highest expression in flowers and the lowest in scales, while LdBBX21 showed the opposite expression pattern. RT-qPCR analysis showed that in scales treated with different light duration, the gene expression of LdBBX21 showed a downward trend with the increase of light duration; the gene expression of LdBBX22 increased with the increase of light, which was basically the same as the change trend of anthocyanin content; the expression of LdBBX24 increased and then decreased of light duration. Conclusion All of LdBBX21, LdBBX22, and LdBBX24 may play important functions in light-regulated anthocyanin glycoside synthesis in lily scales, laying the foundation for subsequent functional verification of the LdBBX gene.

Key words: Lilium davidii var. willmottiae, BBX, gene cloning, expression analysis

刘鑫, 王嘉雯, 李进伟, 牟策, 杨盼盼, 明军, 徐雷锋. 兰州百合三个LdBBXs基因的克隆与表达分析[J]. 生物技术通报, 2025, 41(5): 186-196.

LIU Xin, WANG Jia-wen, LI Jin-wei, MOU Ce, YANG Pan-pan, MING Jun, XU Lei-feng. Cloning and Expression Analysis of Three LdBBXs in Lilium davidii var. willmottiae[J]. Biotechnology Bulletin, 2025, 41(5): 186-196.

图/表 12

图1 兰州百合的不同组织A：花和内花被片；B：茎；C：叶；D：鳞片。标尺＝1 cm

Fig. 1 Different tissues of L. davidii var. willmottiaeA: Flower and inner tepal. B: Stem. C: Leaf. D: Scale. The scale is 1 cm

表1 所用引物信息

Table 1 Information of the primers used in this study

引物 Primer	序列 Sequence （5′‒3′）	退火温度 Annealing temperature （℃）
LdBBX21-F	GGAGCCAGATCGGAGAG	55.5
LdBBX21-R	GCTTGAGGAAACAGAAAGAGG	55.5
LdBBX22-F	GAGAGAGAGGAGAGAGAGA	51.5
LdBBX22-R	TCGACCCATAATAACACATC	51.5
LdBBX24-F	GGGGATTGTCCAACTATATCCC	54.8
LdBBX24-R	TTCTCCGTTCATGTTTTGTGC	54.8
LdBBX21-qRT-F	CTCCATCCACAGGGCCAAC	59.0
LdBBX21-qRT-R	CAAACCGTCGAGGATGAGACG	59.0
LdBBX22-qRT-F	GGAGGCGACGGTGATGTG	60.0
LdBBX22-qRT-R	CGGAAGCGTTGCCAGAGATT	60.0
LdBBX24-qRT-F	CAACACAGCAGTCGCCTTC	57.5
LdBBX24-qRT-R	CCACCAGAAAACTCCTTCTGC	57.5
LilyActin-F	GCACCTGAAGAGCACCCT	60.0
LilyActin-R	TGGCGTAAAGCGACAAAA	60.0
LdBBX21-2300-F	ATTTGGAGAGGACAGGGTACGGAGCCAGATCGGAGAG	55.5
LdBBX21-2300-R	CACCATGGTACTAGTGTCGAGCTTGAGGAAACAGAAAGAGG	55.5
LdBBX22-2300-F	ATTTGGAGAGGACAGGGTAC AGAGAGAGATCTTGTCGGC	55.0
LdBBX22-2300-R	CACCATGGTACTAGTGTCGACATACTGATAGACATGTACAGCAG	55.0
LdBBX24-2300-F	ATTTGGAGAGGACAGGGTACGGGGATTGTCCAACTATATCCC	54.8
LdBBX24-2300-R	CACCATGGTACTAGTGTCGATTCTCCGTTCATGTTTTGTGC	54.8
Detect-2300-F	ATTTGGAGAGGACAGGGTAC	55.0
Detect-2300-R	CACCATGGTACTAGTGTCGA	55.0

图2 LdBBX21、LdBBX22、LdBBX24扩增电泳图及其翻译的氨基酸序列A、C、E：LdBBX21、LdBBX22、LdBBX24扩增电泳图；B、D、F：LdBBX21、LdBBX22、LdBBX24的CDS序列及其翻译的氨基酸序列

Fig. 2 LdBBX21, LdBBX22, and LdBBX24 amplification electropherogram and their translated amino acid sequencesA, C, E: Amplification electropherogram of LdBBX21, LdBBX22, LdBBX24; B, D, F: CDS sequence of LdBBX21, LdBBX22, LdBBX24 and its translated amino acid sequence

表2 LdBBX21、LdBBX22、LdBBX24蛋白基本理化性质分析结果

Table 2 Basic physicochemical properties of LdBBX21, LdBBX22, and LdBBX24 proteins

蛋白 Protein	分子式 Molecular formula	分子量 Molecular weight （kD）	等电点 pI	不稳定系数 Instability index
LdBBX21	C₉₄₈H₁₄₇₇N₂₇₃O₃₀₃S₁₄	22.00	5.87	60.39
LdBBX22	C₁₄₂₅H₂₁₈₅N₃₉₃O₄₅₆S₁₈	32.70	5.28	61.55
LdBBX24	C₁₁₅₁H₁₇₉₂N₃₁₂O₃₅₇S₁₉	26.32	4.91	60.48

图3 LdBBX21（A）、LdBBX22（B）、LdBBX24（C）蛋白亲水性分析

Fig. 3 Hydrophilicity analysis of LdBBX21 (A), LdBBX22 (B), and LdBBX24 (C) proteins

图4 LdBBX21、LdBBX22、LdBBX24蛋白二、三级结构预测A、C、E：LdBBX21、LdBBX22、LdBBX24蛋白的二级结构预测；B、D、F：LdBBX21、LdBBX22、LdBBX24蛋白的三级结构预测

Fig. 4 Prediction of secondary and tertiary structures of LdBBX21, LdBBX22, and LdBBX24 proteinsA, C, E: Secondary structure prediction of LdBBX21, LdBBX22, and LdBBX24 protein. B, D, F: Tertiary structure prediction of LdBBX21, LdBBX22, and LdBBX24 protein

图5 LdBBX21（A）、LdBBX22（B）、LdBBX24（C）蛋白保守结构域分析

Fig. 5 Protein conserved domain analysis of LdBBX21 (A), LdBBX22 (B), and LdBBX24 (C) proteins

图6 LdBBX21、 LdBBX22、LdBBX24蛋白的系统进化树

Fig. 6 Phylogenetic tree of LdBBX21, LdBBX22, and LdBBX24 proteins

图7 LdBBX21、LdBBX22、LdBBX24的亚细胞定位

Fig. 7 Subcellular localization of LdBBX21, LdBBX22, and LdBBX24 proteins

图8 光照处理0 d（A）、2 d（B）、4 d（C）下的兰州百合鳞片

Fig. 8 L. davidii var. willmottiae's scales under light treatment for 0 (A), 2 d (B), and 4 d (C) (Bars = 1 cm)

图9 光照处理0、2、4 d下LdBBX21、LdBBX22、LdBBX24基因的相对表达量及花青素苷含量变化不同字母表示在P<0.05水平差异显著。下同

Fig. 9 Relative expressions and anthocyanin contents of LdBBX21, LdBBX22, and LdBBX24 genes under light treatment for 0, 2, and 4 dDifferent letters above columns indicate significant differences at P<0.05 level. The same below

图10 兰州百合不同组织中LdBBX21、LdBBX22、LdBBX24基因的表达

Fig. 10 Expressions of LdBBX21, LdBBX22, and LdBBX24 genes in different tissues of L. davidii var. willmottiae

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