Genome-wide identification and expression analysis of the MADS- box gene family in Actinidia chinensis

Abstract：【Objective】MADS-box transcription factor plays an important role in plant growth and development, such as regulating abiotic stress response, transition from vegetative growth to reproductive growth, root development and fruit maturation. Members of the MADS-box gene family have been identified in several species. However, knowledge about MADS-box gene family members, and their evolutionary and functional characteristics is limited in kiwifruit. The purpose of this study was to identify and analyze MADS- box gene family and determined its expression during the fruit development and bud dormancy, so as to provide theoretical basis for the regulation mechanism of MADS-box gene family members.【Methods】Based on the Hongyang‘V3’genome data of Actinidia chinensis, MADS-box was identified and its gene structure, chromosome distribution, phylogeny, promoter cis-acting element and expression patterns were analyzed.【Results】68 AcMADS-box genes were identified in the Hongyang genome of A. chinensis, among which 35 belonged to Type I genes and 33 belonged to Type Ⅱ genes. There were 11 subfamilies (Mγ, Mβ, Mδ, Mα, FLM, SOC1, ANR1, SVP, AP3, SHP and SEP). AcMADS-box gene was unevenly distributed on 21 chromosomes except 8, 10, 17, 18, 19, 22 and 24.There were 32 collinear gene pairs, 11, 4 and 3 pairs of MIKC, M, MIKC and M genomes, respectively. The results of the physical and chemical properties showed that the protein encoded by AcMADS-box gene family was composed of 77 to 1703 aa, the molecular weight of the protein was 8 917.01 to 121 149.91 u, and the isoelectric point was 4.55 to 11.25. Except AcMIDS24-MIKC, the protein was fatsoluble hydrophilic protein. AcMADS24-MIKC was a water-soluble hydrophilic protein. The CDS sequence length encoded by AcMADS-box protein was 234 to 1791 bp except for AcMIDS33-M, and the CDS sequence length encoded by AcMIDS33-M gene was 3222 bp, which was significantly higher than other genes of AcMADS- box gene family. In addition, the subcellular localization prediction analysis showed that the AcMADS-box gene family members were located in the nucleus. By predicting the secondary structure of the proteins encoded by 68 members of the MADS gene family, it was found that the secondary structure of the MADS gene family proteins was mainly Alpha helix, in which Type Ⅱ accounted for 45.83%-83.57% and TypeⅠaccounted for 23.75%-72.72%. Random coil of Type Ⅱ accounted for 7.14%-47.06% and TypeⅠaccounted for 16.58%-52.71%. Beta turn of Type Ⅱ accounted for 0%-8.74% and TypeⅠBeta turn accounted for 1.3%-13.1%. Extension strand of Type Ⅱ accounted 1.94%- 18.13% and TypeⅠaccount for 5.88%- 28.22% . The proportion of Beta turn and extension strand had little difference but was significantly lower than Alpha helix and Random coil. 12 motifs were found in AcMADS- box protein sequence named Motif 1-Motif 12 motifs. Motif 1 and Motif 2 were typical domains of MADS, and all proteins contained Motif 1 or Motif 2. Motif 4 was found in all Type Ⅱ proteins except AcMADS1-MIKC and AcMADS30-MIKC. Motif 5, Motif 7, Motif 9 and Motif 10 belonged to the specific domain of TypeⅠ. A total of 47 elements related to light response (28), hormone response (12), stress resistance (4) and growth rhythm (3) were identified in the upstream promoter region of AcMADS-box gene family. 771 components related to optical response were identified, including G-Box, Box4, GT1-motif and CTt-motif, and each AcMADS-box contained at least 5 components related to optical response. Five hormones regulated by AcMADS- box cis- acting element were ABA (ABRE), Auxin (AuxRE, AuxRR-core, TGA-box and TGA-element), MeJA (CGTCA-motif and TGACG-motif), GA3 (GARE-motif, P-box and TATC-box), and SA (SARE and TAT-element). There were stress- related elements in the gene family, including those related to drought induction (MBS), low temperature response (LTR), anaerobic induction (ARE) and stress-resistance (TC-rich repeats). In addition, the AcMADS- box gene family also contained cis- acting element related to meristem expression (CAT-box, NON-box) and circadian rhythms. Transcriptomic analysis showed that the expression of AcMADS -box genes was significantly different in fruit ripening, 18 family members (10 M type and 3 MIKC type) had no expression in the whole development stage, and 16 members of AcMADS- box family had the highest expression level in young fruit period. The expression levels of 20 family members decreased with fruit ripening, while the expression levels of 14 family members increased with fruit ripening. The expression levels of AcMADS35-M and AcMADS21-MIKC genes were the highest at the ripening period, and then decreased. The expression levels of AcMADS26-M genes were opposite to those of AcMADS35-M and AcMADS21-MIKC genes, and increased with fruit ripening. During ripening period, its expression was significantly higher than that in young fruit period and mature-green period. The expression levels of AcMADS10-MIKC, AcMADS12-MIKC and AcMADS6-MIKC were the highest in the mature-green period. The expression level of AcMADS10-MIKC gene was higher in young fruit period, mature-green period and ripening period. The analysis of AcMADS-box expression in different tissues (leaf, root, vine, cane, flower and flower bud) showed that AcMADS-box genes were differentially expressed in different tissues, and 16 family members were highly expressed in leaves, roots, vines,canes and flowers of A. chinensis. Among them, AcMADS10-MIKC, AcMADS11-MIKC, AcMADS12- MIKC, AcMADS17-MIKC, AcMADS21-MIKC, AcMADS23-MIKC and AcMADS7-M were expressed significantly higher in flowers than in other tissues. The expression level of AcMADS2-MIKC in vines and canes was higher than that in leaves, roots and flowers. The expression of AcMADS19- MIKC, AcMADS20-MIKC and AcMADS31-MIKC was significantly higher in leaves, vines and canes than in roots and flowers. Eight family members were highly expressed in the flower buds. Among them, the expression of AcMADS20-MIKC was higher in Jan., Feb., Mar., Apr., Nov. and Dec., while AcMADS26-M was significantly higher in May, Jun., Jul. and Aug. than that of the remaining 6 months.【Conclusion】A total of 68 AcMADS-box genes were identified in this paper. Through the sequence analysis of 2000 bp upstream promoter of AcMADS-box family members, it was found that each gene member of the family contained multiple cis-acting elements related to hormone response and stress response. It is speculated that members of this family may be involved in the regulation of fruit ripening and the resistance to stress such as drought, low temperature and anaerobic response. We found that 17 AcMADS-box genes were highly expressed and had expression specificity among tissues, which were speculated to be the key genes involved in regulating the development of the growth and development of A. chinensis.