柑橘CitPG34的克隆、定位与表达分析
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摘要
【目的】多聚半乳糖醛酸酶是一类参与细胞壁降解的水解酶,在植物生长发育和器官脱落过程中发挥着重要作用。本研究克隆柑橘CitPG34及其启动子(CitPG34-P)并进行表达分析,为深入研究柑橘PG在幼果脱落过程的生物功能奠定基础。【方法】以‘塔罗科’血橙(Citrus sinensis L. Osbeck)为材料,克隆CitPG34及其启动子,利用ProtParam、Cello、CLUSTALX、MEGA5.2、PlantCARE等软件对其蛋白特性及启动子顺式作用元件进行分析预测;利用实时荧光定量PCR(qRT-PCR)分析CitPG34在不同组织以及柑橘幼果脱落过程中的表达水平。采用同源重组的方法构建pCAMBIA1302-CitPG34-GFP融合蛋白表达载体和CitPG34启动子表达载体(CitPG34-P::gus),分别用于亚细胞定位和启动子活性分析。【结果】从‘塔罗科’血橙幼果离层中克隆获得CitPG34,其ORF为1 194 bp,编码397个氨基酸,预测蛋白分子量为41.47 kD,理论等电点为5.19,其不稳定系数为30.23,表明CitPG34属于稳定蛋白;通过在线软件TMHMM分析发现:CitPG34为跨膜蛋白,具有一个跨膜结构,位于第7—29位氨基酸之间。在CitPG34二级结构中,α-螺旋结构约占15.37%,扩展链约占29.72%,无规则卷曲约占54.91%,与其三级结构预测基本一致。NJ树分析显示CitPG34与西洋梨PcPG3(BAF42034)亲缘关系最近,表明CitPG34可能与果实脱落和软化相关。qRT-PCR分析表明,CitPG34在花中表达量最高,在根、叶、离层A、离层C中表达量较低,在幼果中几乎不表达。1-氨基环丙烷羧酸(ACC)处理果梗后能显著提高离层A中CitPG34的表达水平,相反IAA抑制其转录。此外,在柑橘幼果正常脱落过程中,CitPG34表达明显升高。亚细胞定位发现,CitPG34主要位于细胞壁。克隆获取CitPG34起始密码子(ATG)前2 075 bp启动子序列(CitPG34-P),PlantCare预测发现,在CitPG34-P序列上存在多种顺式调控元件,如核心启动元件TATA-box、增强子元件CAAT-box以及脱落酸响应元件ABRE等。将CitPG34-P::gus转入烟草,通过GUS组织化学染色发现,该启动子受乙烯诱导,主要在叶脉和毛状体中表达。【结论】CitPG34的ORF长度为1 194 bp,可编码397个氨基酸,其蛋白主要位于细胞壁;该基因具有明显的组织特异性,在花中表达最高;CitPG34表达量与柑橘幼果脱落显著相关。上述结果表明,CitPG34在柑橘幼果脱落和花发育过程中可能发挥着重要的生物功能。
【Objective】Polygalacturonases(PGs) play important roles in plant growth and development as well as organ abscission by degrading pectin in cell wall. In this study, a citrus PG gene(i.e., CitPG34) and its promoter(CitPG34-P) were cloned and expression analyzed based on our previous data, which would provide a basis for further elucidating the function of Cit PG34 in citrus fruitlet abscission.【Method】The full length of CitPG34 gene and its promoter was cloned from ‘Tarcocco' blood orange(Citrus sinensis L. Osbeck). The protein characteristics and cis-acting elements on promoter were analyzed by ProtParam, Cello, CLUSTALX, MEGA5.2, and PlantCARE, etc. The gene expression level was detected by real-time Quantitative PCR(qRT-PCR). The PCAMBIA1302-CitPG34-GFP fusion protein expression vector for subcellular localization and CitPG34-P expression vector(Cit PG34-P::gus) for promoter activity analysis were constructed by homologous recombination, respectively.【Result】The ORF of CitPG34 was 1 194 bp in length, encoding 397 amino acids. The predicted molecular weight of CitPG34 was 41.47 kD, the theoretical pI was 5.19, and the instability coefficient was 30.23, indicating that CitPG34 belonged to stable protein. TMHMM analysis showed that CitPG34 was a transmembrane protein, the transmembrane domain locating between the amino acid residue 7 and 29. In the secondary structure of Cit PG34, the alpha-helix structure, extended chain and random coil account for 15.37%, 29.72% and 54.91%, respectively, which were nearly consistent with its tertiary structure. NJ tree analysis showed that CitPG34 was close to PcPG3(BAF42034), a pear PG, indicating that it might be related to fruit abscission and softening. qPCR analysis showed that CitPG34 dominantly expressed in flowers, followed by roots, leaves, abscission zone A(AZ A) and C(AZ C), and almost undetected in fruits. In AZ A, the expression level of CitPG34 was significantly up-regulated by ACC, whereas inhibited by IAA, showing the role in citrus fruitlet abscission. Subcellular localization revealed that CitPG34 was mainly located in cell wall. A 2 075 bp promoter sequence of CitPG34 was cloned, which contained several cis-regulatory elements, including TATA-box, enhancer CAAT-box and ABRE, etc. GUS histochemical staining revealed that the GUS activity in vein and trichomes was remarkably up-regulated by ethylene. 【Conclusion】The ORF length of CitPG34 gene was 1 194 bp, encoding 397 amino acids. CitPG34 was mainly located in cell wall. qPCR analysis showed that Cit PG34 dominantly expressed in flowers and was significantly associated with citrus fruitlet abscission. Taken together, these results indicated that Cit PG34 played important roles in citrus fruitlet abscission and flower development.
【Objective】Polygalacturonases(PGs) play important roles in plant growth and development as well as organ abscission by degrading pectin in cell wall. In this study, a citrus PG gene(i.e., CitPG34) and its promoter(CitPG34-P) were cloned and expression analyzed based on our previous data, which would provide a basis for further elucidating the function of Cit PG34 in citrus fruitlet abscission.【Method】The full length of CitPG34 gene and its promoter was cloned from ‘Tarcocco' blood orange(Citrus sinensis L. Osbeck). The protein characteristics and cis-acting elements on promoter were analyzed by ProtParam, Cello, CLUSTALX, MEGA5.2, and PlantCARE, etc. The gene expression level was detected by real-time Quantitative PCR(qRT-PCR). The PCAMBIA1302-CitPG34-GFP fusion protein expression vector for subcellular localization and CitPG34-P expression vector(Cit PG34-P::gus) for promoter activity analysis were constructed by homologous recombination, respectively.【Result】The ORF of CitPG34 was 1 194 bp in length, encoding 397 amino acids. The predicted molecular weight of CitPG34 was 41.47 kD, the theoretical pI was 5.19, and the instability coefficient was 30.23, indicating that CitPG34 belonged to stable protein. TMHMM analysis showed that CitPG34 was a transmembrane protein, the transmembrane domain locating between the amino acid residue 7 and 29. In the secondary structure of Cit PG34, the alpha-helix structure, extended chain and random coil account for 15.37%, 29.72% and 54.91%, respectively, which were nearly consistent with its tertiary structure. NJ tree analysis showed that CitPG34 was close to PcPG3(BAF42034), a pear PG, indicating that it might be related to fruit abscission and softening. qPCR analysis showed that CitPG34 dominantly expressed in flowers, followed by roots, leaves, abscission zone A(AZ A) and C(AZ C), and almost undetected in fruits. In AZ A, the expression level of CitPG34 was significantly up-regulated by ACC, whereas inhibited by IAA, showing the role in citrus fruitlet abscission. Subcellular localization revealed that CitPG34 was mainly located in cell wall. A 2 075 bp promoter sequence of CitPG34 was cloned, which contained several cis-regulatory elements, including TATA-box, enhancer CAAT-box and ABRE, etc. GUS histochemical staining revealed that the GUS activity in vein and trichomes was remarkably up-regulated by ethylene. 【Conclusion】The ORF length of CitPG34 gene was 1 194 bp, encoding 397 amino acids. CitPG34 was mainly located in cell wall. qPCR analysis showed that Cit PG34 dominantly expressed in flowers and was significantly associated with citrus fruitlet abscission. Taken together, these results indicated that Cit PG34 played important roles in citrus fruitlet abscission and flower development.
引文
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[21] MARKOVIC O, JANECEK S. Pectin degrading glycoside hydrolases of family 28:Sequence structural features, specificities and evolution.Protein Engineering Design and Selection, 2001, 14(9):615-631.
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[2]潘小婷,张静,葛廷,马岩岩,邓烈,何绍兰,易时来,郑永强,吕强,谢让金.柑橘CitCEP基因家族的鉴定及对逆境和激素的响应.中国农业科学, 2018, 51(16):3147-3158.PAN X T, ZHANG J, GE T, MA Y Y, DENG L, HE S L, YI S L,ZHENG Y Q, LüQ, XIE R J. Identification of citrus CitCEP genes and their transcriptional response to stress and hormone treatments.Scientia Agricultura Sinica, 2018, 51(16):3147-3158.(in Chinese)
[3] RIOV J. A polygalacturonase from citrus leaf explants:Role in abscission. Plant Physiology, 1974, 53(2):312-316.
[4] NAKANO T, ITO Y. Molecular mechanisms controlling plant organ abscission. Plant Biotechnology, 2013, 30(3):209-216.
[5] CARMEN RODRíGUEZ-GACIO M D C, NICOLáS C, MATILLA A J. Cloning and analysis of a c DNA encoding an endo-polygalacturonase expressed during the desiccation period of the silique-valves of turnip-tops(Brassica rapa L. cv. Rapa). Journal of Plant Physiology,2004, 161(2):219-227.
[6] QUESADA M, BLANCO-PORTALES R, POSéS, GARCíA-GAGO J A, JIMéNEZ-BERMúDEZ S, MUN?OZ-SERRANO A, CABALLERO J L, PLIEGO-ALFARO F, MERCADO J A, MU?OZ-BLANCO J.Antisense down-regulation of the FaPG1 gene reveals an unexpected central role for polygalacturonase in strawberry fruit softening. Plant Physiology, 2009, 150(2):1022-1032.
[7] POSE S, PANIAGUA C, CIFUENTES M, BLANCO-PORTALES R,QUWSADA M A, MERCADO J A. Insights into the effects of polygalacturonase Fa PG1 gene silencing on pectin matrix disassembly,enhanced tissue integrity, and firmness in ripe strawberry fruits.Journal of Experimental Botany, 2013, 64(12):3803-3815.
[8] GONZáLEZ-CARRANZA Z H, ELLIOTT K A, ROBERTS J A.Expression of polygalacturonases and evidence to support their role during cell separation processes in Arabidopsis thaliana. Journal of Experimental Botany, 2007, 58(13):3719-3730.
[9] LYU M L, LIANG Y, YU Y J, MA Z M, SONG L M, YUE X Y, CAO J S. Identification and expression analysis of BoMF25, a novel polygalacturonase gene involved in pollen development of Brassica oleracea. Plant Reproduction, 2015, 28(2):121-132.
[10] ATKINSON R G, SCHRODER R, HALLETT I C, COHEN D,MACRAE E A. Overexpression of polygalacturonase in transgenic apple trees leads to a range of novel phenotypes involving changes in cell adhesion. Plant Physiology, 2002, 129(1):122-133.
[11] ROONGSATTHAM P, MORCILLO F, JANTASURIYARAT C,PIZOT M, MOUSSU S, JAYAWEERA D, COLLIN M, GONZáLEZCARRANZA Z H, AMBLARD P, TREGEAR J W, TRAGOONRUNG S,VERDEIL J, TRANBARGER T J. Temporal and spatial expression of polygalacturonase gene family members reveals divergent regulation during fleshy fruit ripening and abscission in the monocot species oil palm. BMC Plant Biology, 2012, 12(1):150.
[12] JIANG C Z, LU F, IMSABAI W, MEIR S, REID M S. Silencing polygalacturonase expression inhibits tomato petiole abscission.Journal of Experimental Botany, 2008, 59(4):973.
[13] PENG G, WU J Y, LU W J, LI J G. A polygalacturonase gene clustered into clade E involved in lychee fruitlet abscission. Scientia Horticulturae,2013, 150:244-250.
[14] RASCIO N, CASADORO G, RAMINA A, MASIA A. Structural and biochemical aspects of peach fruit abscission(Prunus persica L.Batsch). Planta, 1985, 164(1):1-11.
[15] TAYLOR J E, WEBB S T J, COUPE S A, TUCKER G A, ROBERTS J A. Changes in polygalacturonase activity and solubility of polyuronides during ethylene-stimulated leaf abscission in Sambucus nigra. Journal of Experimental Botany, 1993, 44(1):93-98.
[16] KALAITZIS P, TUCKER S M L. Three different polygalacturonases are expressed in tomato leaf and flower abscission, each with a different temporal expression pattern. Plant Physiology, 1997, 113(4):1303-1308.
[17] CHENG C Z, ZHANG L Y, YANG X L, ZHONG G Y. Profiling gene expression in citrus fruit calyx abscission zone(AZ-C)treated with ethylene. Molecular Genetics and Genomics, 2015, 290(5):1991-2006.
[18] MERELO P, AGUSTíJ, ARBONA V, COSTA M L, ESTORNELL L H, GóMEZ-CADENAS A, COIMBRA S, GóMEZ M D,PéREZ-AMADOR M A, DOMINGO C, TALóN M, TADEO F R.Cell wall remodeling in abscission zone cells during ethylenepromoted fruit abscission in citrus. Frontiers in Plant Science, 2017, 8:126.
[19] XIE R J, GE T, ZHANG J, PAN X T, MA Y Y, YI S L, ZHENG Y Q.The molecular events of IAA inhibiting citrus fruitlet abscission revealed by digital gene expression profiling. Plant Physiology and Biochemistry, 2018,130:192-204.
[20] GONZáLEZ-CARRANZA Z H, WHITELAW C A, SWARUP R,ROBERTS J A. Temporal and spatial expression of a polygalacturonase during leaf and flower abscission in oilseed rape and Arabidopsis.Plant Physiology, 2002, 128(2):534-543.
[21] MARKOVIC O, JANECEK S. Pectin degrading glycoside hydrolases of family 28:Sequence structural features, specificities and evolution.Protein Engineering Design and Selection, 2001, 14(9):615-631.
[22] SITRIT Y, HADFIELD K A, BENNETT A B, BRADFORD K J,DOWNIE A B. Expression of a polygalacturonase associated with tomato seed germination. Plant Physiology, 1999, 121(2):419-428.
[23] SANDER L, CHILD R, ULVSKOV P, ALBRECHTSEN M,BORKHARDT B. Analysis of a dehiscence zone endo-polygalacturonase in oilseed rape(Brassica napus)and Arabidopsis thaliana:Evidence for roles in cell separation in dehiscence and abscission zones, and in stylar tissues during pollen tube growth. Plant Molecular Biology,2001, 46(4):469-479.
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