森林草莓FveD27的表达特性和功能

doi:10.3864/j.issn.0578-1752.2021.10.013

摘要/Abstract

摘要：

【目的】新型植物激素独脚金内酯是调控植物分枝发育的关键因子,但独脚金内酯在草莓生长发育中的作用尚不清楚。揭示森林草莓（Fragaria vesca）中独脚金内酯生物合成关键基因DWARF27（D27）的表达特性及主要功能,探索FveD27在草莓分枝以及生长发育过程中的作用,为研究草莓植株构型奠定理论基础。【方法】本研究选用森林草莓作为试验材料,利用RT-PCR方法克隆FveD27的编码区序列,利用MEGA 6.0分析草莓FveD27与苹果、拟南芥等物种中D27的系统进化关系;构建FveD27与GFP的融合载体,利用注射烟草叶片的方法对FveD27进行亚细胞定位分析;通过qRT-PCR技术对FveD27在森林草莓不同器官的表达水平进行定量分析,同时构建FveD27启动子与GUS融合表达载体并通过农杆菌介导法将其转化到森林草莓中,利用GUS染色进一步分析FveD27的表达特性;构建FveD27过表达载体并通过农杆菌介导的叶盘法进行稳定遗传转化,获得FveD27过表达草莓株系。【结果】从森林草莓中克隆出编码区长度为789 bp的FveD27序列;烟草的亚细胞定位结果表明FveD27定位在叶绿体;FveD27在森林草莓组织器官中的表达量由高至低依次为幼叶、茎尖、叶柄、成熟叶、根;克隆出长度为1 670 bp的FveD27启动子序列,GUS活性分析结果显示转pFveD27::GUS融合基因草莓植株的幼叶和芽等部位GUS活性较强,而成熟叶与叶柄部位GUS活性较弱,根部几乎无GUS活性,GUS染色分析揭示的基因表达结果与qRT-PCR结果相符;构建了FveD27过表达载体并通过农杆菌介导获得了过表达FveD27的草莓转基因株系,表型调查结果表明过表达FveD27能够显著抑制新茎分枝的形成,同时增加花序数量。【结论】FveD27具有调控森林草莓新茎分枝发育、花序数量等功能。研究结果可为调控草莓新茎分枝数量和产量等提供新思路。

关键词: 森林草莓, 独脚金内酯, D27, 亚细胞定位, 分枝

Abstract:

【Objective】It is known that the new plant hormone strigolactone is a key factor regulating plant branching development, but the role of strigolactone in the growth and development of strawberry plants is unclear. In this study, the expression characteristics and function of the key gene DWARF27 (D27) for strigolactone biosynthesis in woodland strawberry (Fragaria vesca) was revealed, and the role of FveD27 in strawberry branching growth and development was explored, which would lay a theoretical foundation for studying strawberry plant architecture. 【Method】The coding sequence region of strigolactone synthesis key gene FveD27 was cloned by RT-PCR from woodland strawberry. The phylogenetic relationship between FveD27 and D27 in apple, Arabidopsis and other species was analyzed by using MEGA 6.0. A fusion vector of FveD27 and GFP was constructed to inject tobacco leaves for analyzing the subcellular location of FveD27. The expression level of FveD27 in different organs of woodland strawberry was analyzed by qRT-PCR technology. The FveD27 promoter and GUS fusion expression vector was constructed and transformed into woodland strawberry by Agrobacterium-mediated method, and the expression characteristic of FveD27 was further analyzed by GUS staining. The overexpression vector of FveD27 was constructed and the FveD27 overexpression strawberry lines were obtained by Agrobacterium-mediated transformation with leaf disks of woodland strawberry as explants. 【Result】The coding sequence region of FveD27 with the length of 789 bp was cloned from woodland strawberry. The subcellular localization in tobacco showed that FveD27 was located in the chloroplast. The expression levels of FveD27 in woodland strawberry organs from high to low were followed by young leaves, shoot tips, petiole, mature leaves and roots. The FveD27 promoter sequence with a length of 1670 bp was cloned, and transgenic plants with pFveD27::GUS gene showed that the young leaves and buds had strong GUS activity, the mature leaves and petioles had weak GUS activity, while the roots had little GUS activity. The result of FveD27 expression revealed by GUS analysis was consistent with the result by qRT-PCR. The FveD27 overexpression vector was constructed and the transgenic woodland strawberry lines overexpressing FveD27 were obtained with Agrobacterium-mediated transformation. Overexpressing FveD27 in woodland strawberry inhibited significantly the formation of branch crowns and increased the number of inflorescences. 【Conclusion】FveD27 had the functions of regulating the development of branch crowns and the number of inflorescences in woodland strawberry, and this study provided new ideas for regulating the number of branch crowns and yield of strawberry.

Key words: Fragaria vesca, strigolactone, D27, subcellular localization, branching

孙洪影,王岩,李伟佳,朱天姝,姜颖,许妍,吴清悦,张志宏. 森林草莓FveD27的表达特性和功能[J]. 中国农业科学, 2021, 54(10): 2179-2191.

SUN HongYing,WANG Yan,LI WeiJia,ZHU TianShu,JIANG Ying,XU Yan,WU QingYue,ZHANG ZhiHong. Expression Characteristics and Function of FveD27 in Woodland Strawberry[J]. Scientia Agricultura Sinica, 2021, 54(10): 2179-2191.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.10.013

https://www.chinaagrisci.com/CN/Y2021/V54/I10/2179

图/表 13

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表2

FveD27启动子相关顺式作用元件的预测"

顺式作用元件 cis-regulatory element	位置（方向） Position（stand）	序列 Sequence	功能 Function
ACA-motif	247(-)	AATCACAACCATA	（gapA-CMA1）中gapA的一部分涉及光响应性 Part of gapA in (gapA-CMA1) involved with light responsiveness
AE-box	405(-)	AGAAACAA	光响应的元件的一部分 Part of a module for light response
GT1-motif	382(+)	GGTTAA	光响应元件 Light responsive element
I-box	345(-), 986(+)	TGATAATGT	光响应元件的一部分 Part of a light responsive element
LAMP-element	181(+)	CTTTATCA	光响应元件的一部分 Part of a light responsive element
TCT-motif	1159(-)	AACGAC	光响应元件的一部分 Part of a light responsive element
MRE	379(-)	AACCTAA	参与光诱导的MYB绑定位点 MYB binding site involved in light responsiveness
CCAAT-box	881(+)	CAACGG	MYBHv1结合位点 MYBHv1 binding site
MBS	1487(-)	CAACTG	参与干旱诱导的MYB绑定位点 MYB binding site involved in drought-inducibility
GC-motif	920(-)	CCCCCG	参与缺氧特异性诱导性的增强子元件 Enhancer-like element involved in anoxic specific inducibility
ARE	1781(+)	AAACCA	厌氧诱导的必要顺式作用调节元件 cis-acting regulatory element essential for the anaerobic induction
TCA-element	-384(-), -1107(-)	CAGAAAAGGA	水杨酸响应的顺式作用元件 cis-acting element involved in salicylic acid responsiveness
P-box	117(+)	CCTTTTG	赤霉素响应元件 Gibberellin-responsive element
TATC-box	1127(-)	TATCCCA	赤霉素响应的顺式作用元件 cis-acting element involved in gibberellin-responsiveness
TGACG-motif	1406(-), 1413(+)	TGACG	MeJA响应的顺式作用元件 cis-acting regulatory element involved in the MeJA-responsiveness
CGTCA-motif	1406(+), 1413(-)	CGTCA	MeJA响应的顺式作用元件 cis-acting regulatory element involved in the MeJA-responsiveness
TATA-box	188(-)	TATAATAAT	转录起始位点上游-30左右位置的核心启动元件 The core activation element at about -30 upstream of the transcription start site

表2

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参考文献 42

[1]	冯丹, 陈贵林. 独脚金内酯调控侧枝发育的研究进展. 生态学杂志, 2011,30(2):349-356.
	FENG D, CHEN G L. Shoot-branching control with strigolactones: Research progress. Chinese Journal of Ecology, 2011,30(2):349-356. (in Chinese)
[2]	胡盼盼, 张香粉, 赵霞, 李刚, 赵凤莉, 李亮杰, 周厚成. 草莓新茎分枝与独脚金内酯的关系. 果树学报, 2019,36(5):578-589.
	HU P P, ZHANG X F, ZHAO X, LI G, ZHAO F L, LI L J, ZHOU H C. Relation betweenship strigolactones and branching in strawberry. Journal of Fruit Science, 2019,36(5):578-589. (in Chinese)
[3]	GOMEZ-ROLDAN V, FERMAS S, BREWER P B, PUECH-PAGÈS V, DUN E A, PILLOT J P, LETISSE F, MATUSOVA R, DANOUN S, PORTAIS J C, BOUWMEESTER H, BÉCARD G, BEVERIDGE C A, RAMEAU C, ROCHANGE S F. Strigolactone inhibition of shoot branching. Nature, 2008,455:189-194. doi: 10.1038/nature07271
[4]	RAMEAU C. Strigolactones, a novel class of plant hormone controlling shoot branching. Comptes Rendus Biologies, 2010,333(4):344-349. doi: 10.1016/j.crvi.2010.01.012
[5]	DOMAGALSKA M A, LEYSER O. Signal integration in the control of shoot branching. Nature Reviews: Molecular Cell Biology, 2011,12(4):211-221. doi: 10.1038/nrm3088
[6]	ALDER A, HOLDERMANN I, BEYER P, AL-BABILI S. Carotenoid oxygenases involved in plant branching catalyse a highly specific conserved apocarotenoid cleavage reaction. The Biochemical Journal, 2008,416(2):289-296. doi: 10.1042/BJ20080568
[7]	BRUNO M, AL-BABILI S. On the substrate specificity of the rice strigolactone biosynthesis enzyme DWARF27. Planta, 2016,243:1429-1440. doi: 10.1007/s00425-016-2487-5
[8]	YAO R F, LI J Y, XIE D X. Recent advances in molecular basis for strigolactone action. Science China: Life Science, 2018,61:277-284. doi: 10.1007/s11427-017-9195-x
[9]	XIONG G S, WANG Y H, LI J Y. Action of strigolactones in plants. Enzyme, 2014,35:57-84. doi: 10.1159/000469319
[10]	LIN H, WANG R X, QIAN Q, YAN M X, MENG X B, FU Z M, YAN C Y, JIANG B, SU Z, LI J Y, WANG Y H. DWARF27, an iron- containing protein required for the biosynthesis of strigolactones, regulates rice tiller bud outgrowth. Plant Cell, 2009,21(5):1512-1525. doi: 10.1105/tpc.109.065987
[11]	WATERS M T, BREWER P B, BUSSELL J D, SMITH S M, BEVERIDGE C A. The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones. Plant Physiology, 2012,159(3):1073-1085. doi: 10.1104/pp.112.196253
[12]	WEN C, ZHAO Q C, NIE J, LIU G Q, SHEN L, CHENG C X, XI L, MA N, ZHAO L J. Physiological controls of chrysanthemum DgD27 gene expression in regulation of shoot branching. Plant Cell Reports, 2016,35:1053-1070. doi: 10.1007/s00299-016-1938-6
[13]	武亭亭. 小麦独脚金内酯合成相关基因TaDWARF27的分离与功能分析[D]. 泰安: 山东农业大学, 2016.
	WU T T. Isolation and functional analysis of TaDWARF27 in Triticum aestivum L[D]. Tai’an: Shandong Agricultural University, 2016. (in Chinese)
[14]	LIU W, KOHLEN W, LILLO A, CAMP R O D, IVANOV S, HARTOG M, LIMPENS E, JAMIL M, SMACZNIAK C, KAUFMANN K, YANG W C, HOOIVELD G, CHARNIKHOVA T, BOUWMEESTER H J, BISSELING T, GEURTS R. Strigolactone biosynthesis in Medicago truncatula and rice requires the symbiotic GRAS-type transcription factors NSP1 and NSP2. Plant Cell, 2011,23(10):3853-3865. doi: 10.1105/tpc.111.089771
[15]	赵晨晨, 范雅丽, 秦岭, 邢宇, 房克凤, 张卿, 曹庆芹. 森林草莓独脚金内酯合成关键基因D27的克隆与表达分析. 园艺学报, 2016,43(5):975-982.
	ZHAO C C, FAN Y L, QIN L, XING Y, FANG K F, ZHANG Q, CAO Q Q. The cloning and expression analysis of D27 gene of Strigolactone biosynthesis pathway in Fragaria vesca. Acta Horticulture Sinica, 2016,43(5):975-982. (in Chinese)
[16]	吴转娣, 刘新龙, 刘家勇, 昝逢刚, 李旭娟, 刘洪博, 林秀琴, 陈学宽, 苏火生, 赵培方, 吴才文. 甘蔗独脚金内酯生物合成关键基因ScD27的克隆与表达分析. 作物学报, 2017,43(1):31-41.
	WU C D, LIU X L, LIU J Y, ZAN F G, LI X J, LIU H B, LIN X Q, CHEN X K, SU H S, ZHAO P F, WU C W. Cloning and expression analysis of the key gene ScD27 in Strigolactones biosynthesis pathway. Acta Agronomica Sinica, 2017,43(1):31-41. (in Chinese)
[17]	李炎坤. 青天葵独脚金内酯合成关键基因D27的克隆与功能分析[D]. 广州: 广州中医药大学, 2019.
	LI Y K. Cloning and function analysis of D27, the key gene for Strigolactones Synthesis from Nervilia fordii [D]. Guangzhou: Guangzhou University of Chinese Medicine, 2019. (in Chinese)
[18]	WU H, LI H H, CHEN H, QI Q, DING Q Q, XUE J, DING J, JIANG X N, HOU X L, LI Y. Identification and expression analysis of strigolactone biosynthetic and signaling genes reveal strigolactones are involved in fruit development of the woodland strawberry (Fragaria vesca). BMC Plant Biology, 2019,19(1):73. doi: 10.1186/s12870-019-1673-6
[19]	李伟佳. 草莓匍匐茎发生基因的定位克隆与鉴定[D]. 沈阳: 沈阳农业大学, 2018.
	LI W J. Positional cloning and identification of runner producing gene in strawberry[D]. Shenyang: Shenyang Agricultural University, 2018. (in Chinese)
[20]	CHANG L L, ZHANG Z H, YANG H, LI H, DAI H Y. Detection of strawberry RNA and DNA viruses by RT-PCR using total nucleic acid as a template. Journal of Phytopathology, 2007,155(7):431-436. doi: 10.1111/jph.2007.155.issue-7-8
[21]	WANG X Q, SHEN X, HE Y M, REN T T, WU W T, XI T. An optimized freeze-thaw method for transformation of Agrobacterium tumefaciens EHA105 and LBA4404. Pharmaceutical Biotechnology, 2011,18(5):382-386.
[22]	于一帆, 朱小彬, 葛会敏, 陈云. 基于绿色荧光蛋白瞬时表达的植物亚细胞定位方法. 江苏农业科学, 2014,42(12):58-61.
	YU Y F, ZHU X B, GE H M, CHEN Y. Subcellular localization in plants based on transient expression of green fluorescent protein. Jiangsu Agricultural Sciences, 2014,42(12):58-61. (in Chinese)
[23]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method. Methods, 2001,25(4):402-408. doi: 10.1006/meth.2001.1262
[24]	LI Y P, WEI W, FENG J, LUO H F, PI M T, LIU Z C, KANG C Y. Genome reannotation of the wild strawberry Fragaria vesca using extensive Illumina-and SMRT-based RNA-seq datasets. DNA Research, 2017,25(1):61-70. doi: 10.1093/dnares/dsx038
[25]	LI W J, ZHANG J X, SUN H Y, MA Y, LIU Y X, LI H, ZHANG Z H. FveRGA1, encoding a DELLA protein, negatively regulates runner production in Fragaria vesca. Planta, 2018,247(4):941-951. doi: 10.1007/s00425-017-2839-9
[26]	孙洪影. 森林草莓FveD27基因分离鉴定及表达特性分析[D]. 沈阳: 沈阳农业大学, 2019.
	SUN H Y. Identification and expression analysis of FveD27 gene in woodland strawberry[D]. Shenyang: Shenyang Agricultural University, 2019. (in Chinese)
[27]	MOUHU K, KUROKURA T, KOSKELA E A, ALBERT V A, ELOMAA P, HYTÖNEN T. The Fragaria vesca homolog of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 represses flowering and promotes vegetative growth. The Plant Cell, 2013,25(9):3296-3310. doi: 10.1105/tpc.113.115055
[28]	BREWER P B, DUN E A, FERGUSON B J, RAMEAU C, BEVERIDGE C A. Strigolactone acts downstream of auxin to regulate bud outgrowth in pea and Arabidopsis. Plant Physiology, 2009,150:482-493. doi: 10.1104/pp.108.134783
[29]	贾昆鹏. 植物激素独脚金内酯和茉莉酸信号与光信号互作的分子机制研究[D]. 上海: 上海交通大学, 2014.
	JIA K P. The molecular mechanism of cross-talking between light and phytohormones Strigolactone- and Jasmonate-signaling[D]. Shanghai: Shanghai Jiao Tong University, 2014. (in Chinese)
[30]	ZHANG J, MAZUR E, BALLA J, GALLEI M, KALOUSEK P, MEDVEĎOVÁ Z, LI Y, WANG Y P, PRÁT T, VASILEVA M, REINÖH V, PROCHÁZKA S, HALOUZKA R, TARKOWSKI P, LUSCHNIG C, BREWER P B, FRIML J. Strigolactones inhibit auxin feedback on PIN-dependent auxin transport canalization. Nature Communications, 2020,11(1):3508. doi: 10.1038/s41467-020-17252-y
[31]	王硕, 葛秀秀. 植物分枝性状研究进展. 生物技术进展, 2017,7(2):98-101.
	WANG S, GE X X. Progress on plant branch characters. Current Biotechnology, 2017,7(2):98-101. (in Chinese)
[32]	DOMAGALSKA M A, LEYSER O. Signal integration in the control of shoot branching. Nature Reviews: Molecular Cell Biology, 2011,12(4):211-221. doi: 10.1038/nrm3088
[33]	黎家, 李传友. 新中国成立70年来植物激素研究进展. 中国科学: 生命科学, 2019,49(10):1227-1281.
	LI J, LI C Y. Seventy-year major research progress in plant hormones by Chinese scholars. Chinese Science: Life Science, 2019,49(10):1227-1281. (in Chinese)
[34]	FERNIE A R. Resolving the role of strigolactone in the early steps of rice axillary bud dormancy. Plant Journal, 2019,97:1003-1005. doi: 10.1111/tpj.2019.97.issue-6
[35]	LUO L, TAKAHASHI M, KAMEOKA H, QIN, R Y, SHIGA T, KANNO Y, SEO M, ITO M, XU G H, KYOZUKA J. Developmental analysis of the early steps in strigolactone-mediated axillary bud dormancy in rice. Plant Journal, 2019,97(6):1006-1021. doi: 10.1111/tpj.2019.97.issue-6
[36]	DUAN J B, YU H, YUAN K, LIAO Z G, MENG X B, JING Y H, LIU G F, CHU J F, LI J Y. Strigolactone promotes cytokinin degradation through transcriptional activation of CYTOKININ OXIDASE/DEHYDROGENASE 9 in rice. Proceedings of the National Academy of Sciences, 2019,116(28):14319-14324.
[37]	WANG L, XU Q, YU H, MA H Y, LI X Q, YANG J, CHU J F, XIE Q, WANG Y H, SMITH S M, LI J Y, XIONG G X, WANG B. Strigolactone and karrikin signaling pathways elicit ubiquitination and proteolysis of SMXL2 to regulate hypocotyl elongation in Arabidopsis thaliana. The Plant Cell, 2020,32(7):2251-2270. doi: 10.1105/tpc.20.00140
[38]	HA C V, LEYVA-GONZÁLEZ M A, OSAKABE Y, TRAN U T, NISHIYAMA R, WATANABE Y, TANAKA M, SEKI M, YAMAGUCHI S, DONG N V, YAMAGUCHI-SHINOZAKI K, SHINOZAKI K, HERRERA-ESTRELLA L, TRAN L S P. Positive regulatory role of strigolactone in plant responses to drought and salt stress. Proceedings of the National Academy of Sciences, 2014,111(2):851-856.
[39]	AL-BABILI S, BOUWMEESTER H J. Strigolactones, a novel carotenoid-derived plant hormone. Annual Review of Plant Biology, 2015,66:161-186. doi: 10.1146/annurev-arplant-043014-114759
[40]	WATERS M T, GUTJAHR C, BENNETT T, NELSON D C. Strigolactone signaling and evolution. Annual Review of Plant Biology, 2017,68:291-322. doi: 10.1146/annurev-arplant-042916-040925
[41]	LI X Y, QIAN Q, FU Z M, WANG Y H, XIONG G S, ZENG D, WANG X Q, LIU X F, TENG S, HIROSHI F, YUAN M, LUO D, HAN B, LI J Y. Control of tillering in rice. Nature, 2003,422(6932):618-621. doi: 10.1038/nature01518
[42]	CHALFUN-JUNIOR A, FRANKEN J, MES J J, MARSCH- MARTINEZ N, PEREIRA A, ANGENENT G C. ASYMMETRIC LEAVES2-LIKE1 gene, a member of the AS2/LOB family, controls proximal-distal patterning in Arabidopsis petals. Plant Molecular Biology, 2005,57(4):559-575. doi: 10.1007/s11103-005-0698-4

引物 Primer	序列（5′→3′） Sequence（5′→3′）	目的 Purpose
FveD27-F FveD27-R	ACGCGTCGACATGGAAGCATCACATTTCT CGCGGATCCCTAACTGGAGCAGTTAT	基因全长克隆 Full-length gene cloning
FveD27-DL-F FveD27-DL-R	AGCAGAATAAGACCGGCAGA TGCAGCTTGGACATTTTGAG	实时荧光定量 Real-time PCR
Fve-26S-F Fve-26S-R	TAACCGCATCAGGTCTCCAA CTCGAGCAGTTCTCCGACAG	内参基因 Reference gene
FveD27(eGFP)-F FveD27(eGFP)-R	TCCCCCGGGATGGAAGCATCACATTT CGGGGTACCACTGGAGCAGTTATTGT	亚细胞定位 Subcellular localization
proFveD27-F proFveD27-R	GCTGCAGGCTGATTCCTAAGGGTATC TGCTCTAGAATGCATGGTGGGGAATTTAAAG	启动子克隆 Promoter cloning