| Citrus is one of the most important cash crops in the world,and fruit aroma is a vital characteristic of citrus fruit quality.Terpenoid is the main substances in citrus fruit aroma.Different varieties of citrus fruits have characteristic aroma,due to the different types and contents of aroma substances in fruits.In recent years,there are abundant researches on large-scale resource evaluation,aroma component analysis and structural gene function identification in citrus fruits,but the regulation of characteristic aroma and key synthesis genes still needs further research.In this paper,electronic nose（E-nose）technology is used in combination with Principal component analysis（PCA）and linear discriminant analysis（LDA）,and headspace solid-phase microextraction（HS-SPME）coupled with gas chromatography-mass spectrometry（GC-MS）were used to study the changes of aroma substances,especially the characteristic aroma of sweet orange,valencene,in‘Newhall’fruit during development and after ethylene treatment.Gene expression was used to further verify the relationship between characteristic aroma valencene and valencene synthetase gene CsTPS1.Finally,bait vector was constructed based on CsTPS1 promoter,and the citrus c DNA library was screened by yeast one-hybrid assay.In order to screen transcription factors related to the synthesis of characteristic aroma valencene of‘Newhall’fruit,and provide scientific basis for the study of the regulation mechanism of the synthesis of valencene.The main results are as follows:（1）A total of 58 volatiles were identified from‘Newhall’fruit during development,including 9 monoterpenes,24 sesquiterpenes,14 monoterpenoids,3 sesquiterpenoids,and 8others.The proportion of sesquiterpenes was small,but compared with the other four aroma substances,the content of sesquiterpenes increased significantly after the color conversion stage.The contents of sesquiterpene aroma substances in different developmental stages were further analyzed.The results showed that the content of valencene in the characteristic aroma of citrus fruit increased significantly after the color break stage.During development of navel orange fruit,the content of characteristic aroma substance valencene and the expression of valencene synthase gene CsTPS1 showed the same trend,and the two showed a positive correlation during development.（2）A total of 64 volatiles were identified from‘Newhall’fruit at color break stage by ethylene treatment(1000 mg·L-1,1 min),including 12 monoterpenes,20 sesquiterpenes,18monoterpenoids,1 sesquiterpenoids,and 13 others.The contents of sesquiterpenoids in citrus fruit in control group and ethylene treatment group were analyzed.It was found that ethylene treatment could increase the content of valencene.The expression level of CsTPS1 after ethylene treatment was verified.The results showed that ethylene treatment could promote the expression of CsTPS1,and there was a positive correlation between the CsTPS1 expression and the content of valencene.（3）Cloning promoter sequence of CsTPS1 and constructing citrus c DNA library.The positive transforters were screened for sequencing by yeast one-hybrid assay,and the sequencing results were compared with NCBI,Plant TFDB and citrus genome database.Three types,a total of 13 candidate transcription factor genes were obtained,including the b HLH family transcription factors,Cit UNE1,Cit UNE2,Cit UNE3,Cit UNE4 and Cit UNE5;MYB family transcription factors,Cit MYB1,Cit MYB2,Cit MYB3,Cit MYB4,Cit MYB5,Cit MYB6;and GARS family transcription factors,Cit SCL1 and Cit SCL13.（4）Among the 13 candidate transcription factors,Cit UNE1,Cit MYB2,Cit MYB4 and Cit MYB5 were validated to bind to the CsTPS1 promoter.Gene expression analysis showed that the expression of Cit UNE1 and Cit MYB2 genes was negatively correlated with the content of valencene and the expression of valencene synthetase CsTPS1.While the gene expression levels of Cit MYB4 and Cit MYB5 were positively correlated with the expression of CsTPS1. |
