桃SERK2 的克隆及其在不同状态愈伤组织中的表达分析

doi:10.3864/j.issn.0578-1752.2019.05.010

摘要/Abstract

摘要：

【目的】分离克隆桃（Prunus persica L.）体细胞胚胎发生相关类受体蛋白激酶（somatic embryogenesis receptor-like kinases,SERKs）基因SERK2 ,检测其在不同状态桃愈伤组织中的表达差异,分析SERK2 与胚性愈伤组织发生的关系,为揭示组织培养困难树种桃胚性愈伤组织产生的分子机理奠定基础。【方法】采用同源克隆法获得PpSERK2 的cDNA全长序列,运用TMPred、DNAMAN和MEGA 5.0等生物信息学软件对序列进行分析;以‘秋蜜红’花药为外植体,接种至添加2.0 mg·L ^-1 6-BA和1.0 mg·L ^-1 2,4-D的NN69培养基诱导愈伤组织;将获得的不同状态愈伤组织制作石蜡切片并进行组织细胞学观察;通过实时荧光定量PCR（qRT-PCR）对PpSERK2 在4种不同状态愈伤组织中的表达进行分析。【结果】克隆获得‘秋蜜红’PpSERK2 的cDNA全长序列1 881 bp,编码626个氨基酸,其蛋白质理论分子量为68.99 kD,理论等电点为5.38,具有完整的SERK蛋白的保守结构域,与牡丹（Paeonia suffruticosa ）等物种的同源性为67.88%—92.71%,且与秘鲁番茄（Solanum peruvianu ）和温州蜜柑（Citrus unshiu ）的相似性最高。在与其他物种的SERK蛋白构建的系统进化树中,PpSERK2与SpSERK1、CitSERK1和PsSERK2等聚在一起,与同源性比对结果一致。以‘秋蜜红’花药为外植体诱导获得4种不同状态的愈伤组织,组织细胞学观察发现黄色疏松状（球形胚出现）、绿色紧实状（心形胚出现）和浅黄色透明状（尚未完全形成的鱼雷形胚结构出现）的3种状态的愈伤组织细胞小、排列紧密;而黄白色水渍状愈伤组织细胞体积大、排列不规则,细胞质稀且染色浅。根据形态学和组织学的鉴定结果,可知黄色疏松状、绿色紧实状和浅黄色透明状的3种状态的愈伤组织均为胚性愈伤组织,而黄白色水渍状愈伤组织为非胚性愈伤组织。实时荧光定量PCR（qRT-PCR）结果表明,PpSERK2 在‘秋蜜红’3种状态的胚性愈伤组织中的表达量显著高于非胚性愈伤组织,且在3种胚性愈伤组织中的表达量有差异,其中在黄色疏松状愈伤组织中表达量最高,绿色紧实状次之,浅黄色透明状中最低。【结论】克隆获得PpSERK2 的cDNA全长序列,其在3种状态的胚性愈伤组织中的表达量明显高于非胚性愈伤组织,且在黄色疏松状胚性愈伤组织中的表达量最高,PpSERK2 在桃体细胞胚发育的早期发挥作用。

关键词: 桃, 体细胞胚发生, SERK2, 胚性愈伤组织

Abstract:

【Objective】The somatic embryogenesis receptor-like kinase 2 (SERK2 ) gene was isolated and cloned from peach (Prunus persica L.). Here, we detected the expression profile of SERK2 in different forms of calli in peach, and analyzed the correlation between SERK2 and the regeneration of embryonic callus. This research will shed on uncovering the molecular mechanism of embryonic callus induction and regeneration in peach with difficulty on tissue culture. 【Method】The full-length cDNA sequence of PpSERK2 gene was obtained by homologous cloning. Then the sequence was analyzed by a series of bioinformatical software packages, including TMPred, DNAMAN and MEGA 5.0 etc. The callus of ‘Qiumihong’ was obtained by using anthers as explants inoculated into NN69 medium supplemented with 2.0 mg·L ^-1 6-BA and 1.0 mg·L ^-1 2,4-D. Then the different forms of calli were observed by production of paraffin section. Real-time quantitative PCR (qRT-PCR) was used to analyze the expression of PpSERK2 gene in four forms of ‘Qiumihong’ calli. 【Result】The full-length cDNA sequence of PpSERK2 gene was 1 881 bp. It encodes 626 amino acids and contains SERK conserved function domains. The theoretical isoelectric point of PpSERK2 is 5.38 and its molecular weight is 68.99 KD. The PpSERK2 has the high homology similarity from 67.88% to 92.71% at protein level, especially the highest similarity to Solanum peruvianu and Citrus unshiu . The phylogenetic analysis of SERK proteins from various plant species indicated that PpSERK2, SpSERK1, CitSERK1 and PsSERK2 were clustered together, which showing the consistent result with above protein similarity analysis. Four different forms of calli were obtained from anther of ‘Qiumihong’. The results of histocytology showed that the cells of three forms of calli, including the yellow, loose callus (global embryo), green, compact callus (heart embryo) and pale yellow, transparent callus (suspected torpedo embryo), were small and closely arranged. While the cells of yellow-white, dropsical callus was large and with irregular shapes. Based on histomorphology results, this demonstrated that the yellow, loose callus, green, compact callus and pale yellow, transparent callus were embryonic calli, while yellow-white, dropsical callus was non-embryonic callus. The results of qRT-PCR showed that transcriptional level of PpSERK2 gene in three forms of embryonic calli was remarkably higher than non-embryonic callus. Meanwhile, the transcriptional level of PpSERK2 gene was highest in the yellow, loose callus, followed by the green, compact callus, and lowest in the pale yellow and transparent callus of ‘Qiumihong’. 【Conclusion】The full-length cDNA of the PpSERK2 gene was successfully obtained. According to the results of PpSERK2 expression in four forms calli of ‘Qiumihong’, we speculated that PpSERK2 gene might play a pivotal role on the early stage during the somatic embryogenesis in peach.

Key words: peach, somatic embryogenesis, SERK2, embryonic callus

谭彬,陈谭星,韩亚萍,张亚如,郑先波,程钧,王伟,冯建灿. 桃SERK2 的克隆及其在不同状态愈伤组织中的表达分析[J]. 中国农业科学, 2019, 52(5): 882-892.

TAN Bin,CHEN TanXing,HAN YaPing,ZHANG YaRu,ZHENG XianBo,CHENG Jun,WANG Wei,FENG JianCan. Cloning and Expression Analysis of SERK2 Gene in Different Forms of Calli on Peach (Prunus persica L.)[J]. Scientia Agricultura Sinica, 2019, 52(5): 882-892.

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参考文献 41

[1]	RAI V R, MCCOMB J .Direct somatic embryogenesis from mature embryos of sandalwood. Plant Cell, Tissue and Organ Culture, 2002,69(1):65-70. doi: 10.1023/A:1015037920529
[2]	吴延军, 徐昌杰, 张上隆 .桃组织培养和遗传转化研究现状及展望. 果树学报, 2002,19(2):123-127. doi: 10.3969/j.issn.1009-9980.2002.02.013
	WU Y J, XU C J, ZHANG S L . Status and prospect of research in peach tissue culture and genetic transformation. Journal of Fruit Science , 2002,19(2):123-127. (in Chinese) doi: 10.3969/j.issn.1009-9980.2002.02.013
[3]	SRINIVASAN C, SCORZA R .The influence of genotype on the induction of somatic embryos fromin vitro cultured zygotic embryos and adventitious shoot regeneration from cotyledons of peach and nectarine. Acta Horticulturae (ISHS) , 2007,738:691-696.
[4]	汤浩茹, 王永清, 任正隆 .核桃体细胞胚发生与转基因研究进展. 林业科学, 2000,36(3):102-110. doi: 10.3321/j.issn:1001-7488.2000.03.017
	TANG H R, WANG Y Q, REN Z L . An overview of progress in somatic embryogenesis and transformation in walnut. Scientia Silvae Sinicae , 2000,36(3):102-110. (in Chinese) doi: 10.3321/j.issn:1001-7488.2000.03.017
[5]	宋跃, 甄成, 张含国, 李淑娟 .长白落叶松胚性愈伤组织诱导及体细胞胚胎发生. 林业科学, 2016,52(10):45-54. doi: 10.11707/j.1001-7488.20161006
	SONG Y, ZHEN C, ZHANG H G, LI S J . Embryogenic callus induction and somatic embryogenesis from immature zygotic embryos ofLarix olgensis . Scientia Silvae Sinicae , 2016,52(10):45-54. (in Chinese) doi: 10.11707/j.1001-7488.20161006
[6]	LI Z X, FAN Y R, DANG S F, LI W F, QI L W, HAN S Y .LaMIR166a -mediated auxin biosynthesis and signalling affect somatic embryogenesis in Larix leptolepis . Molecular Genetics and Genomics , 2018,293(6):1355-1363.
[7]	SEZGIN M,DUMANOĞLU H. Somatic embryogenesis and plant regeneration from immature cotyledons of European chestnut (Castanea sativa Mill.). In Vitro Cellular & Developmental Biology- Plant , 2014,50(1):58-68.
[8]	HOU J Y, WU Y, SHEN Y C, MAO Y J, LIU W B, ZHAO W W, MU Y, LI M H, YANG M L, WU L F .Plant regeneration through somatic embryogenesis and shoot organogenesis from immature zygotic embryos ofSapium sebiferum Roxb. Scientia Horticulturae , 2015,197:218-225. doi: 10.1016/j.scienta.2015.09.040
[9]	KAZMI S K, KHAN S, MIRBAMAR A A, KABIR N .Micropropagation of nucellar embryos and their histological comparative study for regeneration ability with other explants of kinnow mandarin (Citrus reticulata Blanco). Pakistan Journal of Botany , 2018,50(1):345-353.
[10]	JI W, LUO Y X, GUO R R, LI X X, ZHOU Q, MA X H, WANG Y J .Abnormal somatic embryo reduction and recycling in grapevine regeneration. Journal of Plant Growth Regulation, 2017,36(4):912-918. doi: 10.1007/s00344-017-9694-6
[11]	DUTT M, ZAMBON F T, ERPEN L, SORIANO L, GROSSER J .Embryo-specific expression of a visual reporter gene as a selection system for citrus transformation. PLoS ONE, 2018,13(1):e0190413. doi: 10.1371/journal.pone.0190413 pmid: 29293649
[12]	IOCCO P, FRANKS T, THOMAS M R .Genetic transformation of major wine grape cultivars ofVitis vinifera L. Transgenic Research , 2001,10(2):105.
[13]	BOUTILIER K, OFFRINGA R, SHARMA V K, KIEFT H, OUELLET T, ZHANG L, HATTORI J, LIU C M VAN LAMMEREN A A M,MIKI B L A,CUSTERS J B M,VAN LOOKEREN CAMPAGNE M M,. Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth. The Plant Cell, 2002,14(8):1737-1749. doi: 10.1005/Tpc.001941 pmid: 12172019
[14]	STONE S L, KWONG L W, YEE K M, PELLETIER J, LEPINIEC L, FISCHER R L, GOLDBERG R B, HARADA J J .LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development. Proceedings of the National Academy of Sciences of the United States of America , 2001,98(20):11806-11811.
[15]	ZUO J, NIU Q W, FRUGIS G, CHUA N H .The WUSCHEL gene promotes vegetative-to-embryonic transition in Arabidopsis . The Plant Journal , 2002,30(3):349-359.
[16]	SCHMIDT E D L, GUZZO F, TOONEN M A J, DE VRIES S C . A leucine-rich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos. Development, 1997,124(10):2049-2062. doi: 10.1016/S1386-1425(01)00499-1 pmid: 9169851
[17]	HECHT V, VIELLE-CALZADA J P, HARTOG M V, SCHMIDT E D L, BOUTILIER K, GROSSNIKLAUS U, DE VRIES S C. The Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture.Plant Physiology, 2001,127(3):803-816.
[18]	BAUDINO S, HANSEN S, BRETTSCHNEIDER R ,HECHT V F G,DRESSELHAUS T,LÖRZ H,DUMAS C,ROGOWSKY P M. Molecular characterisation of two novel maize LRR receptor-like kinases, which belong to the SERK gene family.Planta, 2001,213(1):1-10. doi: 10.1007/s004250000471 pmid: 11523644
[19]	NOLAN K E, IRWANTO R R, ROSE R J .Auxin up-regulates MtSERK1 expression in both Medicago truncatula root-forming and embryogenic cultures. Plant Physiology , 2003,133(1):218-230.
[20]	THOMAS C, MEYER D, HIMBER C, STEINMETZ A .Spatial expression of a sunflowerSERK gene during induction of somatic embryogenesis and shoot organogenesis. Plant Physiology and Biochemistry , 2004,42(1):35-42. doi: 10.1016/j.plaphy.2003.10.008 pmid: 15061082
[21]	HU H, XIONG L, YANG Y .RiceSERK1 gene positively regulates somatic embryogenesis of cultured cell and host defense response against fungal infection. Planta , 2005,222(1):107-117. doi: 10.1007/s00425-005-1534-4 pmid: 15968510
[22]	SINGH A, KHURANA P .Ectopic expression of Triticum aestivum SERK genes (TaSERKs) control plant growth and development in Arabidopsis . Scientific Reports , 2017,7(1):12368.
[23]	LIU Z J, ZHAO Y P, ZENG L H, ZHANG Y, WANG Y M, HUA J P .Characterization of GhSERK2 and its expression associated with somatic embryogenesis and hormones level in Upland cotton. Journal of Integrative Agriculture , 2018,17(3):517-529. doi: 10.1016/S2095-3119(17)61726-X
[24]	HUANG X, LU X Y, ZHAO J T, ZHAO J T, CHEN J K, DAI X M, XIAO W, CHEN Y P, CHEN Y F, HUANG X L .MaSERK1 Gene expression associated with somatic embryogenic competence and disease resistance response in banana(Musa spp .). Plant Molecular Biology Reporter , 2010,28(2):309-316. doi: 10.1007/s11105-009-0150-z
[25]	AHMADI B, MASOOMI-ALADIZGEH F, SHARIATPANAHI M E, AZADI P, KESHAVARZ-ALIZADEH M .Molecular characterization and expression analysis of SERK1 and SERK2 in Brassica napus L.: Implication for microspore embryogenesis and plant regeneration. Plant Cell Reports , 2016,35(1):185-193. doi: 10.1007/s00299-015-1878-6
[26]	PÉREZNÚÑEZ M T, SOUZA R, SÁENZ L, CHAN J L, ZÚÑIGA-AGUILAR J J, OROPEZA C . Detection of a SERK-like gene in coconut and analysis of its expression during the formation of embryogenic callus and somatic embryos. Plant Cell Reports, 2009,28(1):11-19. doi: 10.1007/s00299-008-0616-8 pmid: 18818928
[27]	DA CUNHA SOARES T, DA SILVA C R C, CARVALHO J M F C, CAVALCANTI J J V, DE LIMA L M, DE ALBUQUERQUE MELO FILHO P, SEVERINO L S, DOS SANTOS R C . Validating a probe from GhSERK1 gene for selection of cotton genotypes with somatic embryogenic capacity. Journal of Biotechnology , 2018,270:44-50.
[28]	王其海, 王裔娜, 刘晓华, 吴文江, 丁宁, 牛洪斌, 王林忠, 吴国良 .桃不同发育时期叶片总RNA提取方法的比较. 河南农业科学, 2014,43(3):116-120. doi: 10.3969/j.issn.1004-3268.2014.03.025
	WANG Q H, WANG Y N, LIU X H, WU W J, DING N, NIU H B, WANG L Z, WU G L . Comparative analysis of total RNA extraction methods for leaves of peach at different development stages. Journal of Henan Agricultural Sciences , 2014,43(3):116-120. (in Chinese) doi: 10.3969/j.issn.1004-3268.2014.03.025
[29]	李和平 .植物显微技术: 第二版. 北京: ALBUQUERQUE科学出版社, 2009.
	LI H P. Plant Microscopy Techniques: 2nd Edition. Beijing: Science Press, 2009. ( in Chinese)
[30]	韩亚萍 .桃体细胞胚发生相关基因SERK2 的克隆与表达分析[D].郑州: 河南农业大学, 2017.
	HAN Y P .Cloning and expression analysis of somatic embryogenesis related gene SERK2 in peach[D].Zhengzhou: Henan Agricultural University, 2017. ( in Chinese)
[31]	LI Y B, LIU C H, GUO G M, HE T, CHEN Z W, GAO R H, XU H W, FAHEEM M, LU R J, HUANG J H .Expression analysis of three SERK-like genes in barley under abiotic and biotic stresses. Journal of Plant Interactions, 2017,12(1):279-285. doi: 10.1080/17429145.2017.1339836
[32]	SHIU S H, BLEECKER A B .Receptor-like kinases fromArabidopsis form a monophyletic gene family related to animal receptor kinases. Proceedings of the National Academy of Sciences of the United States of America , 2001,98(19):10763-10768
[33]	BECRAFT P W .Receptor kinases in plant development. Trends in Plant Science, 1998,3(10):384-388. doi: 10.1016/S1360-1385(98)01301-6
[34]	ZHANG X R .Leucine-rich repeat receptor-like kinases in plants. Plant Molecular Biology Reporter, 1998,16(4):301-311. doi: 10.1023/A:1007540610933
[35]	HAMMERSCHLAG F A, BAUCHAN G, SCOURZA R .Regeneration of peach plant from callus derived from immature embryos. Theoretical and Applied Genetics, 1985,70(3):248-251. doi: 10.1007/BF00304907 pmid: 24252917
[36]	刘航空, 韩明玉, 禹婷, 赵彩平 .影响油桃叶片产生胚性愈伤组织的因素. 果树学报, 2006,23(3):370-374. doi: 10.3969/j.issn.1009-9980.2006.03.012
	LIU H K, HAN M Y, YU T, ZHAO C P . Factors affecting embryonic callus from leaves of early season nectarine cultivars. Journal of Fruit Science , 2006,23(3):370-374. (in Chinese) doi: 10.3969/j.issn.1009-9980.2006.03.012
[37]	鲁娇娇, 严瑞, 何香杉, 靳宏梅, 王锦霞, 王春夏, 孙红梅 .朱顶红‘Red Lion’胚性愈伤组织诱导及体细胞胚发生. 园艺学报, 2016,43(12):2451-2460. doi: 10.16420/j.issn.0513-353x.2016-0495
	LU J J, YAN R, HE X S, JIN H M, WANG J X, WANG C X, SUN H M . The embryonic callus induction and somatic embryogenesis of hippeastrum vittatum‘Red Lion’. Acta Horticulturae Sinica , 2016,43(12):2451-2460. (in Chinese) doi: 10.16420/j.issn.0513-353x.2016-0495
[38]	林丽霞, 屈莹, 徐洋, 林玉玲, 赖钟雄 .龙眼体胚发生过程生长素响应因子DlARF5a 的克隆及表达分析. 西北植物学报, 2014,34(6):1075-1082. doi: 10.7606/j.issn.1000-4025.2014.06.1075
	LIN L X, QU Y, XU Y, LAI Z X . Cloning and expression analysis ofDlARF5a in the process of somatic embryogenesis in Dimocarpus longan Lour. Acta Botanica Boreali-Occidentalia Sinica , 2014,34(6):1075-1082. (in Chinese) doi: 10.7606/j.issn.1000-4025.2014.06.1075
[39]	THOMAS T L .Gene expression during plant embryogenesis and germination: an overview. The Plant Cell, 1993,5(10):1401-1410. doi: 10.2307/3869791 pmid: 8281041
[40]	SCHELLENBAUM P, JACQUES A, MAILLOT P, BERTSCH C, MAZET F, FARINE S, WALTER B .Characterization of VvSERK1 , VvSERK2 , VvSERK3 , and VvL1L , genes and their expression during somatic embryogenesis of grapevine(Vitis vinifera L.). Plant Cell Reports , 2008,27(12):1799-1809.
[41]	OLIVEIRA E J, KOEHLER A D, ROCHA D I, VIEIRA L M , MARQUES PINHEIRO M V, DE MATOS E M, DA CRUZ A C F, FERREIRA DA CRUZ A C, RIBEIRO DA SILVA T C, OSSAMU TANAKA F A, SILVEIRA NOGUEIRA F T, OTONI W C. Morpho-histological, histochemical, and molecular evidences related to cellular reprogramming during somatic embryogenesis of the model grass Brachypodium distachyon . Protoplasma , 2017,254(11):2017-2024. doi: 10.1007/s00709-017-1089-9