Identification, characterization and expression analysis of alcohol acetyl- transferase (AAT) gene family in 10 Rosaceae fruit trees

Abstract: 【Objective】Alcohol acetyltransferase (AAT) is a key enzyme that promotes the formation of ester aroma substances. Genes encoding AAT enzymes exist in different fruits, which can selectively re-lease different substrates and heterologous AATase, and regulate the biosynthesis of volatile esters. In this study, we performed AAT gene family analysis of 10 Rosaceae fruit species to illustrate various properties of their families, in order to provide preliminaries for functional validation of candidate genes for fruit aroma regulation.【Methods】Based on the AATase structural domains, we retrieved the genome database of 10 Rosaceae species (Pyrus bretschneideri, Pyrus communis, Pyrus betulifolia, Ma-lus domestica, Prunus Persica, Prunus mume, Prunus avium, Fragaria vesca, Rubus occidentalis,Fragaria × ananassa) to identify the AAT genes. Then the phylogenetic tree of AAT genes of Rosaceae species was generated with neighbor-joining method using MEGAX software. After that, the AAT gene structures were analyzed by online analysis software GSDS 2.0. And, we used the online tool ExPASy to analyzed physicochemical properties of AAT proteins, including the number of amino acids, molecu-lar weight, isoelectric point, grand average of hydropathicity, aliphatic index and instability index. Sub-cellular localization was predicted by online website CELLO, and protein motif was analyzed by MEME database. Mapdraw was employed to perform the chromosome map, which could help analyz-ing the distribution of AAT genes on the chromosome. MCScanX was used to detect the collinearity of  the AAT genes. Multiple sequence alignment analysis was performed using Muscle, ParaAT, which led to further use of KaKs_Calculator2.0 software to calculate Ks and Ka/Ks values between synteny se-quences. Finally, Dangshansuli and Nanguoli were taken as examples to analyze the relationship be-tween volatile aromatic compounds and AAT activity during the growth and development of pear fruit.【Results】In this study, we totally identified 46 genes, including 4 from Chinese white pear (Pyrus bretschneideri), 4 from plum (Prunus mume), 8 from peach (Prunus Persica), 7 from apple (Malus do-mestica), 4 from European pear (Pyrus communis), 6 from du pear (Pyrus betulifolia), 2 from sweet cher-ry (Prunus avium), 1 from woodland strawberry (Fragaria vesca), 7 from pineapple strawberry (Fragar-ia × ananassa), and 3 from black raspberry (Rubus occidentalis). By means of a dendrogram, these 46 genes were divided into 4 regional groups, which could be found because all branches had AAT genes from different species. Analysis of the gene structure map represented that all 41 sequences had introns,except for five sequences without introns, and the structural composition of each sequence was relative-ly complex. The results of subcellular localization and physicochemical properties revealed that the AAT proteins were mainly localized in the cytoplasm, with an overall range of weakly acidic to weakly basic,hydrophilic proteins and most of them were unstable. Through the chromosome map, it could be found that the AAT genes were randomly distributed on the chromosome 2 AAT genes were distributed on the same chromosome in apple, plum, Chinese white pear, European pear, du pear, sweet cherry, and pineap-ple strawberry. 3 and 4 genes were distributed on one chromosome in peach，and 5 were distributed on one chromosome in pineapple and strawberry. Through MCScanX, we found 37 pairs of collinearity rela-tionships in total. Among them, 8 pairs of colinear relationships were found within species, 1 pair in Chi-nese white pear, 1 pair in plum, 1 pair in sweet cherry, and 5 pairs in pineapple strawberry. Specially, FaAAT1 had the most synteny sequences within species. Furthermore, 29 pairs of collinear relationships were found between species, Chinese white pear and peach had collinear relationships with five species,and European pear, apple, plum and pineapple strawberry had collinear relationships with four species.It could be found that the expansion of AAT gene family had the phenomenon of tandem duplication and fragment duplication in the process of polyploidization. The Ka/Ks values of homologous gene pairs in the Rosaceae AAT gene family were all less than 1, indicating that they were mainly influenced by the ef-fect of purifying selection during the evolutionary process. In the two pear species, the activity of AAT increased with the growth and development of the fruits. Considering former research of Qin, the AAT activity of Dangshansuli with lower content of volatile aroma compounds was lower than that of Nan-guoli with higher content, and the increase of AAT activity increased the production of lactones in the fruits.【Conclusion】46 AAT genes were identified in 10 Rosaceae species, and only 1 was found in woodland strawberry and 8 were found in peach. The structure of AAT genes were relatively conserved and were significantly affected by purification effects. There were differences in AAT activity among different species, and their gene expression was positively correlated with the production of esters.