荠菜生长素极性运输基因1(CbPIN1)的克隆与表达分析
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摘要
为了探讨荠菜PIN1基因的生物学功能,根据Gen Bank中拟南芥PIN1氨基酸序列,设计同源引物,通过RT-PCR技术,克隆了荠菜PIN1基因(CbPIN1)编码区c DNA序列;生物信息学方法分析了荠菜PIN1蛋白结构特征,并进行了亚细胞定位与原核表达;荧光定量PCR方法检测了PIN1组织表达特性;构建了植物表达载体p BI121-CbPIN1,并获得转基因植株。结果表明,CbPIN1 c DNA序列全长1 869 bp,C+G含量为49%。Blast比对结果显示,CbPIN1与拟南芥PIN1基因高度同源,相似性高达93%。进一步分析发现,CbPIN1可编码1条622个氨基酸的多肽,CbPIN1蛋白分子质量为67. 05 ku,等电点为9. 02,碱性氨基酸(K、R)个数为49,酸性氨基酸(D、E)个数为42,疏水氨基酸(A、I、L、F、W、V)个数为241,极性氨基酸(N、C、Q、S、T、Y)个数为157。CbPIN1属于跨膜蛋白,含12个丝氨酸磷酸化位点,1个苏氨酸磷酸化位点。亚细胞定位结果显示,CbPIN1定位于细胞膜; CbPIN1在荠菜不同组织均有表达,其中,根中表达最高,种子中表达最低。原核表达显示CbPIN1蛋白大小87. 05 ku。过表达荠菜植株中CbPIN1基因表达量均显著增加。试验结果为进一步分析CbPIN1在荠菜器官发育中的作用奠定了基础。
To probe the biological function of PIN1 gene in Capsella bursa-pastoris,the cDNA coding sequence of PIN1( CbPIN1) gene was cloned by RT-PCR with the primers designed based on the reported Arabidopsis PIN1 amino acid sequence of Capsella bursa-pastoris in GenBank. The structure of CbPIN1 was analyzed via bioinformatics method. Also,CbPIN1 was expressed in prokaryotic cells. The CbPIN1 expression levels in Capsella bursa-pastoris tissues were detected using q-PCR method. In addition,the plant over-expression vector p BI121-CbPIN1 was constructed,and the transgenetic seedlings were also obtained. Sequencing analysis showed that the length of CbPIN1 gene c DNA was 1 869 bp,and the percentage of G + C was 49%. Further analysis indicated that CbPIN1 shared 93% homology with PIN1 of Arabidopsis. Information analysis demonstrated that the polypeptide encoded by CbPIN1 contained 622 amino acids. The weight and isoelectric point of CbPIN1 were 67. 05 ku and9. 02,respectively. Moreover,CbPIN1 had 49 basic amino acids,42 acidic amino acids,241 hydrophobic amino acids and 157 polar amino acids. As a transmembrane protein,CbPIN1 contained 12 serine phosphorylation sites and one threonine phosphorylation site. The result of subcellular localization indicated that CbPIN1 was located in cytomembrane. q-PCR results suggested that CbPIN1 expressed in all tissues could be detected,and the highest expression site was root,and the lowest site was seed. The data indicated that CbPIN1 protein was 87. 05 ku expressed in prokaryotic cells. In transgenetic seedlings of Capsella bursa-pastoris,CbPIN1 expression levels increased significantly. This research provided the foundation for further studying the function of CbPIN1 in Capsella bursa-pastoris organ development process.
To probe the biological function of PIN1 gene in Capsella bursa-pastoris,the cDNA coding sequence of PIN1( CbPIN1) gene was cloned by RT-PCR with the primers designed based on the reported Arabidopsis PIN1 amino acid sequence of Capsella bursa-pastoris in GenBank. The structure of CbPIN1 was analyzed via bioinformatics method. Also,CbPIN1 was expressed in prokaryotic cells. The CbPIN1 expression levels in Capsella bursa-pastoris tissues were detected using q-PCR method. In addition,the plant over-expression vector p BI121-CbPIN1 was constructed,and the transgenetic seedlings were also obtained. Sequencing analysis showed that the length of CbPIN1 gene c DNA was 1 869 bp,and the percentage of G + C was 49%. Further analysis indicated that CbPIN1 shared 93% homology with PIN1 of Arabidopsis. Information analysis demonstrated that the polypeptide encoded by CbPIN1 contained 622 amino acids. The weight and isoelectric point of CbPIN1 were 67. 05 ku and9. 02,respectively. Moreover,CbPIN1 had 49 basic amino acids,42 acidic amino acids,241 hydrophobic amino acids and 157 polar amino acids. As a transmembrane protein,CbPIN1 contained 12 serine phosphorylation sites and one threonine phosphorylation site. The result of subcellular localization indicated that CbPIN1 was located in cytomembrane. q-PCR results suggested that CbPIN1 expressed in all tissues could be detected,and the highest expression site was root,and the lowest site was seed. The data indicated that CbPIN1 protein was 87. 05 ku expressed in prokaryotic cells. In transgenetic seedlings of Capsella bursa-pastoris,CbPIN1 expression levels increased significantly. This research provided the foundation for further studying the function of CbPIN1 in Capsella bursa-pastoris organ development process.
引文
[1] Gao Y,Zhang Y,Zhang D,Dai X,Estelle M,Zhao Y.Auxin binding protein 1(ABP1)is not required for eitherauxin signaling or Arabidopsis development[J]. Proceed-ings of the National Academy of Sciences of the UnitedStates of America,2015,112(7):2275-2280. doi:10.1073/pnas. 1500365112.
[2] Yue J,Hu X,Huang J. Origin of plant auxin biosynthe-sis[J]. Trends in Plant Science,2014,19(12):764-770. doi:10. 1016/j. tplants. 2014. 07. 004.
[3] Chandler J W. Auxin response factors[J]. Plant,Cell&Environment,2016,39(5):1014-1028. doi:10. 1111/pce. 12662.
[4] Miao Z Q,Zhao P X,Mao J L,Yu L H,Yang Y,HuiT,Liu Z B,Xiang C B. HOMEOBOX PROTEIN52 medi-ates the crosstalk between ethylene and auxin signalingduring primary root elongation by modulating auxin Trans-port-Related gene expression[J]. The Plant Cell,2018,30(11):2761-2778.
[5] Ishida J K,Wakatake T,Yoshida S,Takebayashi Y,Kasahara H,Wafula E,Depamphilis C W,Namba S,Shirasu K. Local auxin biosynthesis mediated by a YUC-CA flavin monooxygenase regulates haustorium develop-ment in the parasitic plant Phtheirospermum japonicum[J]. The Plant Cell,2016,28(8):1795-1814. doi:10.1105/tpc. 16. 00310.
[6] Di Mambro R,De Ruvo M,Pacifici E,Salvi E,SozzaniR,Benfey P N,Busch W,Novak O,Ljung K,Di PaolaL,Mar e A F,Costantino P,Grieneisen V A,SabatiniS. Auxin minimum triggers the developmental Switch fromcell division to cell differentiation in the Arabidopsis root[J]. Proceedings of the National Academy of Sciences ofthe United States of America,2017,114(36):E7641-E7649. doi:10. 1073/pnas. 1705833114.
[7] Paque S,Weijers D. Q&A:auxin:the plant moleculethat influences almost anything[J]. BMC Biology,2016,14(1):67. doi:10. 1186/s12915-016-0291-0.
[8] Strader L C,Zhao Y. Auxin perception and downstreamevents[J]. Current Opinion in Plant Biology,2016,33:8-14. doi:10. 1016/j. pbi. 2016. 04. 004.
[9] Chaiwanon J,Wang Z Y. Spatiotemporal brassinosteroidsignaling and antagonism with auxin pattern stem cell dy-namics in Arabidopsis roots[J]. Current Biology,2015,25(8):1031-1042. doi:10. 1016/j. cub. 2015. 02. 046.
[10] Santos F,Teale W,Fleck C,Volpers M,Ruperti B,Palme K. Modelling polar auxin transport in develop-mental patterning[J]. Plant Biology,2010,12(S1):1438-8677. doi:10. 1111/j. 1438-8677. 2010. 00388. x.
[11] Wang J,Jin Z,Yin H,Yan B,Ren Z Z,Xu J,Mu CJ,Zhang Y,Wang M Q,Liua H. Auxin redistributionand shifts in PIN gene expression during Arabidopsisgrafting[J]. Russian Journal of Plant Physiology,2014,61(5):688-696. doi:10. 1134/S102144371405015X.
[12] Adamowski M,Friml J. PIN-dependent auxin transport:action,regulation,and evolution[J]. The Plant Cell,2015,27(1):20-32. doi:10. 1105/tpc. 114. 134874.
[13] Habets M E,offringa R. PIN-driven polar auxin trans-port in plant developmental plasticity:a key target forenvironmental and endogenous signals[J]. New Phytolo-gist,2014,203(2):362-377. doi:org/10. 1111/nph.12831.
[14]张洁,荆知敏.荠菜的生药鉴定[J].中药材,2012,35(8):1241-1243. doi:10. 13863/j. issn1001-4454. 2012. 08. 022.Zhang J, Jing Z M. Identification of crude drug ofCapsella bursa-pastoris[J]. Journal of Traditional Chi-nese Medicine,2012,35(8):1241-1243. doi:10.13863/j. issn1001-4454. 2012. 08. 022.
[15]刘晓柱,李银凤,赵燕,张学文.生长素对荠菜心皮发育的影响[J].河南农业科学,2018,47(8):1004-3268. doi:10. 15933/j. cnki. 1004-3268. 2018. 08. 015.Liu X Z,Li Y F,Zhao Y,Zhang X W. Effects of auxinon the carpel development of Capsella bursa-pastoris[J].Journal of Henan Agricultural Sciences,2018,47(8):1004-3268. doi:10. 15933/j. cnki. 1004-3268.2018. 08. 015.
[16] Zhang J,Nodzynski T,Pencik A,Rolcik J,Friml J.PIN phosphorylation is sufficient to mediate PIN polarityand direct auxin transport[J]. Proceedings of the Na-tional Academy of Sciences of the United States of Amer-ica,2010,107(2):918-922. doi:org/10. 1073/pnas.0909460107.
[17] Weller B,Zourelidou M,Frank L,Barbosa I C,Fast-ner A,Richter S,Jürqens G,Hammes U Z,Schwech-heimer C. Dynamic PIN-FORMED auxin efflux carrierphosphorylation at the plasma membrane controls auxinefflux-dependent growth[J]. Proceedings of the NationalAcademy of Sciences of the United States of America,2017,114(5):E887-E896. doi:org/10. 1073/pnas.1614380114.
[18] Omelyanchuk N A,Kovrizhnykh V V,Oshchepkova EA,Pasternak T,Palme K,Mironova1 V V. A detailedexpression map of the PIN1 auxin transporter in Arabi-dopsis thaliana root[J]. BMC Plant Biology,2016,16(S1):5. doi:10. 1186/s12870-015-0685-0.
[19] Zhang K X,Xu H H,Gong W,Jin Y,Shi Y Y,YuanT T,Li J,Lu Y T. Proper PIN1 distribution is neededfor root negative phototropism in Arabidopsis[J]. PLo SOne,2014,9(1):e85720. doi:10. 1371/journal. pone.0085720.
[20] Li K,Kamiya T,Fujiwara T. Differential roles of PIN1and PIN2 in root meristem maintenance under Low-Bconditions in Arabidopsis thaliana[J]. Plant&CellPhysiology,2015,56(6):1205-1214. doi:10. 1093/pcp/pcv047.
[2] Yue J,Hu X,Huang J. Origin of plant auxin biosynthe-sis[J]. Trends in Plant Science,2014,19(12):764-770. doi:10. 1016/j. tplants. 2014. 07. 004.
[3] Chandler J W. Auxin response factors[J]. Plant,Cell&Environment,2016,39(5):1014-1028. doi:10. 1111/pce. 12662.
[4] Miao Z Q,Zhao P X,Mao J L,Yu L H,Yang Y,HuiT,Liu Z B,Xiang C B. HOMEOBOX PROTEIN52 medi-ates the crosstalk between ethylene and auxin signalingduring primary root elongation by modulating auxin Trans-port-Related gene expression[J]. The Plant Cell,2018,30(11):2761-2778.
[5] Ishida J K,Wakatake T,Yoshida S,Takebayashi Y,Kasahara H,Wafula E,Depamphilis C W,Namba S,Shirasu K. Local auxin biosynthesis mediated by a YUC-CA flavin monooxygenase regulates haustorium develop-ment in the parasitic plant Phtheirospermum japonicum[J]. The Plant Cell,2016,28(8):1795-1814. doi:10.1105/tpc. 16. 00310.
[6] Di Mambro R,De Ruvo M,Pacifici E,Salvi E,SozzaniR,Benfey P N,Busch W,Novak O,Ljung K,Di PaolaL,Mar e A F,Costantino P,Grieneisen V A,SabatiniS. Auxin minimum triggers the developmental Switch fromcell division to cell differentiation in the Arabidopsis root[J]. Proceedings of the National Academy of Sciences ofthe United States of America,2017,114(36):E7641-E7649. doi:10. 1073/pnas. 1705833114.
[7] Paque S,Weijers D. Q&A:auxin:the plant moleculethat influences almost anything[J]. BMC Biology,2016,14(1):67. doi:10. 1186/s12915-016-0291-0.
[8] Strader L C,Zhao Y. Auxin perception and downstreamevents[J]. Current Opinion in Plant Biology,2016,33:8-14. doi:10. 1016/j. pbi. 2016. 04. 004.
[9] Chaiwanon J,Wang Z Y. Spatiotemporal brassinosteroidsignaling and antagonism with auxin pattern stem cell dy-namics in Arabidopsis roots[J]. Current Biology,2015,25(8):1031-1042. doi:10. 1016/j. cub. 2015. 02. 046.
[10] Santos F,Teale W,Fleck C,Volpers M,Ruperti B,Palme K. Modelling polar auxin transport in develop-mental patterning[J]. Plant Biology,2010,12(S1):1438-8677. doi:10. 1111/j. 1438-8677. 2010. 00388. x.
[11] Wang J,Jin Z,Yin H,Yan B,Ren Z Z,Xu J,Mu CJ,Zhang Y,Wang M Q,Liua H. Auxin redistributionand shifts in PIN gene expression during Arabidopsisgrafting[J]. Russian Journal of Plant Physiology,2014,61(5):688-696. doi:10. 1134/S102144371405015X.
[12] Adamowski M,Friml J. PIN-dependent auxin transport:action,regulation,and evolution[J]. The Plant Cell,2015,27(1):20-32. doi:10. 1105/tpc. 114. 134874.
[13] Habets M E,offringa R. PIN-driven polar auxin trans-port in plant developmental plasticity:a key target forenvironmental and endogenous signals[J]. New Phytolo-gist,2014,203(2):362-377. doi:org/10. 1111/nph.12831.
[14]张洁,荆知敏.荠菜的生药鉴定[J].中药材,2012,35(8):1241-1243. doi:10. 13863/j. issn1001-4454. 2012. 08. 022.Zhang J, Jing Z M. Identification of crude drug ofCapsella bursa-pastoris[J]. Journal of Traditional Chi-nese Medicine,2012,35(8):1241-1243. doi:10.13863/j. issn1001-4454. 2012. 08. 022.
[15]刘晓柱,李银凤,赵燕,张学文.生长素对荠菜心皮发育的影响[J].河南农业科学,2018,47(8):1004-3268. doi:10. 15933/j. cnki. 1004-3268. 2018. 08. 015.Liu X Z,Li Y F,Zhao Y,Zhang X W. Effects of auxinon the carpel development of Capsella bursa-pastoris[J].Journal of Henan Agricultural Sciences,2018,47(8):1004-3268. doi:10. 15933/j. cnki. 1004-3268.2018. 08. 015.
[16] Zhang J,Nodzynski T,Pencik A,Rolcik J,Friml J.PIN phosphorylation is sufficient to mediate PIN polarityand direct auxin transport[J]. Proceedings of the Na-tional Academy of Sciences of the United States of Amer-ica,2010,107(2):918-922. doi:org/10. 1073/pnas.0909460107.
[17] Weller B,Zourelidou M,Frank L,Barbosa I C,Fast-ner A,Richter S,Jürqens G,Hammes U Z,Schwech-heimer C. Dynamic PIN-FORMED auxin efflux carrierphosphorylation at the plasma membrane controls auxinefflux-dependent growth[J]. Proceedings of the NationalAcademy of Sciences of the United States of America,2017,114(5):E887-E896. doi:org/10. 1073/pnas.1614380114.
[18] Omelyanchuk N A,Kovrizhnykh V V,Oshchepkova EA,Pasternak T,Palme K,Mironova1 V V. A detailedexpression map of the PIN1 auxin transporter in Arabi-dopsis thaliana root[J]. BMC Plant Biology,2016,16(S1):5. doi:10. 1186/s12870-015-0685-0.
[19] Zhang K X,Xu H H,Gong W,Jin Y,Shi Y Y,YuanT T,Li J,Lu Y T. Proper PIN1 distribution is neededfor root negative phototropism in Arabidopsis[J]. PLo SOne,2014,9(1):e85720. doi:10. 1371/journal. pone.0085720.
[20] Li K,Kamiya T,Fujiwara T. Differential roles of PIN1and PIN2 in root meristem maintenance under Low-Bconditions in Arabidopsis thaliana[J]. Plant&CellPhysiology,2015,56(6):1205-1214. doi:10. 1093/pcp/pcv047.