Cloning and Expression Analysis of BoRALFA1 Gene in Brassica oleracea

GAO Qi-guo; LI Shuai; YAO Zhi-guang; LIANG Xiao

doi:10.13718/j.cnki.xdzk.2020.11.011

2020 Volume 42 Issue 11

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GAO Qi-guo, LI Shuai, YAO Zhi-guang, et al. Cloning and Expression Analysis of BoRALFA1 Gene in Brassica oleracea[J]. Journal of Southwest University Natural Science Edition, 2020, 42(11): 95-101. doi: 10.13718/j.cnki.xdzk.2020.11.011

Citation:

GAO Qi-guo, LI Shuai, YAO Zhi-guang, et al. Cloning and Expression Analysis of BoRALF_A1 Gene in Brassica oleracea[J]. Journal of Southwest University Natural Science Edition, 2020, 42(11): 95-101. doi: 10.13718/j.cnki.xdzk.2020.11.011

Cloning and Expression Analysis of BoRALF_A1 Gene in Brassica oleracea

School of Horticulture and Landscape Architecture, Southwest University/Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education, Chongqing 400715, China

More Information

Received Date: 30/03/2020
Available Online: 20/11/2020
MSC: Q786;635.1

Abstract

A rapid alkalinization factor (RALF) gene, named as BoRALF_A1, has been cloned from the self-incompatible 'A1' line of Brassica oleracea based on our former transcriptome analysis result. BoRALF_A1 has no intron, its full length of CDS is 240 bp, encoding 79 amino acids. It contains 1 signaling peptide and 5 post-translational modification sites. The mature BoRALF_A1 peptide comprises four highly conserved cysteine residues characteristic of the RALF family, a conservative YIXY motif, a YXRGC motif and a RCG motif. BoRALF_A1 gene is mainly expressed in pollen. Homologous sequence analysis shows that their sequences are highly conserved in Brassica species, and their expression patterns are identical. It is speculated that BoRALF_A1 protein may function in pollen development and pollen fertility.
- Brassica oleracea,
- BoRALF_A1,
- homologous gene,
- sequence analysis,
- expression analysis

References

[1]	MURPHY E, SMITH S, DE SMET I. Small Signaling Peptides in Arabidopsis Development: How Cells Communicate over a Short Distance [J]. The Plant Cell, 2012, 24(8): 3198-3217. doi: 10.1105/tpc.112.099010 CrossRef Google Scholar
[2]	CZYZEWICZ N, YUE K, BEECKMAN T, et al. Message in a Bottle: Small Signalling Peptide Outputs during Growth and Development [J]. Journal of Experimental Botany, 2013, 64(17): 5281-5296. doi: 10.1093/jxb/ert283 CrossRef Google Scholar
[3]	PEARCE G, MOURA D S, STRATMANN J, et al. RALF, a 5-kDa Ubiquitous Polypeptide in Plants, Arrests Root Growth and Development [J]. PNAS, 2001, 98(22): 12843-12847. doi: 10.1073/pnas.201416998 CrossRef Google Scholar
[4]	CAMPBELL L, TURNER S R. A Comprehensive Analysis of RALF Proteins in Green Plants Suggests there are Two Distinct Functional Groups [J]. Frontiers in Plant Science, 2017, 8: 37. DOI: 10. 3389/fpls. 2017. 00037. CrossRef Google Scholar
[5]	WU J S, KURTEN E L, MONSHAUSEN G, et al. NaRALF, a Peptide Signal Essential for the Regulation of Root Hair Tip Apoplastic pH in Nicotiana attenuata, is Required for Root Hair Development and Plant Growth in Native Soils [J]. The Plant Journal, 2007, 52(5): 877-890. doi: 10.1111/j.1365-313X.2007.03289.x CrossRef Google Scholar
[6]	COMBIER J P, KVSTER H, JOURNET E P, et al. Evidence for the Involvement in Nodulation of the Two Small Putative Regulatory Peptide-Encoding Genes MtRALFL1 and MtDVL1 [J]. Molecular Plant-Microbe Interactions^®, 2008, 21(8): 1118-1127. doi: 10.1094/MPMI-21-8-1118 CrossRef Google Scholar
[7]	MINGOSSI F B, MATOS J L, RIZZATO A P, et al. SacRALF1, a Peptide Signal from the Grass Sugarcane (Saccharum SPP.), is Potentially Involved in the Regulation of Tissue Expansion [J]. Plant Molecular Biology, 2010, 73(3): 271-281. doi: 10.1007/s11103-010-9613-8 CrossRef Google Scholar
[8]	ATKINSON N J, LILLEY C J, URWIN P E. Identification of Genes Involved in the Response of Arabidopsis to Simultaneous Biotic and Abiotic Stresses [J]. Plant Physiology, 2013, 162(4): 2028-2041. doi: 10.1104/pp.113.222372 CrossRef Google Scholar
[9]	MURPHY E, VU L D, VAN DEN BROECK L, et al. RALFL34 Regulates Formative Cell Divisions in Arabidopsis Pericycle during Lateral Root Initiation [J]. Journal of Experimental Botany, 2016, 67(16): 4863-4875. doi: 10.1093/jxb/erw281 CrossRef Google Scholar
[10]	STEGMANN M, MONAGHAN J, SMAKOWSKA-LUZAN E, et al. The Receptor Kinase FER is a RALF-regulated Scaffold Controlling Plant Immune Signaling [J]. Science, 2017, 355(6322): 287-289. doi: 10.1126/science.aal2541 CrossRef Google Scholar
[11]	COVEY P A, SUBBAIAH C C, PARSONS R L, et al. A Pollen-specific RALF from Tomato that Regulates Pollen Tube Elongation [J]. Plant Physiology, 2010, 153(2): 703-715. doi: 10.1104/pp.110.155457 CrossRef Google Scholar
[12]	MORATO DO CANTO A, CECILIATO P H O, RIBEIRO B, et al. Biological Activity of Nine Recombinant AtRALF Peptides: Implications for Their Perception and Function in Arabidopsis [J]. Plant Physiology and Biochemistry, 2014, 75: 45-54. doi: 10.1016/j.plaphy.2013.12.005 CrossRef Google Scholar
[13]	GE Z X, BERGONCI T, ZHAO Y L, et al. Arabidopsis Pollen Tube Integrity and Sperm Release are Regulated by RALF-mediated Signaling [J]. Science, 2017, 358(6370): 1596-1600. doi: 10.1126/science.aao3642 CrossRef Google Scholar
[14]	SHI F Y, ZHOU X, LIU Z Y, et al. Rapid Alkalinization Factor (RALF) Genes are Related to Genic Male Sterility in Chinese Cabbage (Brassica rapa L. SSP. pekinensis) [J]. Scientia Horticulturae, 2017, 225: 480-489. doi: 10.1016/j.scienta.2017.07.041 CrossRef Google Scholar
[15]	高启国, 雷镇泽, 梁云飞, 等.基于家族基因分析的甘蓝MLPK互作PUB蛋白的筛选[J].园艺学报, 2019, 46(4): 714-722. Google Scholar
[16]	GAO Q G, SHI S M, LIU Y D, et al. Identification of a Novel MLPK Homologous Gene MLPKn1 and Its Expression Analysis in Brassica oleracea [J]. Plant Reproduction, 2016, 29(3): 239-250. doi: 10.1007/s00497-016-0287-5 CrossRef Google Scholar
[17]	WANG X, WANG H, WANG J, et al. The genome of the mesopolyploid crop species Brassica rapa [J]. Nat Genet, 2011, 43: 1035-1039. doi: 10.1038/ng.919 CrossRef Google Scholar
[18]	SHARMA A, HUSSAIN A, MUN B G, et al. Comprehensive Analysis of Plant Rapid Alkalization Factor (RALF) Genes [J]. Plant Physiology and Biochemistry, 2016, 106: 82-90. doi: 10.1016/j.plaphy.2016.03.037 CrossRef Google Scholar
[19]	FREDERICK R O, HARUTA M, TONELLI M, et al. Function and Solution Structure of the Arabidopsis thaliana RALF₈ Peptide [J]. Protein Science, 2019, 28(6): 1115-1126. doi: 10.1002/pro.3628 CrossRef Google Scholar
[20]	PEARCE G, YAMAGUCHI Y, MUNSKE G, et al. Structure-Activity Studies of RALF, Rapid Alkalinization Factor, Reveal an Essential-YISY-Motif [J]. Peptides, 2010, 31(11): 1973-1977. doi: 10.1016/j.peptides.2010.08.012 CrossRef Google Scholar
[21]	XIAO Y, STEGMANN M, HAN Z F, et al. Mechanisms of RALF Peptide Perception by a Heterotypic Receptor Complex [J]. Nature, 2019, 572(7768): 270-274. doi: 10.1038/s41586-019-1409-7 CrossRef Google Scholar
[22]	HARUTA M, CONSTABEL C P. Rapid Alkalinization Factors in Poplar Cell Cultures. Peptide Isolation, cDNA Cloning, and Differential Expression in Leaves and Methyl Jasmonate-Treated Cells [J]. Plant Physiology, 2003, 131(2): 814-823. Google Scholar
[23]	SRIVASTAVA R, LIU J X, GUO H Q, et al. Regulation and Processing of a Plant Peptide Hormone, AtRALF23, in Arabidopsis [J]. The Plant Journal, 2009, 59(6): 930-939. doi: 10.1111/j.1365-313X.2009.03926.x CrossRef Google Scholar
[24]	李焰焰, 刘瑞娇, 范亚丽, 等.油菜花粉发育相关基因RALFbn的克隆与表达分析[J].西北植物学报, 2013, 33(2): 235-239. Google Scholar
[25]	ZHANG G Y, WU J, WANG X W. Cloning and Expression Analysis of a Pollen Preferential Rapid Alkalinization Factor Gene, BoRALF1, from Broccoli Flowers [J]. Molecular Biology Reports, 2010, 37(7): 3273-3281. doi: 10.1007/s11033-009-9912-9 CrossRef Google Scholar
[26]	MECCHIA M A, SANTOS-FERNANDEZ G, DUSS N N, et al. RALF4/19 Peptides Interact with LRX Proteins to Control Pollen Tube Growth in Arabidopsis [J]. Science, 2017, 358(6370): 1600-1603. doi: 10.1126/science.aao5467 CrossRef Google Scholar
[27]	HARUTA M, SABAT G, STECKER K, et al. A Peptide Hormone and Its Receptor Protein Kinase Regulate Plant Cell Expansion [J]. Science, 2014, 343(6169): 408-411. doi: 10.1126/science.1244454 CrossRef Google Scholar
[28]	WANG L, YANG T, LIN Q L, et al. Receptor Kinase FERONIA Regulates Flowering Time in Arabidopsis [J]. BMC Plant Biology, 2020, 20(1): 26. doi: 10.1186/s12870-019-2223-y CrossRef Google Scholar

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小分子分泌型多肽是重要的胞间信号分子，广泛地参与调节植物生长发育、抗病和抗逆等生理过程^[1-2].快速碱化因子(Rapid alkalinization factor，RALF)属于植物小分子多肽的一种，最早是由Pearce等^[3]从烟草叶片提取物中发现，该蛋白能引起烟草悬浮细胞的培养基快速碱化，过表达时能抑制番茄和拟南芥根的生长和发育.至今已在51种植物中鉴定出795个RALF基因^[4].研究表明烟草NaRALF基因沉默后主根更长且毛状体细胞发育成不正常的根毛^[5].蒺藜苜蓿根中过表达MtRALF1基因时结节发育不正常^[6].在细胞悬浮培养基中加入纯化的甘蔗SacRALF1蛋白能抑制愈伤组织细胞的延伸，在培养基加入AtRALF1导致黑暗条件下萌发的拟南芥幼苗下胚轴伸长受到抑制，幼苗放到没有AtRALF1的培养基时下胚轴的伸长得到恢复^[7].拟南芥中过表达AtRALK8基因可以显著增强植株抗干旱和线虫侵染的能力^[8].拟南芥AtRALF34调控中柱鞘形成细胞的分化进而影响侧根发育^[9].拟南芥AtS1P切割内源的AtRALF23以抑制植物免疫响应，而AtFER可以促进配体诱导的AtEFR，AtFLS2和AtBAK1形成复合体进而激活免疫信号^[10].

在生殖发育方面，人工合成的番茄SlPRALF蛋白能抑制花粉管的伸长，但不影响花粉水化和花粉活力^[11].拟南芥AtRALF4可强烈抑制花粉萌发^[12].拟南芥花粉AtRALF4/19与BUPS1/2和ANX1/2形成复合体维持花粉管的完整性，柱头AtRALF34与AtRALF4/19竞争性结合BUPS-ANX复合体，在纳摩尔水平就可以引起花粉管破裂^[13]. Shi等^[14]从白菜中鉴定出38个RALF基因，有14个在核雄性不育和可育性材料间差异表达.前期笔者通过甘蓝转录组测序从自交不亲和甘蓝“A1”花粉中获取了一个RALF基因的编码序列，命名为BoRALF_A1，在甘蓝02-12系基因组CDS数据库(http://brassicadb.org/brad/)中检索不到同源序列.本文从甘蓝“A1”克隆了BoRALF_A1的CDS序列，进行了生物信息学分析，对BoRALF_A1及其同源基因组织表达模式进行了分析，以期为深入研究的BoRALF_A1基因的生物学功能提供基础.

1. 材料与方法

1.1. 供试材料

甘蓝高度自交不亲和系“A1”种植在西南大学十字花科歇马实验基地隔离网内. 2019年3月底甘蓝花期，采集开花当天且长势一致的柱头、花粉、萼片、花瓣和叶片，置于-80 ℃冰箱保存备用.

利用RNAprep Pure Plant kit(天根，北京)提取RNA，经琼脂糖电泳检测RNA的完整性，用NanoVue Plus超微量分光光度计(Gen，美国)测定RNA的浓度，然后采用Primescript RT Reagent Kit(TaKaRa，日本)进行反转录合成cDNA的第一条链，-80 ℃冰箱保存备用.以叶片为材料，利用Plant Genomic DNA Kit(天根，北京)提取甘蓝基因组DNA，-20 ℃冰箱保存备用.

1.2. 引物设计和目的基因克隆

利用前期甘蓝转录组测序获得的BoRALF_A1序列在NCBI(http://www.ncbi.nlm.nih.gov/)和BRAD(http://brassicadb.org/brad/)数据库进行BlastN检索，依据检索结果用Primer Primer 5.0软件设计基因特异性引物RALF-1和RALF-2. RALF-1的序列为ATTAATAATAATATGGGGATGTCTGAAAG，RALF-2的序列为TGGTACTCTACTTGGTCGATTCACA.以甘蓝花粉cDNA和基因组DNA为模板，参照PrimerSTAR Max DNA polymerase说明书配制25 μL反应体系. PCR循环参数为：98 ℃预变形3 min；98 ℃变性15 s，55 ℃退火15 s，72 ℃延伸30 s，35个循环；72 ℃延伸5 min. PCR扩增产物经1%琼脂糖电泳，回收目的条带送重庆擎科公司测序.

1.3. 甘蓝BoRALF_A1基因生物信息学分析

利用在线软件MultAlin(http://multalin.toulouse.inra.fr/multalin/multalin.html)进行cDNA和gDNA序列比对，用DNAstar软件推导BoRALF_A1基因编码的氨基酸序列及其理化性质.利用在线软件SOSUI(http://harrier.nagahama-i-bio.ac.jp/sosui/sosui_submit.html)分析蛋白的跨膜运域.利用在线软件SignalP-5.0(http://www.cbs.dtu.dk/services/SignalP/)分析信号肽序列，利用在线Prosite(https://prosite.expasy.org/)分析蛋白活性位点.用BRAD在线数据库进行BlastN检索，分析甘蓝02-12系基因组BoRALF_A1基因编码位点，检索白菜、油菜、拟南芥和琴叶拟南芥基因组中高度同源序列.利用在线数据库Phytozome 12(https://phytozome.jgi.doe.gov/pz/portal.html#)查找Brassica oleracea capitata V1.0基因组中同源序列.多序列比对利用MultAlin软件进行，参照高启国等^[15]的方法利用MEGA软件并采用邻接法构建系统发育树.利用在线软件SWISS-MODEL(https://swissmodel.expasy.org/)，以AtRALF8空间结构为模板，构建甘蓝BoRALF_A1d的空间结构，用DeepView软件查看和分析生成的空间结构.

1.4. 甘蓝BoRALF_A1和白菜Bra027081基因表达分析

以Ubiquitin作为内参基因，RT-PCR分析甘蓝BoRALF_A1基因在柱头、花粉、萼片、花瓣和叶片5个组织的表达情况，PCR反应体系和循环参数参照1.2，其中循环为30个. Ubiquitin基因的循环参照Gao等^[16]的方法，循环数为22.基于Wang等^[17]对白菜Chiifu-401-42的愈伤组织、根、茎、叶、花和果荚6个组织的转录组分析，从GEO数据库(http://www.ncbi.nlm.nih.gov/geo/)下载转录组分析结果(收录号：GSE43245)，利用FPKM值绘制白菜Bra027081基因在6个组织中表达的柱形图.

3. 结论与讨论

RALF蛋白作为重要的胞间信号因子广泛参与植物的多种生理过程.然而至今关于甘蓝RALF蛋白还鲜有报道.本文从甘蓝“A1”材料中克隆的1个RALF蛋白编码基因，命名为BoRALF_A1，其全长CDS为240 bp，编码79个氨基酸，包含1个信号肽和5个翻译后修饰位点.在甘蓝02-12系基因组C09号染色体上存在6个串联重复编码位点.序列比对表明BoRALF_A1具有RALF蛋白家族典型保守基序^[4]，包含4个保守的半胱氨酸残基、N-端YIXY基序、C-端YXRGC和RCG基序.与已经功能鉴定的拟南芥AtRALF4，AtRALF19和AtRALF34^[13]，烟草NaRALF和NtRALF^[3-5]，及杨树PtdRALF1和PtdRALF2^[22]的氨基酸序列相比，BoRALF1蛋白N-端明显较短，且缺失保守的RRXL基序，该基序是RALF前体蛋白加工过程中丝氨酸蛋白酶AtSIP1的识别位点^{[18, 23]}，由此推测BoRALF_A1可能存在不同的加工过程.

甘蓝BoRALF_A1与白菜Bra027081、油菜RALFbn和青花菜BoRALF1序列高度一致且在进化上高度保守，表明它们可能具有相似的生物学功能.李焰焰等^[24]分析表明油菜RALFbn主要在雄蕊中表达，而在雌蕊、花瓣和萼片中没有表达. Zhang等^[25]从青花菜中克隆了BoRALF1基因，发现其主要在花粉中表达. Wang等^[17]转录组分析显示白菜Bra027081主要在花中表达，Shi等^[14]进一步分析表明白菜Bra027081在可育材料花蕾中高量表达，在核雄性不育材料花蕾中不表达.本文结果显示甘蓝BoRALF_A1主要在花粉中表达.因此推测BoRALF_A1可能在花粉发育和育性方面具有重要的功能. BoRALF_A1蛋白具有保守的N-端YIXY基序，在三维空间结构上裸露于表面.研究表明YIXY基序是RALF蛋白与受体结合的核心功能域^[20-21].在已经鉴定的与生殖有关的RALF蛋白中，AtRALF4/19与BUPS-ANX受体复合体结合维持花粉管的完整性，AtRALF34与AtRALF4/19竞争性结合BUPS-ANX复合体，致使花粉管破裂^[13-26].先前研究显示拟南芥FEROINA (FER)与AtRALF1作用调控主根细胞伸长，与AtxRALF23作用调控植物免疫反应^[10-27]，最新研究表明AtRALF1-FER调控拟南芥的开花时间^[28].因此，接下来工作的重点是进一步明确BoRALF_A1蛋白的功能，分离其互作蛋白并探究其作用机理.

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Cloning and Expression Analysis of BoRALF_A1 Gene in Brassica oleracea