RNA-seq Transcriptome Analysis Based On The Difference Of Sanshool Contents In Different Varieties Of Zanthoxylum Bungeanum

Zanthoxylum bungeanum Maxim is one of the important cash crops in China belongs to the Zanthoxylum genus of the family Rutaceae.Sanshool is a series of long chain unsaturated alkylamides represented by hydroxyl-α-sanshool.It is the main source of the taste of numbing,but its chemical properties are extremely unstable and the reason for the difference in the taste of numbing among different varieties of Z.bungeanum has not been clearly reported.Therefore,in order to analyze the difference source of numbing of different varieties of Z.bungeanum,the study on the separation,purification and structural identification of sanshool,analyzed the dynamic difference of the content of hydroxyl-α-sanshool in different varieties of Z.bungeanum,constructed the HPLC fingerprint,RNA-seq analysis of 3 varieties of Z.bungeanum with significant difference in contents of sanshool were carried out,and the key differentially expressed genes（DEGs）related to the synthesis of sanshool were identified.The main conclusions are as follows:1.A sanshool was separated from Fengxian Dahongpao,and it was identified as hydroxyl-α-sanshool by ¹H-NMR and ¹³C-NMR.A qualitative and quantitative analytical method of HPLC was also established,and the precision,repeatability,recovery rate and stability of the method were investigated.2.The contents of hydroxyl-α-sanshool in different varieties of Z.bungeanum peels were determined.The results showed that the contents of the 12 varieties of Z.bungeanum were significant（p<0.05）,You Huajiao had the highest content of hydroxyl-α-sanshool（46.936±1.122 mg/g）and the lowest content was in Fugu Huajiao（2.235±0.077 mg/g）.On this basis,HPLC fingerprint combined with chemometrics of 12 varieties of Z.bungeanum peel was performed.12 common peaks were identified and the similarities between different varieties were within a range of 0.424-1.12 varieties of peels were classified into 4 quality groups（G1-G4）,G1 consists of S3（Fengxian Dahongpao）,S5（Hancheng Dahongpao）,S7（Maoxian Dahongpao）,S8（Qinan Yihao）and S10（Wudu Dahongpao）,G2 consists of S1（Hancheng Dangcun stingless）,S4（Hancheng Gelao stingless）,S6（Hancheng stingless）,S9（Hancheng Shizitou）and S11（Xinong stingless）;S12（You Huajiao）was in G3 and S2（Fugu Huajiao）was in G4.Using PCA and DA,it was found that G2 had higher quality,and discrimination functions for further discriminate and classify the unknown membership were also constructed.Then,peak 9（hydroxyl-α-sanshool）and peak 11（unknown）were chosen as critical chemical markers for quality control and assessment of Z.bungeanum peels.3.The biosynthesis accumulation dynamics of hydroxyl-α-sanshool in different varieties of Z.bungeanum peel and leaves were revealed.On the whole,it presented a trend of rapidly increasing and then decreasing slowly.Among them,the contents of hydroxyl-α-sanshool in S1,S4,S7,S9 and S10 were the highest in July.S3,S5,S6,S8,S11 and S12 were the highest in August.Except for S3,S4 and S12,the contents of hydroxyl-α-sanshool in the other 9varieties reached the highest values in May and June.4.RNA sequencing for 3 varieties of Z.bungeanum（Hancheng stingless,Fengxian Dahongpao,and Fugu Huajiao）with significant difference in the content of sanshool were carried out by Illumina Hiseq 2500.83522 Unigene were obtained by de novo and 40668（48.69%）Unigene obtained annotated results.A total of 123 Unigene were related to the biosynthesis of sanshool.Expression pattern of the DEGs showed that a total of 6656 DEGs were obtained,and 10 DEGs of biosynthesis of unsaturated fatty acid and 9 DEGs of valine biosynthesis were identified by functional annotation results.Further screened the 6 DEGs may be the key genes responsible for the differentiation of the sanshool between different varieties of Z.bungeanum.They are mainly responsible for the desaturation of fatty acids and the amino transfer of the branched chain amino acids.The relative expression profiles of 6key DEGs were verified by q RT-PCR,indicating that the results of RNA-seq were accurate and reliable.The results of q RT-PCR showed that the relative expression of the other 5 genes were consistent with the results of RNA-seq except c102796.graph＿c0,indicating that the results of RNA-seq were accurate and reliable.