Cloning and Functional Study of the PgSPL2 Gene Related to the Development of Pomegranate Flowers

doi:10.13560/j.cnki.biotech.bull.1985.2022-0665

Abstract

Abstract:

This study is aimed to investigate the role of the pomegranate（Punica granatum L.）SPL family gene PgSPL2（XP_031375027.1）in pomegranate flower development and lay a foundation for further research on the mechanism of pomegranate flower development. Using ‘Tunisian’ soft-seeded pomegranate as experimental material，we used homologous cloning technique to clone the SPL family gene PgSPL2，analyze the physicochemical properties of the gene and the expression differences in different tissues of pomegranate. Then we applied RT-qPCR to analyze the spatio-temporal expression characteristics of the gene upon growth regulator treatment. Finally we transformed Arabidopsis and verified the role of the gene in flower development. The results showed that the gene contained a 591 bp open reading frame encoding 196 amino acids，and multiple sequence alignment revealed that the gene contained a conserved SBP structural domain and nuclear localization signal，belonged to the SBP transcription factor gene family. Phylogenetic tree analysis showed that PgSPL2 clustered with Arabidopsis AtSPL3，AtSPL4 and AtSPL5. The RT-qPCR results showed that the expression of PgSPL2 was relatively high in fruits and flower buds，and the expression of PgSPL2 in complete flowers was significantly down-regulated after spraying with growth regulator，while the ratio of complete flowers significantly increased simultaneously，and the first flowering and blooming periods delayed. Overexpression of PgSPL2 in Arabidopsis presented early flowering and significantly higher expression of inflorescence downstream regulatory genes. These results indicate that PgSPL2 plays a role in regulating flowering time in pomegranate flower development.

Key words: pomegranate, SPL, growth regulator, gene cloning, gene expression

GAN Cheng-yan, ZHANG Xin-hui, WANG Sha, FAN Yao-yu-wei, ZHAO Xue-qing, YUAN Zhao-he. Cloning and Functional Study of the PgSPL2 Gene Related to the Development of Pomegranate Flowers[J]. Biotechnology Bulletin, 2022, 38(12): 194-203.

Figures/Tables 11

Fig. 1 Incomplete（a）and complete（b）flowers at different developmental stages of pomegranate（Punica granatum L.） P1：Longitudinal diameter 3.0-5.0 mm. P2：Longitudinal diameter 5.1-13.0 mm. P3：Longitudinal diameter 13.1-25.0 mm

Table 1 Primers for the gene cloning，functional verification and specific expressions

引物Primer	引物序列Primer sequence（5'-3'）	试验用途Test purpose
PgSPL2	F：ATGAATGCCAATGG R：TTATCTTATCTGGAAGTGC	基因克隆 Gene clone
GUS-PgSPL2	F：gagaacacgggggactctagaATGAATGCCAATGG R：gcccttgctcaccatggatccTTATCTTATCTGGAAGTGC	功能验证 Functional verification
qRT-PgSPL2	F：GACTTCACCACCGAGCTGT R：CTCAGCCCAGCAACTATCACC	荧光定量引物 Fluorescent quantitative PCR primer
qRT-AtFUL	F：GAGAAGCGGGTCAGCAAG R：GCCATCTCTGGAGGAGGTTA	荧光定量引物 Fluorescent quantitative PCR primer
qRT-AtLFY	F：ATCGCTTGTCGTCATGGCTG R：GCAACCGCATTGTTCCGCTC	荧光定量引物 Fluorescent quantitative PCR primer
qRT-AtSOC1	F：TGTTCCAGGTCCTAGTGACCCCT R：ACGTAGCATCTGTTGTGCCGGG	荧光定量引物 Fluorescent quantitative PCR primer
AtActin	F：ACTTGAACGAGTAGGAGTGAAG R：CAGCCGACTTTACAAGAAC	拟南芥内参引物 Internal primer of Arabidopsis thaliana
PgActin	F：AGTCCTCTTCCAGCCATCTC R：CACTGAGCACAATGTTTCCA	石榴内参引物 Internal primer of pomegranate

Fig. 2 Phylogenetic analysis of SPL proteins in Pomegra-nate，Arabidopsis，Solanum lycopersicum and Vitis vinifera

Fig. 3 Multiple sequence alignment of protein VvSBP4：Vitis vinifera，GSVIVT01003836001；SlySBP4：Solanum lycopersicun，Solyc07g053810；PgSPL14：Punica granatum，Pg029352.1；VvSBP11：Vitis vinifera，GSVIVT01020578001；AtSPL4：Arabidopsis thaliana，AT1G5316；AtSPL5：Arabidopsis thaliana，AT3G15270；VvSBP18：Vitis vinifera，GSVIVT01014302001；PgSPL13：Punica granatum，Pg029123.1；AtSPL3：Arabidopsis thaliana，AT2G33810；PgSPL2：Punica granatum，XP_031375027.1；VvSBP9：Vitis vinifera，GSVIVT01021087001；CNR：Solanum lycopersicun，Solyc02g077920；SlySBP3：Solanum lycopersicun，Solyc10g009080

Fig. 4 PCR amplification of PgSPL2 in pomegranate

Fig. 5 CDS sequence and deduced amino acid sequence of PgSPL2 gene The red box is the SBP conserved domain

Table 2 Effects of growth regulators on pomegranate flo-wer stage

处理种类 Types of processing	初花期 First flowering period/d	盛花期 Blooming period/d	末花期 Final flowering period/d
CK	5-10	5-25	6-20
PP333	5-13	5-29	6-17
6-BA	5-15	5-27	6-15
IBA	5-15	5-29	6-17

Table 3 Effects of growth regulators on complete flowering rate of pomegranate

处理Treatment	完全花比率Complete flower ratio/%
CK	28.64±2.13b
PP333	36.17±2.75a
6-BA	33.43±1.86a
IBA	33.04±1.72a

Fig. 6 Relative expressions of PgSPL2 gene in different tissues of pomegranate and at different developmental stages after different treatments A：Analysis of PgSPL2 expression in different tissues；B：PP333 treatment；C：6-BA treatment；D：IBA treatment. In the illustration (X) and (Y) represent incomplete flowers and complete flowers respectively. Different lowercase letters indicate significant differences at the 0.05 level. The same below

Fig. 7 Detection and performance observation of the PgSPL2 transgenic plants A：The T0 transgenic plants were screened，red arrows indicate the transgenic Arabidopsis plants screened for Kana resistance. B：PCR characterization of overexpressed PgSPL2 Arabidopsis plants. C：The T2 transgenic plants were screened；red arrows indicate the transgenic Arabidopsis plants screened for Kana resistance. D：Phenotype of wild-type versus transgenic Arabidopsis. E：GUS staining of seedlings of T2 transgenic plants. F：GUS staining of the inflorescence of T3 transgenic plants

Fig. 8 PgSPL2 transgenic plants and downstream associated gene expressions

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