烟草HD-ZIP转录因子HD20的克隆及纤维分化功能的研究
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摘要
旨在揭示烟草HD20在植物纤维分化中的作用。基于前期烟草基因组数据库设计特异性引物,通过RT-PCR克隆获得基因cDNA序列,将其命名为HD20,NCBI登录号为KC339003。该基因全长762 bp,编码253个氨基酸,亚细胞定位于细胞核,其编码的HD-ZIP蛋白由2个不同的结构域组成,包括N末端含有60个氨基酸的HD结构域和其后28个氨基酸组成的ZIP基序。构建植物表达载体,通过农杆菌蘸花法遗传转化拟南芥,经筛选获得阳性转基因植株,其石蜡切片显示,茎部木质部较对照发达。HD20对烟草及拟南芥的纤维分化具有调控作用。
To understand the role of HD20 in the specification of fiber differentiation,we cloned the HD20 gene from tobacco by RT-PCR method. The cDNA sequence was cloned according to specific primers designed by the genomic database in tobacco and named as HD20 (GenBank accession number :KC339003). Sequence analysis showed that the cDNA sequence of HD20 was 762 bp and encoded 253 amino acids. Subcellular localization assays showed that it appeared in the nuclei. The HD-ZIP protein contained two significant domains,including the HD domain with 60 amino acids at the N-terminus and a ZIP matrix with 28 amino acids. We introduced HD20 into the expression vector pSH-737,transferred it by Agrobacterium mediated-dip flower method,and obtained positive transgenic plants for paraffin sections. The results of paraffin section showed that the xylem of stem was more developed than the control group. In conclusion,the result provided evidence that HD20 plays a role in the spatial control of fiber differentiation. The analysis of the regulatory effect of HD20 on tobacco and Arabidopsis helps to create a theoretical base for studying HD20 function.
To understand the role of HD20 in the specification of fiber differentiation,we cloned the HD20 gene from tobacco by RT-PCR method. The cDNA sequence was cloned according to specific primers designed by the genomic database in tobacco and named as HD20 (GenBank accession number :KC339003). Sequence analysis showed that the cDNA sequence of HD20 was 762 bp and encoded 253 amino acids. Subcellular localization assays showed that it appeared in the nuclei. The HD-ZIP protein contained two significant domains,including the HD domain with 60 amino acids at the N-terminus and a ZIP matrix with 28 amino acids. We introduced HD20 into the expression vector pSH-737,transferred it by Agrobacterium mediated-dip flower method,and obtained positive transgenic plants for paraffin sections. The results of paraffin section showed that the xylem of stem was more developed than the control group. In conclusion,the result provided evidence that HD20 plays a role in the spatial control of fiber differentiation. The analysis of the regulatory effect of HD20 on tobacco and Arabidopsis helps to create a theoretical base for studying HD20 function.
引文
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[21]Zhong R,Ye ZH.Alteration of auxin polar transport in the Arabidopsis ifl1 mutants[J].Plant Physiology,2001,126(2):549-563.
[22]Manavella PA,Dezar CA,Bonaventure G,et al.HAHB4,a sunflower HD-Zip protein,integrates signals from the jasmonic acid and ethylene pathways during wounding and biotic stress responses[J].Plant Journal for Cell&Molecular Biology,2008,56(3):376-388.
[2]Baima S,Possenti M,Matteucci A,et al.The Arabidopsis ATHB-8 HD-Zip protein acts as a differentiation-promoting transcription factor of the vascular meristems[J].Plant Physiology,2001,126(2):643-655.
[3]Ariel FD,Manavella PA,Dezar CA,et al.The true story of the HD-Zip family[J].Trends in Plant Science,2007,12(9):419-426.
[4]Harris JC,Hrmova M,Lopato S,et al.Modulation of plant growth by HD-Zip class I and II transcription factors in response to environmental stimuli[J].New Phytology,2011,190(4):823-837.
[5]单淳敏.棉纤维伸长的关键调控因子Gh HOX3的研究[D].北京:中国科学院大学,2014.
[6]M.阿拉姆,M.S.伊斯拉姆,B.艾哈迈德,等.编码来自长蒴黄麻和圆果种黄麻的同源框-亮氨酸拉链蛋白hat22(hd-zip蛋白22)的核苷酸序列和使用方法:中国,105143247 A[P].2015-12-09.
[7]Du J,Miura E,Robischon M,et al.The populus class III HD ZIPtranscription factor,POPCORONA,affects cell differentiation during secondary growth of woody stems[J].PLo S One,2011,6(2):e17458.
[8]Meijer AH,Kam RJD,D’Erfurth I,et al.HD-Zip proteins of families I and II from rice:interactions and functional properties[J].Molecular Genetics and Genomics,2000,263(1):12-21.
[9]秦永芳,李登弟,李学宝.植物HD-Zip转录因子研究进展[J].细胞生物学杂志,2009,31(4):514-520.
[10]Zhong R,Taylor JJ,Ye ZH.Transformation of the collateral vascular bundles into amphivasal vascular bundles in an Arabidopsis mutant[J].Plant Physiology,1999,120(1):53-64.
[11]王瑞,鲁丽丽,刘拓,等.棉花中HD-ZipⅣ家族基因的全基因组鉴定及特征分析[J].分子植物育种,2017,15(8):2921-2936.
[12]Raud B,Chan RL,Baldwin IT,et al.RNAi-mediated silencing of the HD-Zip gene HD20 in Nicotiana attenuata affects benzyl acetone emission from corollas via ABA levels and the expression of metabolic genes[J].BMC Plant Biol,2012,12(1):60-75.
[13]吴拥军,赵德刚,许文钊,等.转Ch IFN-γ基因烟草抗虫作用初步研究[J].云南大学学报:自然科学版,2009,31(6):629-631.
[14]Wu YJ,Wu YJ,Luo X,et al.Identification of differentially expressed genes that potentially confer pest resistance in transgenic Ch IFN-?3tobacco[J].Gene,2014,543(2):181-189.
[15]Li D,Ren W,Wang X,et al.A modified method using TRIzol?,reagent and liquid nitrogen produces high-quality RNA from rat pancreas[J].Applied Biochemistry&Biotechnology,2009,158(2):253-261.
[16]徐锦涛.八棱海棠actin基因的克隆与GO基因转化拟南芥研究[D].南京:南京农业大学,2008.
[17]蔡雁峰.拟南芥/柴胡高度不对称体细胞杂种的研究[D].济南:山东大学,2007.
[18]RéDA,Dezar CA,Chan RL,et al.Nicotiana attenuata Na HD20plays a role in leaf ABA accumulation during water stress,benzylacetone emission from flowers,and the timing of bolting and flower transitions[J].J Exp Bot,2011,62(1):155-166.
[19]Sachs T.The induction of fibre differentiation in peas[J].Annals of Botany,1972,36(144):189-197.
[20]Aloni R.Polarity of induction and pattern of primary phloem fiber differentiation in coleus[J].American Journal of Botany,1976,63(6):877-889.
[21]Zhong R,Ye ZH.Alteration of auxin polar transport in the Arabidopsis ifl1 mutants[J].Plant Physiology,2001,126(2):549-563.
[22]Manavella PA,Dezar CA,Bonaventure G,et al.HAHB4,a sunflower HD-Zip protein,integrates signals from the jasmonic acid and ethylene pathways during wounding and biotic stress responses[J].Plant Journal for Cell&Molecular Biology,2008,56(3):376-388.