橡胶草(Taraxacum kok-saghyz Rodin)小橡胶粒子蛋白基因的克隆与表达分析
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摘要
以橡胶草(Taraxacum kok-saghyz Rodin)为试材,在茉莉酸处理的根转录组数据分析的基础上,采用PCR的方法克隆获得小橡胶粒子蛋白(small rubber particle protein,SRPP)基因。通过生物信息学分析研究了该基因的特征,并检测了该基因的时空和原核表达情况。结果表明:SRPP3蛋白分子质量24.431 kDa,理论等电点(pI)4.64,属于酸性蛋白,二级结构以β-折叠为主,具有REF保守结构域,属于REF/SRPP家族成员之一。系统进化树分析显示TkSRPP3基因与短角蒲公英同源性最近;时空表达分析表明,TkSRPP3基因在六月龄的根中表达最高,推测该基因可能与天然橡胶的合成有关。TkSRPP3原核表达最佳诱导条件为浓度1 mmol·L~(-1) IPTG诱导表达6 h。该研究为深入了解REF/SRPP家族中SRPP3在天然橡胶中的作用提供了参考依据。
Using Tarexacunm kok-saglvg Rodin as experimental material,based on jasmonic acid-treated root transcriptome data analysis,PCR was used to clone the Small Rubber Particle Protein(SRPP) gene,and the open reading frame was long 690 bp,encoding 229 amino acids.The bioinformatics,spatiotemporal expression analysis and heterologous expression analysis of the gene were studied.The results showed that the molecular weight of SRPP3 protein was 24.431 kDa,the theoretical isoelectric point(pI) was 4.64,which was acidic protein,and the secondary structure was mainly β-sheet.It had a REF conserved domain and was a member of the REF/SRPP family.Phylogenetic tree analysis showed that the TkSRPP3 gene was the most homologous to the short-horned dandelion;the spatio-temporal expression analysis showed that the TkSRPP3 gene was the highest in the roots of June age,and the prediction was related to the synthesis of natural rubber.The prokaryotic expression of TkSRPP3 was induced at a concentration of 1 mmol·L~(-1) IPTG for 6 h,which was the best induction condition.The aim is to lay the foundation for a preliminary study of the role of SRPP3 in natural rubber in the REF/SRPP family.
Using Tarexacunm kok-saglvg Rodin as experimental material,based on jasmonic acid-treated root transcriptome data analysis,PCR was used to clone the Small Rubber Particle Protein(SRPP) gene,and the open reading frame was long 690 bp,encoding 229 amino acids.The bioinformatics,spatiotemporal expression analysis and heterologous expression analysis of the gene were studied.The results showed that the molecular weight of SRPP3 protein was 24.431 kDa,the theoretical isoelectric point(pI) was 4.64,which was acidic protein,and the secondary structure was mainly β-sheet.It had a REF conserved domain and was a member of the REF/SRPP family.Phylogenetic tree analysis showed that the TkSRPP3 gene was the most homologous to the short-horned dandelion;the spatio-temporal expression analysis showed that the TkSRPP3 gene was the highest in the roots of June age,and the prediction was related to the synthesis of natural rubber.The prokaryotic expression of TkSRPP3 was induced at a concentration of 1 mmol·L~(-1) IPTG for 6 h,which was the best induction condition.The aim is to lay the foundation for a preliminary study of the role of SRPP3 in natural rubber in the REF/SRPP family.
引文
[1] 罗士苇,冯午,吴相钰.橡膠草的研究部分Ⅰ.新疆产橡膠草的形态观察[J].中国科学,1951(3):125-131.
[2] 袁彬青,王秀珍,罗成华,等.农杆菌介导的橡胶草遗传转化体系的建立[J].西北农业学报,2014,23(9):98-105.
[3] BEILEN J B V,POIRIER Y.Establishment of new crops for the production of natural rubber[J].Trends in Biotechnology,2007,25(11):522-529.
[4] 鞠岩峰,张剑,吴润,等.天然橡胶种植现状及市场需求预测分析[J].林业资源管理,2014(1):152-157.
[5] 罗明武,邓柳红,易小平,等.巴西橡胶树顺式异戊烯基转移酶基因cDNA克隆及其序列特征分析[J].热带亚热带植物学报,2009,17(3):223-228.
[6] YAMASHITA S,MIZUNO M,HAYASHI H,et al.Purification and characterization of small and large rubber particles from Hevea brasiliensis[J].Bioscience Biotechnology & Biochemistry,2018,82(6):1011-1020.
[7] LIN T,XU X,RUAN J,et al.Genome analysis of Taraxacum kok-saghyz Rodin provides new insights into rubber biosynthesis[J].National Science Review,2018,5(1):78-87.
[8] 吴华玲,段翠芳,聂智毅,等.巴西橡胶树橡胶粒子研究进展[J].热带作物学报,2009,30(7):1044-1049.
[9] OH S K,KANG H,SHIN D H,et al.Isolation,characterization,and functional analysis of a novel cDNA clone encoding a small rubber particle protein from Hevea brasiliensis[J].Journal of Biological Chemistry,1999,274(24):17132-17138.
[10] WADEESIRISAK K,CASTANO S,BERTHELOT K,et al.Rubber particle proteins REF1 and SRPP1 interact differently with native lipids extracted from Hevea brasiliensis Latex[J].Biochimica et Biophysica Acta (BBA)-Biomembranes,2016,1859(2):201-210.
[11] SANDO T,HAYASHI T,TAKEDA T,et al.Histochemical study of detailed laticifer structure and rubber biosynthesis-related protein localization in Hevea brasiliensis using spectral confocal laser scanning microscopy[J].Planta,2009,230(1):215-225.
[12] HILLEBRAND A,POST J J,WURBS D,et al.Down-regulation of small rubber particle protein expression affects integrity of rubber particles and rubber content in Taraxacum brevicorniculatum[J].PLoS One,2012,7(7):e41874.
[13] COLLINS-SILVA J,NURAL A T,SKAGGS A,et al.Altered levels of the Taraxacum kok-saghyz (Russian dandelion) small rubber particle protein,TkSRPP3,result in qualitative and quantitative changes in rubber metabolism[J].Phytochemistry,2012,79:46-56.
[14] CAO X W,YAN J,LEI J L,et al.De novo transcriptome sequencing of MeJA-induced Taraxacum koksaghyz Rodin to identify genes related to rubber formation[J].Scientific Reports,2017,7(1):15697.
[15] 罗成华,闫洁,祝建波.橡胶草高频再生体系的建立[J].北方园艺,2012(7):115-119.
[16] WITITSUWANNAKUL R,RUKSEREE K,KANOKWIROON K,et al.A rubber particle protein specific for Hevea latex lectin binding involved in latex coagulation[J].Phytochemistry,2008,69(5):1111-1118.
[17] BERTHELOT K,PERUCH,FRéDéRIC,LECOMTE S.Highlights on Hevea brasiliensis (pro) hevein proteins[J].Biochimie,2016,127:258-270.
[18] CHUNTAI W,LI L,YU L,et al.The relationship between latex metabolism gene expression with rubber yield and related traits in Hevea brasiliensis[J].BMC Genomics,2018(19):897.
[19] BROWN D,FEENEY M,AHMADI M,et al.Subcellular localization and interactions among rubber particle proteins from Hevea brasiliensis[J].Journal of Experimental Botany,2017,68(18):5045-5055.
[20] FRICKE J,HILLEBRAND A,TWYMAN R M,et al.Abscisic acid-dependent regulation of small rubber particle protein gene expression in Taraxacum brevicorniculatum is mediated by TbbZIP1[J].Plant & Cell Physiology,2013,54(4):448-464.
[21] KIM E Y,PARK K Y,SEO Y S,et al.Arabidopsis small rubber particle protein homolog SRPs play dual roles as positive factors for tissue growth and development and in drought stress responses[J].Plant Physiology,2016,170(4):2494-2510.
[2] 袁彬青,王秀珍,罗成华,等.农杆菌介导的橡胶草遗传转化体系的建立[J].西北农业学报,2014,23(9):98-105.
[3] BEILEN J B V,POIRIER Y.Establishment of new crops for the production of natural rubber[J].Trends in Biotechnology,2007,25(11):522-529.
[4] 鞠岩峰,张剑,吴润,等.天然橡胶种植现状及市场需求预测分析[J].林业资源管理,2014(1):152-157.
[5] 罗明武,邓柳红,易小平,等.巴西橡胶树顺式异戊烯基转移酶基因cDNA克隆及其序列特征分析[J].热带亚热带植物学报,2009,17(3):223-228.
[6] YAMASHITA S,MIZUNO M,HAYASHI H,et al.Purification and characterization of small and large rubber particles from Hevea brasiliensis[J].Bioscience Biotechnology & Biochemistry,2018,82(6):1011-1020.
[7] LIN T,XU X,RUAN J,et al.Genome analysis of Taraxacum kok-saghyz Rodin provides new insights into rubber biosynthesis[J].National Science Review,2018,5(1):78-87.
[8] 吴华玲,段翠芳,聂智毅,等.巴西橡胶树橡胶粒子研究进展[J].热带作物学报,2009,30(7):1044-1049.
[9] OH S K,KANG H,SHIN D H,et al.Isolation,characterization,and functional analysis of a novel cDNA clone encoding a small rubber particle protein from Hevea brasiliensis[J].Journal of Biological Chemistry,1999,274(24):17132-17138.
[10] WADEESIRISAK K,CASTANO S,BERTHELOT K,et al.Rubber particle proteins REF1 and SRPP1 interact differently with native lipids extracted from Hevea brasiliensis Latex[J].Biochimica et Biophysica Acta (BBA)-Biomembranes,2016,1859(2):201-210.
[11] SANDO T,HAYASHI T,TAKEDA T,et al.Histochemical study of detailed laticifer structure and rubber biosynthesis-related protein localization in Hevea brasiliensis using spectral confocal laser scanning microscopy[J].Planta,2009,230(1):215-225.
[12] HILLEBRAND A,POST J J,WURBS D,et al.Down-regulation of small rubber particle protein expression affects integrity of rubber particles and rubber content in Taraxacum brevicorniculatum[J].PLoS One,2012,7(7):e41874.
[13] COLLINS-SILVA J,NURAL A T,SKAGGS A,et al.Altered levels of the Taraxacum kok-saghyz (Russian dandelion) small rubber particle protein,TkSRPP3,result in qualitative and quantitative changes in rubber metabolism[J].Phytochemistry,2012,79:46-56.
[14] CAO X W,YAN J,LEI J L,et al.De novo transcriptome sequencing of MeJA-induced Taraxacum koksaghyz Rodin to identify genes related to rubber formation[J].Scientific Reports,2017,7(1):15697.
[15] 罗成华,闫洁,祝建波.橡胶草高频再生体系的建立[J].北方园艺,2012(7):115-119.
[16] WITITSUWANNAKUL R,RUKSEREE K,KANOKWIROON K,et al.A rubber particle protein specific for Hevea latex lectin binding involved in latex coagulation[J].Phytochemistry,2008,69(5):1111-1118.
[17] BERTHELOT K,PERUCH,FRéDéRIC,LECOMTE S.Highlights on Hevea brasiliensis (pro) hevein proteins[J].Biochimie,2016,127:258-270.
[18] CHUNTAI W,LI L,YU L,et al.The relationship between latex metabolism gene expression with rubber yield and related traits in Hevea brasiliensis[J].BMC Genomics,2018(19):897.
[19] BROWN D,FEENEY M,AHMADI M,et al.Subcellular localization and interactions among rubber particle proteins from Hevea brasiliensis[J].Journal of Experimental Botany,2017,68(18):5045-5055.
[20] FRICKE J,HILLEBRAND A,TWYMAN R M,et al.Abscisic acid-dependent regulation of small rubber particle protein gene expression in Taraxacum brevicorniculatum is mediated by TbbZIP1[J].Plant & Cell Physiology,2013,54(4):448-464.
[21] KIM E Y,PARK K Y,SEO Y S,et al.Arabidopsis small rubber particle protein homolog SRPs play dual roles as positive factors for tissue growth and development and in drought stress responses[J].Plant Physiology,2016,170(4):2494-2510.