Functional Analysis of Aroma Biosynthesis Related Genes DXS Gene and CHS Gene in Tobacco
|School||Chinese Academy of Agricultural Sciences|
|Course||Crop Genetics and Breeding|
|Keywords||transgenic tobacco Subcelluar localization over-expression vectors RNAi vectors HPLC|
As an important index for evaluating the qualities of tobacco leaves and their products, aroma is animportant goal that the breeders seeked to get, with the contradiction between the lowering tar andreducing harm of tobacco being intensified increasingly. Polyphenols and terpenes generated from thephenylalanine metabolic pathways and the armour hydroxyl pentanoic acid (MEP) pathway areimportant aroma precursors in tobacco. Chalcone Synthase (CHS) gene and1-deoxidizationxylulose-5-phosphate synthetase (DXS) gene were the two key genes in the metabolic pathways.therefore, the over expression of these genes and their silencing or mutations could directly or indirectlyaffect the plant phenylalanine metabolic pathways and the MEP pathways,and thus affect the formationof aroma precursors. In this paper, the functions of CHS gene and DXS gene were studied from thepoint of specific genes regulations with the variety “Dabaijin599”, which was characterized of higharoma. Our research laid a foundation for further studies on the molecular mechanism of aromasubstances formation and high aroma breeding, the main results were as follows:(1) CHS gene and DXS gene separation and subcelluar localization. CHS gene (AF311783.1) andDXS gene (EU650419.1) were separated from the cDNA of the variety “Dabaijin599” through PCRtechnology. Recombinant of DXS gene (without the termination codon TAA) and green fluorescentprotein (GFP) gene in expression vectors was to used for the localization of expression vectors in thesubcellular and finally it was located to chloroplasts.(2) Construction of RNAi vectors and over-expression vectors and genetic transformation. TheDXS gene RNAi vectors and the over-expression vectors of CHS gene were obtained through theenzyme digestion method and CHS gene RNAi vectors were constructed through Gateway method.Allof the expression vectors were used for tobacco genetic transformation via agrobacterium-mediatedmethod. Detection of transgenic tobacco plants were carried out by using the PCR assay and GUSstaining analysis. Results showed that the numbers of tobacco plants with DXS gene silence, the CHSover-expression and CHS gene silence were12,13and24, respectively.(3) Study on the physiological-biochemical characteristic of transgenic tobacco plants and controltobacco plants. It was indicated that the leaf color of the plant with gene silence in the seeding stagebecame shallow and yellow. The total content of chlorophyll and carotenoid were reduced by45.3%and40.2%, respectively compared with the control plants. The result demonstrated that DXS gene played asignificant role in the MEP pathway. Tobacco plants with the over expression or silence of CHS genehad a normal morphological characteristics and normal growth, however, the enzyme activities of4CLand C4H increased in tobacco plants with the over expression of CHS gene. On the contrary; the twoenzyme activities decreased.(4) Quantity analysis of polyphenols of the transgenic tobacco plants via high performance liquidchromatography (HPLC). The polyphenols were extracted through ultrasonic technology and theextraction condition was optimized, of which the optimal extraction temperature and time were55℃and25min, respectively. Results of polyphenols content determination showed that the content of polyphenols increased in the tobacco plants with over expression of CHS gene and the content ofpolyphenols decreased in the tobacco plants with CHS gene silence, which indicated that the overexpression of CHS gene promoted the synthesis and accumulation of polyphenols and the synthesis ofpolyphenols was blocked when the CHS gene was silenced. Based on these results it was concluded thatCHS gene played an important role in the formation of aroma precursors which was generated from thephenylalanine metabolic pathways.