Analysis Of BAHD Gene Family And The Function Of PbrAAT Genes In Regulation Aroma Accumulation In Pear Frutis

The aroma content in pear fruit is an important factor to measure fruit quality.Screening genes related to aroma synthesis in pear fruits and conducting certain researches are of great significance for understanding the mechanism of aroma accumulation and improving fruit quality.In this study,based on the released whole-genome sequencing results,by using bioinformatics analysis methods,the relevant information of pear and six other Rosaceae species acyltransferase(BAHD)gene families were identified and analyzed.The alcohol acyltransferase gene(AAT),a member of the pear BAHD gene family,which may play an important role in the accumulation of fruit aroma,was screened out.By performing experiments such as tobacco-mediated subcellular localization,homologous transient expression in pear fruit,yeast two-hybrid and tomato heterologous genetic transformation,the functions of Pbr AATs were preliminarily identified.The main contents and conclusions are as follows:1.A total of 717 BAHD genes were obtained from the genomes of 7 species of Rosaceae plants(Pyrus bretschneideri,Malus domestica,Prunus avium,Prunus persica,Fragaria vesca,Pyrus communis and Rubus occidentalis)using the hidden Markov model.The gene structure of BAHD gene family members is highly conserved,with two very typical conserved motifs(HXXXD and DFGWG).Combining bioinformatics methods and software to analyze its Ka,Ks,Ka/Ks,chromosome location,gene duplication events,phylogeny and so on.The results show that the members are unevenly distributed on their chromosomes randomly.The BAHD superfamily gene members are mainly divided into 7 groups: I-a,I-b,II-a,II-b,III-a,IV and V.Among them,group I-a and group IV may play a major role in the synthesis and accumulation of aroma.Collinearity analysis shows that among the 7 species of Rosaceae,the BAHD superfamily has 78 pairs of synteny genes,which is an ancient origin.Ka/Ks computational analysis shows that purification selection dominates the evolution of BAHD genes.2.Combining the transcriptome data from different tissues of pears,we determined that most of Pbr AATs in white pears are expressed at a high level in the roots.A total of 37 BAHD genes are expressed in the four stages of fruit development.The expression levels of Pbr020016.1,Pbr027303.1,Pbr029551.1,Pbr014028.1 and Pbr006821.1 were the highest in the late fruit development period(Fruit_S4).Further analysis showed that the four genes Pbr020016.1,Pbr014028.1,Pbr029551.1 and Pbr019034.1 were significantly positively correlated with changes in total ester content during fruit development.In addition,these four genes were used to construct a phylogenetic tree with the reported alcohol acyltransferase(AAT)members in apples,strawberries,melons and other species,and the results showed that the homology of these genes is considerably high.The result implied that the four members are important candidate genes involved in aroma formation during pear fruit development.3.Pbr AAT1-GFP,Pbr AAT2-GFP,Pb AAT3-GFP overexpression vectors,and Pbr AAT1-TRV2,Pbr AAT2-TRV2,Pbr AAT3-TRV2 silencing expression vectors were constructed to infect ‘Dangshan Suli’ and ‘Nanguo’.The experimental results showed that the esters content of ‘Dangshan Suli’ after infection increased significantly,and the esters content of ‘Nanguo’after infection decreased significantly.The Pbr AAT1-GFP,Pbr AAT2-GFP,and Pbr AAT3-GFP fusion proteins were transformed into tomatoes by the Agrobacterium transformation method.The analysis of physiological traits indicated that the esters content of overexpressed tomato was significantly higher than that of wild-type tomatoes.4.According to the above experimental results,the potential functional gene Pbr AAT2 was selected to construct the yeast two-hybrid plasmid p GBKT7-Pbr AAT2.The toxicity and self-activation analyses show that it is non-toxic and does not self-activate,and the library screening experiment can be carried out.Three proteins(Pbr013139.1,Pbr022402.1 and Pbr013141.1)that may interact with Pbr AAT2 were screened from the‘Nanguo’ c DNA library using yeast two-hybrid technology,and a p GADT7-candidate gene fusion vector was constructed.Point-to-point experimental verification was carried out in the yeast system.And the authenticity of the experimental results was verified by the Luciferase Complementary Imaging(LCI)experiment.