| 摘要: |
| 【目的】克隆小黑杨（Populus×xiaohei T.S.Hwang et Liang）RAV1和RAV2基因，分析这2个基因在非生物胁迫下的表达模式，为进一步研究小黑杨基因功能提供理论依据。【方法】以抗逆性强的小黑杨根为试材，克隆2个RAV家族成员。利用生物信息学方法对小黑杨2个RAV基因的理化性质、保守基序、保守结构域、系统进化进行分析，并采用半定量 RT-PCR方法探讨这2个基因在小黑杨顶芽、叶、木质部、韧皮部、根、第1～14片叶、第1～11茎节中的表达情况，以及在CdCl2、NaCl、NaHCO3、PEG和ABA胁迫下的表达特性。【结果】从小黑杨中克隆出2个RAV基因,命名为PsnRAV1和PsnRAV2。PsnRAV1编码405个氨基酸，预测分子质量约为99.46 ku，等电点5.06，不存在信号肽；PsnRAV2编码407个氨基酸，预测分子质量约为99.33 ku，等电点5.06，存在信号肽；2个基因均位于细胞核上。保守结构域和保守基序分析结果表明，小黑杨RAV蛋白具有RAV家族特有的AP2和B3这2个保守蛋白结构域，基序1、2、3是构成2个结构域的主要基序。系统进化分析表明，20种植物的RAV蛋白分为3组，其中PsnRAV1和PsnRAV2与毛果杨（Populus trichocarpa）RAV同源性很高。半定量RT-PCR分析结果显示，2个PsnRAVs基因在小黑杨的顶芽、叶、木质部、韧皮部和根中均有表达；且随着叶和茎的发育，2个基因的表达量均呈现升高的趋势。在CdCl2、NaCl、NaHCO3模拟的重金属及盐胁迫下，2个基因的表达模式虽不相同，但在根部均受到诱导；在PEG模拟的干旱胁迫下，PsnRAV1和PsnRAV2在叶片和根部均有响应，其中在根部受到的诱导更为明显，尤以PsnRAV2的表达更为显著；此外，PsnRAV1和PsnRAV2也受到ABA的诱导，其在叶中的表达量在初期表现为上升，而后期下降。【结论】2个PsnRAVs基因与小黑杨的抗逆性密切相关，可能参与了小黑杨的非生物胁迫应答，其中PsnRAV2在胁迫下的作用更为显著。 |
| 关键词: 小黑杨；RAV；生物信息学 非生物胁迫；基因表达分析 林木遗传育种 |
| DOI： |
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| 基金项目:国家转基因生物新品种培育科技重大专项（2018ZX08020002）；东北林业大学大学生创新项目（201910225527） |
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| Cloning and expression analysis of PsnRAV1 and PsnRAV2 in Populus×xiaohei T.S. Hwang et Liang |
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JIN Chunhui,WEN Xiaohui,LIU Yuting,et al
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| Abstract: |
| 【Objective】The RAV1 and RAV2 genes of Populus×xiaohei T.S.Hwang et Liang were cloned and their expression patterns under abiotic stress were analyzed to provide basis for further studies on their functions.【Method】Two members of the RAV family were cloned from roots of the highly resistant Populus×xiaohei T.S. Hwang et Liang.Bioinformatics was used to analyze their physicochemical properties,conserved motifs,conserved structural domains and phylogeny.The semi-quantitative RT-PCR was used to investigate their expressions in terminal buds,leaves,xylem,bast,roots,1st-14th leaves and 1st-11th stem segments of Populus×xiaohei T.S.Hwang et Liang,as well as their expression characteristics under CdCl2,NaCl,NaHCO3,PEG and ABA stresses.【Result】Two RAV genes were cloned from Populus×xiaohei T.S. and named as PsnRAV1 and PsnRAV2.PsnRAV1 encoded 405 amino acids with a predicted molecular weight of 99.46 ku,an isoelectric point of 5.06,and no signal peptide.PsnRAV2 encoded 407 amino acids with a predicted molecular weight of 99.33 ku,an isoelectric point of 5.06,and a signal peptide.Both genes were located on the nucleus.Analysis of the conserved structural domains and conserved motifs showed that the RAV proteins of Populus×xiaohei T.S had two conserved protein structural domains of AP2 and B3,which are unique to the RAV family.The motifs 1,2 and 3 were the main motifs constituting the two structural domains.Phylogenetic analysis showed that the RAV proteins of 20 plants were divided into 3 groups,of which PsnRAV1 and PsnRAV2 were highly homologous to the RAV of Populus trichocarpa.Semi-quantitative RT-PCR analysis showed that the two PsnRAVs genes were expressed in terminal buds,leaves,xylem,bast and roots of small black poplar.Their expression showed an increasing trend with the development of leaves and stems.Under heavy metal and salt stresses simulated by CdCl2,NaCl and NaHCO3,their expression patterns were different,except they were all induced in roots.Under drought stress simulated by PEG,PsnRAV1 and PsnRAV2 responded in both leaves and roots,with more pronounced induction of PsnRAV2 in roots.The expressions of PsnRAV1 and PsnRAV2 were also induced by ABA,and their expressions in leaves decreased after initial increase.【Conclusion】These two PsnRAVs genes closely related to the stress resistance of Populus×xiaohei T.S.Hwang et Liang and may be involved in the abiotic stress responses,with PsnRAV2 playing a more significant role under stress. |
| Key words: Populus×xiaohei T.S.Hwang et Liang RAV bioinformattics abiotic stress gene expression analysis tree genetic breeding |
