Cloning and Expression Analysis of Three LdBBXs in Lilium davidii var. willmottiae

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

Abstract

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

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.

Figures/Tables 12

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

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

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

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

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

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

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

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

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

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

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

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

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