石榴花发育相关基因PgSPL2的克隆及功能研究

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

摘要/Abstract

摘要：

探究石榴SPL家族基因PgSPL2（XP_031375027.1）在石榴花发育中的作用，为进一步研究石榴花发育的机制奠定基础。以‘突尼斯’软籽石榴为试材，采用同源克隆技术克隆SPL家族基因PgSPL2，分析该基因的理化性质，以及在石榴不同组织中的表达差异，利用RT-qPCR分析生长调节剂处理时该基因的时空表达特性，转化拟南芥，验证该基因在花发育中的作用。结果表明，该基因包含一个长为591 bp开放阅读框，编码196个氨基酸，多重序列比对发现，该基因含有一个保守的SBP结构域和核定位信号，属于SBP转录因子基因家族；系统进化树分析表明，PgSPL2与拟南芥AtSPL3、AtSPL4和AtSPL5聚为一簇。RT-qPCR结果表明，PgSPL2在果实和花蕾中的表达量相对较高，且生长调节剂喷施处理后，该基因在完全花中表达显著下调，同时，石榴完全花比率同步显著提高，初花、盛花期延迟。过量表达PgSPL2拟南芥出现早花现象，花序下游调控基因表达量显著提高。PgSPL2在石榴花发育中起调控开花时间的功能。

关键词: 石榴, SPL, 生长调节剂, 基因克隆, 基因表达

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

甘诚燕, 张心慧, 王沙, 樊瑶羽薇, 招雪晴, 苑兆和. 石榴花发育相关基因PgSPL2的克隆及功能研究[J]. 生物技术通报, 2022, 38(12): 194-203.

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.

图/表 11

图1 石榴不同发育阶段的不完全花（a）与完全花（b） P1：花蕾纵径3.0-5.0 mm；P2：花蕾纵径5.1-13.0 mm；P3：花蕾纵径13.1-25.0 mm

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

表1 基因克隆、功能验证以及特异性表达分析引物表

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

图2 石榴、拟南芥、番茄和葡萄SPL系统进化分析

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

图3 蛋白多重序列比对 VvSBP4：葡萄，GSVT01003836001；SlySBP4：番茄，Solyc07g053810；PgSPL14：石榴，XP_031389713.1；VvSBP11：葡萄，GSVIVT01020578001；AtSPL4：拟南芥，AT1G53160；AtSPL5：拟南芥，AT3G15270；VvSBP18：葡萄，GSVIVT01014302001；PgSPL13：石榴，XP_031394973.1；AtSPL3：拟南芥，AT2G33810；PgSPL2：石榴，XP_031375027.1；VvSBP9：葡萄，GSVIVT01021087001；CNR：番茄，Solyc02g077920；SlySBP3：番茄，Solyc10g009080

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

图4 石榴PgSPL2的PCR扩增

Fig. 4 PCR amplification of PgSPL2 in pomegranate

图5 PgSPL2的CDS序列及其编码氨基酸序列红色框为SBP保守结构域

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

表2 生长调节剂对石榴花期的影响

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

表3 生长调节剂对石榴完全花成花率的影响

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

图6 PgSPL2在石榴不同组织及不同处理后不同发育阶段的相对表达量 A：PgSPL2在不同组织中的表达分析；B：PP333处理；C：6-BA处理；D：IBA处理。图例中(X)、(Y)分别代表不完全花与完全花。不同小写字母表示在0.05水平差异显著。下同

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

图7 PgSPL2转基因植株的检测与表型 A：T0转基因植株筛选，红色箭头指示为Kana抗性筛选出来的转基因拟南芥植株；B：过量表达PgSPL2拟南芥植株的PCR鉴定；C：T2转基因植株筛选，红色箭头指示为Kana抗性筛选出来的转基因拟南芥植株；D：野生型与转基因拟南芥的表型；E：T2转基因植株幼苗的GUS染色；F：T3转基因植株花序的GUS染色

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

图8 PgSPL2转基因植株与下游相关基因的表达量

Fig. 8 PgSPL2 transgenic plants and downstream associated gene expressions

参考文献 29

[1]	Klein J, Saedler H, Huijser P. A new family of DNA binding proteins includes putative transcriptional regulators of the Antirrhinum majus floral meristem identity gene SQUAMOSA[J]. Mol Gen Genet, 1996, 250(1):7-16.
[2]	Birkenbihl RP, Jach G, Saedler H, et al. Functional dissection of the plant-specific SBP-domain:overlap of the DNA-binding and nuclear localization domains[J]. J Mol Biol, 2005, 352(3):585-596. pmid: 16095614
[3]	田晶, 赵雪媛, 谢隆聖, 等. SPL转录因子调控植物花发育及其分子机制研究进展[J]. 南京林业大学学报:自然科学版, 2018, 42(3):159-166.
	Tian J, Zhao XY, Xie LS, et al. Research advances and molecular mechanism on SPL transcription factors in regulating plant flower development[J]. J Nanjing For Univ Nat Sci Ed, 2018, 42(3):159-166.
[4]	Yamaguchi A, Wu MF, Yang L, et al. The microRNA-regulated SBP-Box transcription factor SPL3 is a direct upstream activator of LEAFY, FRUITFULL, and APETALA1[J]. Dev Cell, 2009, 17(2):268-278. doi: 10.1016/j.devcel.2009.06.007 pmid: 19686687
[5]	Wang JW, Czech B, Weigel D. miR156-regulated SPL transcription factors define an endogenous flowering pathway in Arabidopsis thaliana[J]. Cell, 2009, 138(4):738-749. doi: 10.1016/j.cell.2009.06.014 URL
[6]	Hou H, Jia H, Yan Q, et al. Overexpression of a SBP-box gene(VpS-BP16)from Chinese wild Vitis species in Arabidopsis improves sal-inity and drought stress tolerance[J]. Int J Mol Sci, 2018, 19(4):E940.
[7]	Preston JC, Hileman LC. SQUAMOSA-PROMOTER BINDING PROTEIN 1 initiates flowering in Antirrhinum majus through the activation of meristem identity genes[J]. Plant J, 2010, 62(4):704-712. doi: 10.1111/j.1365-313X.2010.04184.x URL
[8]	陈晓博. 参与番茄花柄离区发育的转录因子SPL3的基因功能研究[D]. 北京: 中国农业科学院, 2010.
	Chen XB. Functional study of a transcription factor Squamosa promoter binding protein like 3 in tomato flower abscission zone development[D]. Beijing: Chinese Academy of Agricultural Sciences, 2010.
[9]	Yuan ZH, Fang YM, Zhang TK, et al.The pomegranate(Punica granatum L. )genome provides insights into fruit quality and ovule developmental biology[J]. Plant Biotechnol J, 2018, 16(7):1363-1374. doi: 10.1111/pbi.12875 URL
[10]	曹尚银, 谭洪花, 刘丽, 等. 中国石榴栽培历史、生产与科研现状及产业化方向[C]// 中国石榴研究进展(一). 北京: 中国农业出版社, 2010.
	Cao SY, Tan HH, Liu L, et al. Chinese history, status of production and scientific research, industrialization orientation of Chinese pomegranate[C]// Research Progress on China Pomegranate(Ⅰ). Beijing: China Agricultural Publishing House, 2010.
[11]	Wetzstein HY, Ravid N, Wilkins E, et al. A morphological and histological characterization of bisexual and male flower types in pomegranate[J]. J Amer Soc Hort Sci, 2011, 136(2):83-92. doi: 10.21273/JASHS.136.2.83 URL
[12]	Holland D, Hatib K, Bar-Ya’akov I. Pomegranate:botany, horticulture, breeding[M]// Horticultural Reviews. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2009:127-191.
[13]	赵静, 王昱, 何倩茹. 石榴生长主要物候期气象服务指标分析[J]. 现代农业科技, 2015(19):263-264, 266.
	Zhao J, Wang Y, He QR. Analysis of the meteorological service index during the main phenological period of pomegranate growth[J]. Mod Agric Sci Technol, 2015(19):263-264, 266.
[14]	李绍稳, 张如锋, 钟家煌, 等. 石榴花芽形态分化的研究[J]. 中国南方果树, 1996(3):39-41.
	Li SW, Zhang RF, Zhong JH, et al. Study on the morphological differentiation of flower bud of pomegranate[J]. South China Fruits, 1996(3):39-41.
[15]	Guerriero R, Scalabrelli G, Fiocchi C. Influence of light and chilling conditions on apricot bud opening[J]. Acta Hortic, 1991(293):327-330.
[16]	马翠云. 梨花器官冻害生理机理的研究[D]. 南京: 南京农业大学, 2013.
	Ma CY. The study on the physiological mechanism of frost injury to apricot floral organs[D]. Nanjing: Nanjing Agricultural University, 2013.
[17]	赵莲花, 王富河, 夏玉宝, 等. PBO等4种生长调节剂对石榴坐果和产量的影响[C]// 中国石榴研究进展(一). 北京: 中国农业出版社, 2010: 216-218.
	Zhao LH, Wang FH, Xia YB, et al. The influence on set fruit and output of pomegranate on four plant growth regulators[C]// Research Progress on China Pomegranate(I). Beijing:China Agricultural Publishing House, 2010:216-218.
[18]	宋雪娜. 6个苹果品种花期冻害调查及抗冻性分析[D]. 杨凌: 西北农林科技大学, 2019.
	Song XN. Investigation of freezing injury and freezing resistance of six varieties during apple florescence[D]. Yangling: Northwest A & F University, 2019.
[19]	陈利娜. 石榴花雌蕊败育相关microRNAs发掘与pg-miR166a-3p调控雌蕊发育的功能分析[D]. 北京: 中国农业科学院, 2020.
	Chen LN. Exploration of microRNAs related to pistil abortion in pomegranate flowers and functional analysis of Pg-miR166a-3p regulating pistil development[D]. Beijing: Chinese Academy of Agricultural Sciences, 2020.
[20]	Kim JJ, Lee JH, Kim W, et al. The microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 module regulates ambient temperature-responsive flowering via FLOWERING LOCUS T in Arabidopsis[J]. Plant Physiology, 2012, 159(1):461-478. doi: 10.1104/pp.111.192369 pmid: 22427344
[21]	侯鸿敏. 葡萄转录因子SBP家族基因克隆、表达及功能分析[D]. 杨凌: 西北农林科技大学, 2013.
	Hou HM. Cloninq expression and function analysis of the SBP-box family genes in grape[D]. Yangling: Northwest A & F University, 2013.
[22]	Schwarz S, Grande AV, Bujdoso N, et al. The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis[J]. Plant Mol Biol, 2008, 67(1/2):183-195. doi: 10.1007/s11103-008-9310-z URL
[23]	陈文文, 吴怀通, 陈赢男. SPL家族基因复制及功能分化分析[J]. 南京林业大学学报:自然科学版, 2020, 44(5):55-66.
	Chen WW, Wu HT, Chen YN. Gene duplications and functional divergence analyses of the SPL gene family[J]. J Nanjing For Univ Nat Sci Ed, 2020, 44(5):55-66.
[24]	孔佑涵, 苑平, 谢新华, 等. 多效唑对突尼斯软籽石榴开花结实的影响[J]. 湖南农业科学, 2017(3):10-12.
	Kong YH, Yuan P, Xie XH, et al. Effects of paclobutrazol on the flowering and the fruiting ability of‘Tunisiruanzi’ pomegranate(Punica granatum)[J]. Hunan Agric Sci, 2017(3):10-12.
[25]	Pan BZ, Xu ZF. Benzyladenine treatment significantly increases the seed yield of the biofuel plant Jatropha curcas[J]. J Plant Growth Regul, 2011, 30(2):166-174. doi: 10.1007/s00344-010-9179-3 URL
[26]	Villar L, Lienqueo I, Llanes A, et al. Comparative transcriptomic analysis reveals novel roles of transcription factors and hormones during the flowering induction and floral bud differentiation in sweet cherry trees(Prunus avium L. cv. Bing)[J]. PLoS One, 2020, 15(3):e0230110. doi: 10.1371/journal.pone.0230110 URL
[27]	冯贝贝. 欧洲李生长发育生物学特性研究[D]. 乌鲁木齐: 新疆农业大学, 2017.
	Feng BB. Biological characteristics of growth and development of Prunus domestica L[D]. Urumqi: Xinjiang Agricultural University, 2017.
[28]	孙云才. 提高果树树体营养促进花芽分化[J]. 果树实用技术与信息, 2011(6):18.
	Sun YC. Improve nutrition of fruit trees and promote flower bud differentiation[J]. Guoshu Shiyong Jishu Yu Xinxi, 2011(6):18.
[29]	Jung JH, Lee HJ, Ryu JY, et al. SPL3/4/5 integrate developmental aging and photoperiodic signals into the FT-FD module in Arabidopsis flowering[J]. Mol Plant, 2016, 9(12):1647-1659. doi: 10.1016/j.molp.2016.10.014 URL