7个苹果KNOX转录因子基因克隆、表达和蛋白互作分析

Cloning,expression and protein-protein interaction analysis of seven KONX transcription factors in apple

【目的】KNOTTED1likehomeobox(KNOX)蛋白在植物生长发育等多个生物学过程中发挥着重要作用。以紫弘富士为材料,分离了多个苹果(Malusdomestica)KONX基因,研究其结构域、进化分析、组织表达、非生物胁迫响应及其与MdOFP相互作用情况。【方法】使用RT-PCR技术克隆获得7个MdKNOX基因并进行生物信息学分析。利用Array技术检测MdKNOX基因在苹果不同组织中的表达模式。使用RT-qPCR技术检测MdKNOX基因在盐胁迫和渗透胁迫下的表达模式。通过Y2H实验检测了MdKNOX蛋白与MdOFP蛋白的互作情况。【结果】测序结果表明,获得了7个KNOX转录因子cDNA:MdKNOX1、MdKNOX2、MdKNOX5、MdKNOX10、MdKNOX13、MdKNOX16和Md-KNOX22(GenBank登录号:MG021644~MG021650)。结构域分析表明,获得的7个MdKNOX蛋白序列均含有MEI-NOX、HD和ELK结构域。进化分析表明,MdKNOX1、MdKNOX2和MdKNOX5属于KNOXⅡ亚组;MdKNOX10、MdKNOX13、MdKNOX16和MdKNOX22属于KNOXⅠ亚组。启动子顺式作用元件分析结果表明,7个MdKNOX基因启动子上包含多个顺式作用元件。Array分析结果显示,MdKNOX基因具有不同的组织表达模式。实时荧光定量PCR分析结果表明,盐胁迫处理下,MdKNOX13的相对表达水平上调,而MdKNOX1、MdKNOX2和MdKNOX5的相对表达水平下调;渗透胁迫处理下,MdKNOX2的转录水平下调。酵母双杂交结果显示,MdKNOX1/22蛋白能与MdOFP6蛋白相互作用,MdKNOX5蛋白能与多个MdOFP蛋白相互作用,且MdKNOX5蛋白与MdOFP蛋白相互作用,HD区域是互作必需的。【结论】这些结果为苹果KNOX转录因子在生长、发育和逆境下生物学功能的解析、调控网络的构建提供了强有力的理论基础和参考。

【Objective】KNOTTED1 like homeobox(KNOX)proteins are a class of transcription fac-tors that can regulate gene expression via binding with promoters of down-stream target genes.KNOX genes belong to the TALE(Three Amino acid Loop Extension)homeodomain subfamily,and contain multiple family members in plants.KNOX proteins contain four conserved domains:the KNOX1 do-main,which is a conserved region of about 39 amino acids at the N-terminal of the KNOX protein,has shown to be important for generating the altered phenotypes caused by ectopic KNOX gene expression;the KNOX2 domain,which is critical for dimer formation and transactivation,is essential for the gener-ation of abnormal phenotypes in transgenics;the HD domain,which is located in the C-terminal of the KNOX protein,is involved in DNA binding and possibly in homodimer formation;and the ELK do-main,which is located between the KNOX2 domain and the HD domain,is involved in nuclear localiza-tion and transcriptional repression.The various studies have shown that KNOX proteins play important roles in many biological processes such as plant growth and development.In this study,various apple(Malus domestica)KONX genes were isolated using Zihong Fuji as plant material,and their domains,evolutionary analysis,tissue expression,abiotic stress response and interaction with MdOFP proteins were studied.【Methods】The total RNA was extracted from Zihong Fuji leaves using the CTAB meth-od and the first strand cDNAwas synthesized by PrimeScriptTM 1st Strand cDNASynthesis Kit.The full-length cDNA sequences of the MdKNOXs were isolated by RT-PCR method,the obtained cDNA se-quences and the deduced amino acid sequences were analyzed with DNAMAN 6.0.3.The phylogenetic tree was constructed using the MEGA 6.0 software to investigate the evolutional relationship between MdKNOXs and other KNOX proteins from Arabidopsis and rice.The PlantCARE(https://bioinformat-ics.psb.ugent.be/webtools/plantcare/html/)was used to annotate elements,and elements related to growth,development,hormones,and stress were selected for location distribution mapping.The expres-sion levels of the MdKNOXs were detected in 16 different tissues using array from NCBI GEO database.The expression levels of the MdKNOXs were detected under 150 mmol·L-1 NaCl and 300 mmol·L-1 mannitol treatments using qRT-PCR method with BIO-RAD IQ5 Real-time PCR Detection Systems(USA).The interaction between MdKNOX proteins and MdOFP proteins was detected by Y2H.【Re-sults】Totally seven MdKNOX genes(designated as MdKNOX1,MdKNOX2,MdKNOX5,MdKNOX10,MdKNOX13,MdKNOX16 and MdKNOX22;GenBank Accession No.MG021644-MG021650)were isolated from Zihong Fuji leaves using RT-PCR method.The cDNAs of the MdKNOXs contained open reading frame(ORF)of 1083,867,867,1005,990,1062 and 1002 bp in length which encoded proteins of 360,288,288,333,329,353 and 332 amino acid residues with calculated molecular weight(MW)of 40.78,32.89,32.86,37.72,36.92,40.21 and 37.67 kD and predicted isoeletric point(pI)of 5.15,6.65,6.78,6.61,5.20,5.65 and 6.57,respectively.Conserved domain analysis showed that all the 7 Md-KNOX protein contained MEINOX,HD and ELK domains.The Phylogenetic analyses revealed that KNOX proteins were divided into three groups:KNOXⅠgroup,KNOXⅡgroup and KNOX M group.The KNOXⅠgroup was classed into STM-like subgroup,KNAT2/6-like subgroup and KNAT1/BP-like subgroup.The KNOXⅡgroup was classed into KNAT7-like subgroup and KANT3/4/5-like subgroup.MdKNOX1 and MdKNOX19 belonged to KANT3/4/5-like subgroup.MdKNOX2 and Md-KNOX5 belonged to KNAT7-like subgroup.MdKNOX10 and MdKNOX22 belonged to STM-like sub-group.MdKNOX13,MdKNOX15 and MdKNOX16 belonged to KNAT2/6-like subgroup.The results of cis acting elements showed that promoters of the MdKNOX genes contained multiple cis acting ele-ments,including methyl jasmonate,salicylic acid,auxin,gibberellin,ethylene,abscisic acid,anaerobic induction,wound,defense and stress,MYB binding site was involved in drought-inducibility,heat stress,low-temperature and fungal elicitor responsive elements.The expression profiles of the 16 differ-ent tissues of apple(GSE42873)were downloaded using the NCBI GEO database to detect the expres-sion of the MdKNOXs in different tissues.The array results indicated that the MdKNOX genes were ex-pressed at different levels in the detected tissues.Among these MdKNOX genes,the MdKNOX1,Md-KNOX2 and MdKNOX5 were relatively highly expressed in the detected tissues;The MdKNOX10,Md-KNOX13,MdKNOX16 and MdKNOX22 were relatively highly expressed in the leaf(M49),flower(M49)and fruit(M20-100DAM and M20-harvest).RT-qPCR results showed that the transcription level of the MdKNOX13 was induced,while the transcription level of the MdKNOX1,MdKNOX2 and Md-KNOX5 were down-regulated under the salt stress;the transcription level of the MdKNOX2 was down-regulated under the osmotic stress.Y2H experiment showed that MdKNOX1 and MdKNOX22 proteins interacted with MdOFP6 protein,MdKNOX5 protein interacted with MdOFP1,MdOFP4,MdOFP14 and MdOFP16 proteins,and HD domain of MdKNOX5 protein was essential for its interaction with MdOFP1,MdOFP4,MdOFP14 and MdOFP16 proteins.【Conclusion】Seven MdKNOX genes were iso-lated and constitutively expressed in all examined tissues,and they showed different expression patterns under salt or mannitol treatment.In addition,MdKNOX1,MdKNOX5 and MdKNOX22 could interact with multiple MdOFPproteins.These results would provide a strong theoretical basis and a valuable ref-erence for analysis of the biological functions of the MdKNOX transcription factors in apple growth,de-velopment and stress and also for construction of regulatory networks.