| 摘要: |
| 【目的】克隆小麦TaWRKY28基因，通过农杆菌浸染法将其转化至拟南芥，研究转基因拟南芥植株生理生化指标变化，探究其在抵御非生物胁迫中的作用。【方法】采用RT-PCR方法，从小麦百农207 cDNA中克隆得到TaWRKY28基因，利用ProtParam等软件对其氨基酸序列进行生物信息学分析；利用实时荧光定量PCR方法，分析TaWRKY28基因的组织表达特异性及在NaCl、ABA、H2O2、干旱和低温逆境胁迫下的表达情况；构建基因超表达载体，通过花序浸染法转化拟南芥，选取TaWRKY28基因相对表达量较高的转基因T3拟南芥株系，测定拟南芥超氧化物歧化酶（SOD）和过氧化物酶（POD）的活性以及丙二醛（MDA）、H2O2和脯氨酸含量，研究TaWRKY28对拟南芥抗旱性的影响。【结果】成功克隆获得小麦TaWRKY28基因，该基因ORF全长为978 bp，编码325个氨基酸，相对分子质量为34.87 ku，理论等电点为9.27，含有1个WRKY保守结构域和1个C2H2锌指结构域，属于WRKY基因家族的第Ⅱ类成员。序列系统进化分析结果显示，小麦TaWRKY28与大麦HvWRKY2亲缘关系最近。亚细胞定位显示该基因位于细胞核中。qRT-PCR结果表明，TaWRKY28在小麦根、茎、叶、雌蕊和雄蕊组织中均有表达，但具有组织特异表达性，在叶中表达量最高，雄蕊中次之，雌蕊中表达量最低，且受NaCl、ABA、H2O2、干旱和低温胁迫后TaWRKY28表达会增强。对TaWRKY28转基因拟南芥植株的抗旱性分析表明，在干旱条件下，转基因植株的表型优于野生型，SOD、POD活性和脯氨酸含量显著增加，而H2O2和MDA含量显著降低，转基因植株抗旱性显著提高。【结论】克隆了小麦TaWRKY28基因，该基因可以增强转基因拟南芥植株的抗旱能力。 |
| 关键词: 小麦 转录因子 TaWRKY28基因 抗旱性 |
| DOI： |
| 分类号: |
| 基金项目:河南省科技攻关项目（192102110031，212102110067） |
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| Cloning and function against drought stress of TaWRKY28 transcription factor gene |
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YU Yongang,SUI Xiaotian,ZHANG Lei,et al
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| Abstract: |
| 【Objective】This study cloned sequences of TaWRKY28 gene in wheat and transformed TaWRKY28 gene into Arabidopsis by Agrobacterium infection to study the changes of physiological and biochemical indexes of transgenic plants and their role in resisting abiotic stress.【Method】Based on the information of TaWRKY28 gene,the full-length cDNA of TaWRKY28 was isolated from wheat leaves by RT-PCR and analysis of bioinformatics and phylogenetic tree were carried out.The qRT-PCR method was used to analyze the tissue expression specificity and expression of TaWRKY28 under NaCl,ABA,H2O2,cold and drought stresses.Expression vectors carrying TaWRKY28 were constructed and transformed into wild Arabidopsis via floral dip method.Independent lines of T3 with high relative expression of TaWRKY28 were selected for further analysis based on the activities of superoxide dismutase (SOD),peroxidase (POD) and MDA as well as H2O2 and proline contents of transgenic Arabidopsis plant under drought stress.【Result】The TaWRKY28 gene was cloned successfully and its ORF was 978 bp,encoding 325 amino acid residues.Its molecular weight was 34.87 ku and theoretical pI was 9.27.The amino acid sequence contained a conserved WRKY domain and a zinc finger structure (C2H2),belonging to the class Ⅱ protein of WRKY transcription factor family.Phylogenetic analysis revealed that TaWRKY28 was mostly close to HvWRKY2.Subcellular localization showed that the gene was located in the nucleus.Spatiotemporal expression analysis showed that TaWRKY28 constitutively expressed in roots,stems,leaves,pistils and stamens.However,the expression was tissue specific with the highest expression in leaves,followed by stamens,and the lowest expression in pistils.Meanwhile,its expression was enhanced under stress of low temperature,drought,salt,ABA and H2O2.The TaWRKY28 transgenetic plants under drought stress were better than control.The activities of SOD and POD and content of proline were significantly increased,while contents of H2O2 and MDA were significantly reduced.The drought resistance of transgenic plants was significantly improved.【Conclusion】TaWRKY28 was isolated from wheat and it was verified that TaWRKY28 plays an important role in increasing plants tolerance against drought stress. |
| Key words: wheat transcription factor TaWRKY28 gene drought resistance |
