苹果MdWRKY35基因克隆与表达分析

Cloning and expression analysis of MdWRKY35 gene in apple

WRKY转录因子是植物中发现的一种新型转录调控因子,在响应非生物胁迫、调控生长发育等方面具有重要作用。然而, WRKY基因如何调控开花及通过调控植物发育从而提高抗逆等方面的研究鲜有报道。本研究以新疆红肉苹果(Malus neidzwetzkyana)杂种二代群体(M. niedzwetzkyana/M. pumila ‘Fuji’/M. pumila ‘Gala’)的幼嫩叶片为试材,克隆得到一个新的WRKY转录因子,命名为MdWRKY35,测序发现其包含804 bp的完整开放阅读框(ORF),编码267个氨基酸,预测其编码蛋白质分子质量为30.83 kDa,理论等电点为4.81,定位于苹果基因组的第11号染色体上,由3个外显子和2个内含子构成。MdWRKY35与白梨(Pyrus×bretschneideri)的同源性高达93.28%,具有WRKY转录因子家族典型的保守结构域,属于第II组WRKY蛋白家族。组织特异性分析表明MdWRKY35在根中表达最高,在茎尖及叶片的表达次之。多种逆境胁迫因子如高盐、干旱、低温以及脱落酸(ABA)等诱导后,‘嘎啦’苹果(M. pumila ‘Gala’)组培苗中的MdWRKY35呈上调表达; MdWRKY35蛋白可能定位于细胞核中;荧光素酶(LUC)活性检测试验表明, MdWRKY35可能促进MdFT的表达。研究MdWRKY35在抗逆调节及成花进程中的功能可以为进一步完善红肉苹果育种的理论与技术体系提供参考。

WRKY transcription factor is a novel transcriptional regulator found in plants,which plays an important role in responding to abiotic stress and regulating growth and development.However,there are few reports on how WRKY gene regulates flowering and enhances stress resistance by regulating plant development.In this study,the young leaves of hybrid second generation group of Malus neidzwetzkyana(M.niedzwetzkyana/M.pumila‘Fuji’/M.pumila‘Gala’)were used as test materials,a new WRKY transcription factor was cloned and named MdWRKY35.It was sequenced and found to contain a complete 804 bp open reading frame(ORF),encoding 267 amino acids.It was predicted that the protein molecular mass is 30.83 kDa and the isoelectric point is 4.81,which was located on chromosome 11 of the apple genome and consisted of three exons and two introns.MdWRKY35 shares 93.28%homology with Pyrus×bretschneideri,has a typical conserved domain of WRKY transcription factor family,and belongs to group II WRKY protein family.Tissue specificity analysis showed that MdWRKY35 was the highest in roots and highly expressed in stem tips and leaves.The expression of MdWRKY35 in‘Gala’plantlet was significantly increased after induction by many stress factors such as high salt,drought,low temperature and abscisic acid(ABA)treatment.MdWRKY35 protein might be localized in the nucleus.Luciferase activity assay showed that MdWRKY35 might promote the expression of MdFT.Studying the function of MdWRKY35 in the process of stress tolerance and flowering could provide a reference for further improving the theory and technical system of red-fleshed apple breeding.