Changes Of Aroma Components And Expression Levels Of Related Genes In The Fruit Development Of Zanthoxylum Bungeanum Maxim.

Chinese prickly ash(Zanthoxylum bungeanum Maxim.)is a plant of the rutaceae family,widely cultivated in China.It’s outstanding characteristic is rich aroma.Volatile terpene are the main components of this aroma.Aroma is one of the most important characteristics of Z.bungeanum and also an important index to evaluate its quality.However,the molecular synthesis mechanism of Z.bungeanum is still in a blank stage.In this study,we used fruits of different development stages of Hanchengdahongpao and Fugu huajiao as research materials.Sixteen key genes on the terpene metabolic pathway of plants were selected from KEGG database for primer design.We used headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry(HS-SPME-GC-MS),gene cloning,transient expression of tobacco,in vitro enzymatic activity detection of prokaryotic expression,RT-qPCR and bioinformatics to carry out the research.The main research results are as follows:1.GC-MS results showed that the variation trend of volatile terpenes of the two varieties was consistent.As the fruit ripens,terpenoids accumulate,peak at the third stage(middle development),and then decrease.The most abundant terpenes are monoterpenes,among which,the highest content of a-pinene is 24.74%and 20.78%respectively in Hanchengdahongpao and Fugu huajiao.In general,the contents of volatile terpene in Hanchengdahongpao were 62%and 41%in Fugu huajiao.2.RT-qPCR detected the expression profiles of selected pathway genes and found that most genes were up-regulated.Among them,ZbDXS has the highest relative expression,which is a key rate-limiting enzyme in the MEP pathway.The high expression of genes in the MEP pathway is relative to the MVA pathway,indicating that the MEP pathway may be the main terpene metabolism in Z.bungeanum.3.Based on the above results,we performed functional analysis on the MEP upstream key speed limiting enzyme,1-deoxyxylose-5-phosphate synthetase(DXS)and downstream key speed limiting enzyme,Farnesyl pyrophosphate synthase(FPS)of the MVA pathway.Based on the previous transcriptome data,we successfully cloned CDS sequences of ZbDXS and ZbFPS from Z.bungeanum,with lengths of 2172 bp and 1029 bp,encoding 724 and 343 amino acids,respectively.4.Bioinformatics analysis showed that ZbDXS was a hydrophilic protein without signal peptide and transmembrane domain,and its localization in mitochondria was predicted by subcellular localization.Phylogenetic tree analysis showed that ZbDXS clustered with DXS2 family members of a variety of plants on a large branch,which was highly clustered with citrus DXS2 in a small branch.It was speculated that this gene sequence had the function of DXS2 type genes and was involved in the metabolism of terpene compounds in Z.bungeanum.ZbFPS belongs to the stability of protein and hydrophilic protein,containing no signal peptide and the membrane structure domain,phylogenetic tree analysis showed that ZbFPS was most closely related to pepper,tomato,potato and citrus,clustered in a large branch with a support rate of 100%,and its citrus plants were highly clustered in a small branch,which was consistent with the phylogenetic relationship of plants.5.In vitro enzyme activity experiments showed that the purified proteins ZbDXS and ZbFPS had the functions of DXS enzyme and FPS enzyme.The transient expressions of ZbDXS and ZbFPS in the tobacco leaves were found that increase the terpene content of tobacco.RT-qPCR was used to analyze the expression patterns of the two genes.The relative expression of ZbDXS and ZbFPS was the highest in the fruit,among which the relative expression of ZbDXS was the lowest in the root,and the relative expression of ZbFPS was the lowest in the stem.