Cloning and Functional Analysis of LlLBD18 in Lilium lancifolium

doi:10.16420/j.issn.0513-353x.2022-0766

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (10): 2117-2127.doi: 10.16420/j.issn.0513-353x.2022-0766

• Genetic & Breeding · Germplasm Resources · Molecular Biology • Previous Articles Next Articles

Cloning and Functional Analysis of LlLBD18 in Lilium lancifolium

YIN Xiaoyu¹^,², HE Guoren³, BI Mengmeng², TANG Yuchao², HAO Chunlian¹^,², QU Yuxiao², HAO Zehui⁴, XU Leifeng², HU Fengrong¹^,^*(), YANG Panpan²^,^*(), MING Jun²^,^*()

¹ College of Landscape Architecture，Nanjing Forestry University，Nanjing 210037，China
² State Key Laboratory of Vegetable Biobreeding，Institute of Vegetables and Flowers，Chinese Academy of Agricultural Sciences，Beijing 100081，China
³ College of Life Sciences，Shanghai Normal University，Shanghai 200233，China
⁴ College of Landscape Architecture，Beijing University of Agriculture，Beijing 102206，China

Received:2023-05-19 Revised:2023-10-13 Online:2023-10-25 Published:2023-10-30

Abstract

Abstract:

In this study，a LBD transcription factor gene，named LlLBD18（GenBank accession number ON455210）was cloned from Lilium lancifolium. Its open reading frame was 684 bp，which encoded 227 amino acids，and contained a typical LOB domain. The phylogenetic tree analysis showed that LlLBD18 had the highest homology with Zingiber officinale ZoLBD18. Subcellular localization results showed that LlLBD18 was localized in the cytoplasm and nucleus. Quantitative real-time PCR analysis showed that the expression of LlLBD18 was the highest in primary bulbils，followed by roots，and the lowest in leaves. In the process of bulbil formation，the overall expression of LlLBD18 was first increased，then decreased，and then increased，and the highest expression was at the early stage of bulbil primordium initiation（the 4th day）. Functional studies found that overexpression of LlLBD18 in L. lancifolium can promote bulbil formation，while the formation of bulbils after VIGS silencing of LlLBD18 was significantly inhibited. This research suggested that LlLBD18 plays a positive regulatory role in bulbil formation of L. lancifolium.

Key words: Lilium lancifolium, lateral organ, bulbil formation, gene cloning, function analysis

YIN Xiaoyu, HE Guoren, BI Mengmeng, TANG Yuchao, HAO Chunlian, QU Yuxiao, HAO Zehui, XU Leifeng, HU Fengrong, YANG Panpan, MING Jun. Cloning and Functional Analysis of LlLBD18 in Lilium lancifolium[J]. Acta Horticulturae Sinica, 2023, 50(10): 2117-2127.

Figures/Tables 7

Table 1 Primer sequences in this study

用途 Usage	引物名称 Primer name	引物序列（5′-3′） Primer sequence
全长克隆Full length cloning	LBD18-F	ATGAGCTGCAGTACGAGC
	LBD18-R	TCACCTAGAGACAGGAGGAGTA
实时荧光定量PCR Quantitative real-time PCR	q-LBD18-F	TCCTGTGTATGGATGTGTTGC
	q-LBD18-R	AAGCGGCTGTCGGAATTGAA
亚细胞定位Subcellular localization	2300-LBD18-F	tggagaggacagggtacccgggATGAGCTGCAGTACGAGCAG
	2300-LBD18-R	ccatggtactagtgtcgactctagaCCTAGAGACAGGAGGAGTATCCAG
过表达试验Overexpression experiment	3301-LBD18-F	cggtacccggggatcctctagaATGAGCTGCAGTACGAGCAG
	3301-LBD18-R	acgacggccagtgccaagcttTCACCTAGAGACAGGAGGAG
VIGS沉默试验VIGS silence experiment	TRV2-LBD18-F	gtgagtaaggttaccgaattcCCTAGAGCTACCATCTCCTCC
	TRV2-LBD18-R	tccccatggaggccttctagaTCACCTAGAGACAGGAGGAG

Fig.1 PCR amplification and bioinformatics analysis of LlLBD18 in Lilium lancifolium

Fig. 2 Sequence alignment of LBD18 from Lilium lancifolium and Arabidopsis thaliana The red box is the main component of LOB domain，the red triangle is highly conserved amino acids.

Fig. 3 Phylogenetic tree of LlLBD18 and LBD18 from other 15 species The numbers at node represent the distance of the kinship.

Fig. 4 The subcellular localization of LlLBD18

Fig. 5 The expression analysis of LlLBD18 in different tissues (A) and during bulbil formation (B) in Llium lancifolium Different lowercase letters indicate significant differences at P < 0.05.

Fig. 6 Leaf axil phenotype, bulbil induction rate, and relative expression of LlLBD18 transient overexpression（A-C）and VIGS silencing（D-F）plants in Lilium lancifolium Control was treated with pCAMBIA-3301 and OE was treated with pCAMBIA-3301-LlLBD18. TRV was empty vector treatment and TRV::LlLBD18 was transient silencing treatment. Different capital letters indicate significant difference at the level of P < 0.01.

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Cloning and Functional Analysis of LlLBD18 in Lilium lancifolium

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