Aroma And Terpene Synthase Gene Mining Of Hong Xiangya Mango

Mango is an important tropical cash crop in China.Mango fruit maturation is accompanied by physiological and biochemical processes,including hardness,color,and flavor changes.The flavor is an important criterion to determine the market value of Mango,and fruit odor is one of the crucial factors affecting the flavor.However,the current mango industry in China is facing the challenge that with the development of breeding and cultivation,the aroma of mango fruits is lost.Among the odor substances of Mango,terpenes occupy the most crucial position with the highest content and the most abundant species.The whole life of Mango is closely related to terpenes,and terpenes have significant changes at each stage of fruit maturation.According to the recent research background of mango genome data publication,we employ the key gene for terpene synthesis,TPS,in Hong Xiangya as the research object in this study,and the reasons for the decrease of mango aroma are explored by molecular biologytechnics.To understand the biological mechanism of the basic metabolic process of terpenes during the mango maturation process,the volatile compounds of Mango fruits at three different post-maturation stages,3 d,5 d and 7 d after harvest,are detected by GC-MS using Hong Xiangya leaves as the reference sample.As the ripeness of the fruit increases,the flavor of the fruit is richer,and the content of most terpenes rises.Among them,Terpinolene is the most abundant volatile in Hong Xiangya fruits,with a relative content of about 70%.There is no α-Terpinene in the fruits in the early after-ripening stage,but α-Terpinene begins to appear in the pulp of the 5th day.In contrast to the changing trend of α-Terpinene,Caryophyllene is detected only in fruits at an early stage of post-ripening.In addition,we find that the leaves contain specific volatiles,Menogene and 1-methyl-4-cyclohexane.In mango fruits,terpenes exhibit developmental stage specificity and tissue specificity.We screen 35 TPS genes from the Hong Xiangya genome and analyze their bioinformatics characteristics and expression patterns.At the same time,we systematically clone and verify the functions of 35 TPS.Most of these 35 genes are between 520-600 amino acids in length,which is generally shorter than the TPS(600-900 amino acids)reported in previous studies.In addition to the most of these TPS located in the cytoplasm and plastid,some TPS are predicted to be located in the nucleus,endoplasmic reticulum.It shows that TPS may have independent evolution in the mango system.The phylogenetic tree,functional verification results,and gene localization on the chromosome further confirmed the suspect that terpene synthase could not predict its function based on sequence,and even the function of TPS genes clustered on the chromosome was not regular.The expression level of most TPS in fruits is deficient,and the expression level in leaves is generally higher than that in fruits.The functional verification results of Hong Xiangya TPS shows that 13 TPS do not show terpene synthase activity.Compared with tomato,grape,and other species,the number of TPS genes in Hong Xiangya is significantly less.There are 10 TPS that can catalyze GPP to produce monoterpenes,of which 7 TPS can only catalyze GPP;15 TPS can catalyze NPP to produce monoterpenes,of which 10 TPS can only catalyze NPP;3 TPS can catalyze FPP to produce sesquiterpenes,and These three TPS can simultaneously catalyze GPP or NPP to produce monoterpenes;no TPS can catalyze GGPP to produce diterpenes.About one-third of TPS has no functional activity,and the number of pseudo-TPS genes is more than that of other species that have been characterized by gene families,indicating that there is a possibility of loss of function in the Hong Xiangya TPS family.Through in vitro functional verification,combine with the predicted results of gene expression patterns and subcellular localization,we respectively analyze the TPS genes that play a significant role in the biosynthesis of the major terpene compounds in fruits and leaves.Terpinolene is mainly synthesized by Mi TPS013687.There are 6 TPS genes that have the function of synthesizing 3-Carene.The content of 3-Carene in Hong Xiangya is the result of the collaboration of multiple genes.Mi TPS016652 is the main gene for the synthesis of pulpspecific volatiles(S)-(1)-β-Pinene.Mi TPS029492 is mainly responsible for the biosynthesis of D-Limonene in fruits,and the biosynthesis of D-Limonene in leaves is mainly participated by Mi TPS029492 and Mi TPS030550.Mi TPS013695 can participate in the biosynthesis of1-methylene-4-cyclohexane in leaves.Mi TPS029716 and Mi TPS012261 are mainly responsible for the biosynthesis of Menogene in leaves.The speculation of related genes of these main volatiles will provide a molecular biological basis for the selection of core germplasm for mango fragrance breeding.According to the experimental results,we speculate that the main reasons for the loss of mango aroma are as follows: there may be changes in the function loss of the TPS gene in the long-term production and breeding of mangos;because the changes in gene function require long-term accumulation,the loss of mango aroma is more likely the reason that,many TPS in the fruit are not expressed,or the expression level is very low.In this study,the family analysis of key enzymes for biosynthesis of terpene,the most abundant compound in Mango,TPS was performed for the first time,and its functional activities were identified in vitro.This study not only contributes to understand the mango TPS family,but also provides a molecular biological basis for the breeding of high-quality and aromatic mango.More importantly,this has conducted a preliminary exploration of the reasons for the loss of mango aroma,which will help to further solve the quality problem of mango aroma.