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

Abstract:【Objective】KNOTTED1 like homeobox (KNOX) proteins are a class of transcription factors 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 domain, 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 generation 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 domain, which is located between the KNOX2 domain and the HD domain, is involved in nuclear localization 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 method and the first strand cDNA was synthesized by PrimeScript^TM 1^st Strand cDNA Synthesis Kit. The fulllength cDNA sequences of the MdKNOXs were isolated by RT- PCR method, the obtained cDNA sequences 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://bioinformatics.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 expression 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.【Results】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 MdKNOX protein contained MEINOX, HD and ELK domains. The Phylogenetic analyses revealed that KNOX proteins were divided into three groups: KNOX Ⅰ group, KNOX Ⅱ group and KNOX M group. The KNOX Ⅰ group was classed into STM-like subgroup, KNAT2/6-like subgroup and KNAT1/ BP-like subgroup. The KNOX Ⅱ group was classed into KNAT7-like subgroup and KANT3/4/5-like subgroup. MdKNOX1 and MdKNOX19 belonged to KANT3/4/5-like subgroup. MdKNOX2 and MdKNOX5 belonged to KNAT7-like subgroup. MdKNOX10 and MdKNOX22 belonged to STM-like subgroup. 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 elements, 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 different tissues of apple (GSE42873) were downloaded using the NCBI GEO database to detect the expression of the MdKNOXs in different tissues. The array results indicated that the MdKNOX genes were expressed at different levels in the detected tissues. Among these MdKNOX genes, the MdKNOX1, MdKNOX2 and MdKNOX5 were relatively highly expressed in the detected tissues; The MdKNOX10, MdKNOX13, 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 MdKNOX5 were down-regulated under the salt stress; the transcription level of the MdKNOX2 was downregulated 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 isolated 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 MdOFP proteins. These results would provide a strong theoretical basis and a valuable reference for analysis of the biological functions of the MdKNOX transcription factors in apple growth, development and stress and also for construction of regulatory networks.