Identification and expression analysis of PIN gene family during adventitious root formation in apple

Abstract: 【Objective】Apple (Malus×domestica Borkh.) is one of the most economically important tree fruits in China, and the intensive cultivation with dwarfing rootstock is popular in the world. The use of dwarfing rootstocks is the main way to realize the intensive cultivation of apple. The formation of adventitious roots is one of the key steps in the vegetative propagation of dwarfing rootstocks. The auxin transporter PIN-FORMED (PIN) protein is a typical transmembrane transport protein, which plays an important role in adventitious root formation by participating in the polar transport of auxin in plants. In this study, the characteristics of the apple PIN gene family at the whole genome and its expression in the process of adventitious root formation were investigated, so as to better understand its structural characteristics and potential functions, and lay a foundation for studying the molecular mechanism of adventitious root formation.【Methods】The apple PIN gene family members were identified using the apple genome database GDR and HMMER. The gene mapping, phylogenetic relationship, gene structure, conserved motif analysis and protein conserved domain of the PIN gene family members were analyzed using bioinformatics software such as MEGA, TBtools and MEME. qRT-PCR was used to ana-lyze the expression pattern of the PIN gene family members in adventitious root formation.【Results】 There were 13 members of the apple PIN gene family, and they were distributed on 8 chromosomes. The amino acid lengths ranged from 357 to 660 aa, the molecular weights of protein ranged from 38.84 to 71.61 ku, and the isoelectric points ranged from 6.93 to 9.35. The sequence analysis showed the 13 PIN genes were unevenly distributed on 8 chromosomes of apple (Chr 4, Chr 6, Chr 9, Chr 10, Chr 12, Chr 13, Chr 14 and Chr 16), among which there were two PIN genes on Chr 4, 12, 13, 14 and 16 respectively, there was only one PIN gene on Chr 6, 9 and 10. The collinear analysis showed that there was segment replication between the MdPIN2 and the MdPIN7, the MdPIN3 and the MdPIN11, the MdPIN6 and the MdPIN10, the MdPIN8 and the MdPIN12, and the MdPIN9 and the MdPIN13, and no tandem duplication was found, suggesting that gene segment replication was the main driving force for the amplification of this family. The apple PIN genes contained multiple introns and exons, and the structures were relatively complex. The conserved motifs analysis showed that the MdPIN1, MdPIN2, MdPIN4 and MdPIN7 had 8 conserved motifs, and all the other genes had 10 conserved motifs. In order to study the evolutionary relationships of the members of the PIN gene family, MEGA software was used to conduct phylogenetic analysis of the apple PIN family was mainly distributed in the G1 and G3 subfamilies, with 10 in the G3 subfamily, three in the G1 subfamily. The analysis of promoter-acting elements showed that 13 PIN promoters contained cis-acting elements related to hormone, growth and stress response, and 8 of them contained auxin response elements, suggesting that they might be involved in auxin regulation. The transcriptome analysis showed that the expression levels of the MdPIN3, MdPIN4, MdPIN5, MdPIN6, MdPIN8, MdPIN10, MdPIN11 and MdPIN12 under IBA treatment were higher than those of the control. There was no change in the MdPIN1 and MdPIN13 compared with the control, while the expression of the MdPIN7 was lower than that of the control under IBA treatment. Six genes with significantly different expressions (the MdPIN3, MdPIN4, MdPIN6, MdPIN7, MdPIN11 and MdPIN12) were screened and their dynamic expression patterns in adventitious root formation were further verified by qRT-PCR. The expression of the MdPIN3, MdPIN4, MdPIN6 and MdPIN11 was significantly induced by IBA treatment, which was consistent with the results of the transcriptome. The expression level of the MdPIN3 and MdPIN4 was the highest on the 5th day, the expression level of the MdPIN4 was 2.9 times as high as that of the control on the 5th day, and the expression level of the MdPIN6 was the highest on the 10th day.【Conclusion】A total of 13 PIN genes in apple were identified and they were distributed on 8 chromosomes. The transcriptome and qRT-PCR analysis suggested that the MdPIN4 might play an important role in regulating the adventitious root formation of apple rootstocks. These findings might lay a foundation to functionally characterize the MdPIN genes to unravel their exact role in adventitious root formation in apple.