怀黄菊CmPAN基因的克隆与表达分析
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摘要
怀菊花(Chrysanthemum morifolium)是著名的四大怀药之一,具有很高的观赏、食用及药用价值,但其花器官发育缺乏相关研究。为探究PERIANTHIA (PAN)基因在怀菊花花发育中的功能,首先利用同源克隆与cDNA末端快速扩增(rapid amplification of cDNA ends, RACE)技术成功克隆获得怀菊花优良种质怀黄菊(Ch. morifolium cv. Huaihuang)中CmPAN的cDNA序列,全长为1 484 bp,其中CDS区为1 314bp,5'UTR和3'UTR分别为64和106 bp。预测其编码437个氨基酸,与其他植物的PAN序列具有较高同源性,且C末端具有碱性亮氨酸拉链(basic leucine zipper motif, bZIP)家族D亚族的特征区和2个谷氨酰胺丰富区,与拟南芥(Arabidopsis thaliana) AtPAN的亲缘关系最近,故命名为CmPAN (GenBank No.KX380854)。生物信息学分析显示,CmPAN蛋白质的预测分子量和等电点分别为48.265 k D和8.82;CmPAN为非分泌性蛋白,并具有核定位序列,无序化程度小于AtPAN。qRT-PCR检测结果表明,CmPAN在花与茎中的表达量高于根与叶;在花发育的现蕾期至透色期表达量较高,之后呈下降趋势;在花器官中表达水平从高至低依次为管状花瓣、管状花雄蕊、舌状花瓣、舌状花心皮、花托。上述结果提示,CmPAN可能在花发育初期发挥重要作用,并且与管状花的花瓣和雄蕊的发育密切相关。本研究为进一步探究CmPAN基因的功能提供了参考依据。
Chrysanthemum morifolium originated from the ancient Huaiqingfu is one of the Four Famous Huai Medicines, which has high ornamental, edible and medicinal value. But it is rare reported about the research on its flower organ development. To study the function of PERIANTHIA(PAN) gene in flower development of Ch. morifolium, a 1 484 bp full-length cDNA sequence of homologous PAN gene was cloned by employing homology gene cloning and Rapid amplification of cDNA ends(RACE) from Ch. morifolium cv.Huaihuang. The cDNA sequence contained 64 bp 5' UTR, 106 bp 3' UTR and 1 314 bp CDS which encoded437 amino acids. The protein of CmPAN had very high similarity with PANs from other plant species. Cterminal amino acids sequence of CmPAN included group D structural features with a well characterized family of plant bZIPs(basic leucine zipper motif) and 2 glutamine-rich regions. CmPAN was most close to AtPAN, so the cloned sequence was named CmPAN(GenBank No. KX380854). Putative protein molecular weight was 48.265 kD, the theoretical isoelectric point was 8.82, and it was non-secretory protein with nuclear localization sequence, its disordered degree was smaller than that of AtPAN. The qRT-PCR detection showed that CmPAN was higher expressed in flowers and stems than that in roots and leaves, and the CmPAN expession in floral development increased from bud emergence stage to visible color stage, then decreased at late phase.The CmPAN expession in floral organ ranked from high to low was petal of tubular flower, stamen of tubular flower, petal of ray floret, carpel of ray floret and receptacle. As a result, CmPAN might have important roles in early phase of floral development, and be closely related to the development of petals and stamens of tubular flower. The study provides some reference for further exploring the function of CmPAN gene.
Chrysanthemum morifolium originated from the ancient Huaiqingfu is one of the Four Famous Huai Medicines, which has high ornamental, edible and medicinal value. But it is rare reported about the research on its flower organ development. To study the function of PERIANTHIA(PAN) gene in flower development of Ch. morifolium, a 1 484 bp full-length cDNA sequence of homologous PAN gene was cloned by employing homology gene cloning and Rapid amplification of cDNA ends(RACE) from Ch. morifolium cv.Huaihuang. The cDNA sequence contained 64 bp 5' UTR, 106 bp 3' UTR and 1 314 bp CDS which encoded437 amino acids. The protein of CmPAN had very high similarity with PANs from other plant species. Cterminal amino acids sequence of CmPAN included group D structural features with a well characterized family of plant bZIPs(basic leucine zipper motif) and 2 glutamine-rich regions. CmPAN was most close to AtPAN, so the cloned sequence was named CmPAN(GenBank No. KX380854). Putative protein molecular weight was 48.265 kD, the theoretical isoelectric point was 8.82, and it was non-secretory protein with nuclear localization sequence, its disordered degree was smaller than that of AtPAN. The qRT-PCR detection showed that CmPAN was higher expressed in flowers and stems than that in roots and leaves, and the CmPAN expession in floral development increased from bud emergence stage to visible color stage, then decreased at late phase.The CmPAN expession in floral organ ranked from high to low was petal of tubular flower, stamen of tubular flower, petal of ray floret, carpel of ray floret and receptacle. As a result, CmPAN might have important roles in early phase of floral development, and be closely related to the development of petals and stamens of tubular flower. The study provides some reference for further exploring the function of CmPAN gene.
引文
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庄静,周熙荣,孙超才,等.2008.甘蓝型油菜中一类AP2/ERF转录因子的克隆和生物信息学分析[J].中国生物工程杂志,28(5):29-40.(Zhuang J,Zhou X R,Sun CC,et al.2008.Cloning and bioinformatic analyzing of AP2/REF transcription factor genes from Brassica napus L.[J].China Biotechnology,28(5):29-40.)
Aida R,Komano M,Saito M,et al.2008.Chrysanthemum flower shape modification by suppression of chrysanthemum-AGAMOUS gene[J].Plant Biotechnology,25(1):55-59.
Becker A,Ehlers K.2016.Arabidopsis flower development of protein complexes,targets,and transport[J].Protoplasma,253(2):219-230.
Chuang C F,Running M P,Williams R W,et al.1999.The PERIANTHIA gene encodes a bZIP protein involved in the determination of floral organ number in Arabidopsis thaliana[J].Genes&Development,13(3):34-44.
Courey A J,Tjian R.1988.Analysis of Sp1 in vivo reveals multiple transcriptional domains,including a novel glutamine-rich activation motif[J].Cell,55(5):887-898.
Das P,Ito T,Wellmer F,et al.2009.Floral stem cell termination involves the direct regulation of AGAMOUS by PERIANTHIA[J].Development,136(10):1605-1611.
Fei J,Tan S,Zhang F,et al.2016.Morphological and physiological differences between dehiscent and indehiscent anthers of Chrysanthemum morifolium[J].Journal of Plant Research,129(6):1069-1082.
Fink A L.2005.Natively unfolded proteins[J].Current Opinion in Structural Biology,15(1):35-41.
Jakoby M,Weisshaar B,Dr?ge-Laser W,et al.2002.b ZIPtranscription factors in Arabidopsis[J].Trends in Plant Science,7(3):106-111.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTMethod[J].Methods,25(4):402-408.
Maier A T,Stehlingsun S,Wollmann H,et al.2009.Dual roles of the bZIP transcription factor PERIANTHIA in the control of floral architecture and homeotic gene expression[J].Development,136(10):1613-1620.
Murmu J,Bush M J,Delong C,et al.2010.Arabidopsis basic leucine-zipper transcription factors TGA9 and TGA10interact with floral glutaredoxins ROXY1 and ROXY2and are redundantly required for anther development[J].Plant Physiology,154(3):1492-1504.
Running M P,Meyerowitz E M.1996.Mutations in the PERI?ANTHIA gene of Arabidopsis specifically alter floral organ number and initiation pattern[J].Development,122(4):1261-1269.
Schlessinger A,Punta M B.2007.Natively unstructured regions in proteins identified from contact predictions[J].Bioinformatics,23(18):2376-2384.
Shearer H L,Wang L,Delong C,et al.2009.NPR1 enhances the DNA binding activity of the Arabidopsis bZIP transcription factor TGA7[J].Botany-botanique,87(6):561-570.
Tuskan G A,Difazio S,Jansson S,et al.2006.The genome of black cottonwood,Populus trichocarpa(Torr.&Gray)[J].Science,313(5793):1596-1604.
李超.2010.DgDREB1A基因分离及对切花菊的遗传转化[D].硕士学位论文,东北林业大学,导师:洪波,pp.11-17.(Li C.2010.Isolation and transformation of Dg?DREB1A in cut chrysanthemum[D].Thesis for M.S.,Northeast Forestry University,Supervisor:Hong B,pp.11-17.)
李冉辉.2013.一种大豆固有无序蛋白GmASR蛋白的多重保护功能研究[D].博士学位论文,东北师范大学,导师:郑易之,pp.9-10.(Li R H.2013.Multi-protective functions of GmASR protein-a soybean intrinsically disordered protein[D].Thesis for Ph.D.,Northeast Normal University,Supervisor:Zheng Y Z,pp.9-10.)
刘国宝.2011.二种大豆LEA4蛋白的金属离子结合特性及对生物大分子的保护作用[D].博士学位论文,东北师范大学,导师:郑易之,pp.25-28.(Liu G B.2011.The metal-binding properties and the protective effect on biological macromolecules of two soybean LEA4 proteins[D].Thesis for Ph.D.,Northeast Normal University,Supervisor:Zheng Y Z,pp.25-28.)
杨艳歌,吕维涛,孙冬梅,等.2013.玉米D亚族bZIP转录因子基因的数据库挖掘、分析、克隆与表达[J].作物学报,39(12):2115-2122.(Yang Y G,Lu W T,Sun D M,et al.2013.Database mining,bioinformatics,cloning,and expression analyses of D subfamily bZIP genes in Maize[J].Acta Agronomica Sinica,39(12):2115-2122.)
曾丽,赵梁军,张华丽,等.2010.万寿菊花器官发生与发育的观察[J].园艺学报,37(5):785-793.(Zeng L,Zhao LJ,Zhang H L,et al.2010.Observation of floral organogenesis and development of Tagets erecta L.[J].Acta Horticulturae Sinica,37(5):785-793.)
周小东,沈富兵.2013.MAP30蛋白结构功能的生物信息学研究[J].广西植物33(4),560-563.(Zhou X D,Shen FB.2013.Research on bioinformatics of MAP30 protein structural function[J].Guihaia,33(4):560-563.)
庄静,周熙荣,孙超才,等.2008.甘蓝型油菜中一类AP2/ERF转录因子的克隆和生物信息学分析[J].中国生物工程杂志,28(5):29-40.(Zhuang J,Zhou X R,Sun CC,et al.2008.Cloning and bioinformatic analyzing of AP2/REF transcription factor genes from Brassica napus L.[J].China Biotechnology,28(5):29-40.)
Aida R,Komano M,Saito M,et al.2008.Chrysanthemum flower shape modification by suppression of chrysanthemum-AGAMOUS gene[J].Plant Biotechnology,25(1):55-59.
Becker A,Ehlers K.2016.Arabidopsis flower development of protein complexes,targets,and transport[J].Protoplasma,253(2):219-230.
Chuang C F,Running M P,Williams R W,et al.1999.The PERIANTHIA gene encodes a bZIP protein involved in the determination of floral organ number in Arabidopsis thaliana[J].Genes&Development,13(3):34-44.
Courey A J,Tjian R.1988.Analysis of Sp1 in vivo reveals multiple transcriptional domains,including a novel glutamine-rich activation motif[J].Cell,55(5):887-898.
Das P,Ito T,Wellmer F,et al.2009.Floral stem cell termination involves the direct regulation of AGAMOUS by PERIANTHIA[J].Development,136(10):1605-1611.
Fei J,Tan S,Zhang F,et al.2016.Morphological and physiological differences between dehiscent and indehiscent anthers of Chrysanthemum morifolium[J].Journal of Plant Research,129(6):1069-1082.
Fink A L.2005.Natively unfolded proteins[J].Current Opinion in Structural Biology,15(1):35-41.
Jakoby M,Weisshaar B,Dr?ge-Laser W,et al.2002.b ZIPtranscription factors in Arabidopsis[J].Trends in Plant Science,7(3):106-111.
Livak K J,Schmittgen T D.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTMethod[J].Methods,25(4):402-408.
Maier A T,Stehlingsun S,Wollmann H,et al.2009.Dual roles of the bZIP transcription factor PERIANTHIA in the control of floral architecture and homeotic gene expression[J].Development,136(10):1613-1620.
Murmu J,Bush M J,Delong C,et al.2010.Arabidopsis basic leucine-zipper transcription factors TGA9 and TGA10interact with floral glutaredoxins ROXY1 and ROXY2and are redundantly required for anther development[J].Plant Physiology,154(3):1492-1504.
Running M P,Meyerowitz E M.1996.Mutations in the PERI?ANTHIA gene of Arabidopsis specifically alter floral organ number and initiation pattern[J].Development,122(4):1261-1269.
Schlessinger A,Punta M B.2007.Natively unstructured regions in proteins identified from contact predictions[J].Bioinformatics,23(18):2376-2384.
Shearer H L,Wang L,Delong C,et al.2009.NPR1 enhances the DNA binding activity of the Arabidopsis bZIP transcription factor TGA7[J].Botany-botanique,87(6):561-570.
Tuskan G A,Difazio S,Jansson S,et al.2006.The genome of black cottonwood,Populus trichocarpa(Torr.&Gray)[J].Science,313(5793):1596-1604.