| Pentacyclic triterpenes are a group of structurally diverse plant metabolites that exist in the free or conjugated state.Studies have shown that pentacyclic triterpenes have a wide range of biological activities and pharmacological effects,such as anti-inflammatory,anti-tumour and anti-HIV.Loquat(Eriobotrya japonica Lindl.),as a plant with high triterpene acid content in nature,contains triterpene acids in its fruit skin,pulp and leaves,but the triterpene acid content varies significantly between different tissues of loquat.At present,there are few studies on the regulation of pentacyclic triterpene metabolism in loquat.Studying the key genes regulating pentacyclic triterpene metabolism in loquat will both enhance the value of loquat and lay the physiological and molecular foundation for more in-depth studies on loquat in the future.To this end,in this study,by using the flesh and peel of loquat ’Baiyu’ as materials,firstly,the process of ultrasonic extraction of total triterpene acids from the peel and flesh of loquat was optimised,total triterpene and antioxidant activities were determined for different periods of loquat,and different fractions were identified by LC-MS to analyse the content of triterpene acids in different tissue parts at different stages Secondly,the key genes regulating the metabolism of pentacyclic triterpenoids in loquat were screened based on transcriptome sequencing;cDNA libraries of different tissues of loquat were constructed,EjCYP716A gene and promoter were cloned and EjCYP716A yeast monohybrid vector was constructed for subsequent screening library assays.The main results are as follows:(1)The effects of material-liquid ratio,ethanol concentration,sonication time,sonication power and sonication temperature on the yield of triterpenic acid in the peel and pulp of loquat fruit were investigated.The optimal sonication conditions for loquat fruit peel triterpene were obtained by mathematical model and regression equation:ethanol concentration 71%,sonication time 45 min,sonication power 160 W,material-liquid ratio 1:10 and sonication temperature 30℃.The extracted fruit peel total triterpene content The best sonication conditions were:85%ethanol concentration,51 min sonication time,43℃,160 W sonication power,1:8 ratio,and 11.69±0.25 mg/g of total triterpenes in loquat flesh.A total of 29 triterpenes were detected in the flesh of loquat fruit and 49 triterpenes were detected in the peel,mainly ursolic acid derivatives and oleanolic acid derivatives.(2)Transcriptome sequencing and biological analysis of loquat peel and pulp using high-throughput sequencing technology yielded a total of 39.29 Gb Clean Data,uncovering 7421 new genes,5158 of which were functionally annotated,and 6435 differential genes in peel vs.pulp,2660 of which were up-regulated in peel.Based on KEGG and GO analyses,eight pathways and nodes related to terpene synthesis and metabolism were identified,from which a total of nine key enzymes related to triterpenoid saponin biosynthesis were found to be significantly more expressed in the peel than in the pulp.Eight differentially expressed genes related to pentacyclic triterpene metabolism were postulated and validated by qRT-PCR.The results showed that only two of the screened UGTs were significantly up-regulated in the fruit pulp,while the rest of the screened genes(APS,CYP716,OSC)were significantly up-regulated in the fruit peel,which was consistent with the transcriptome sequencing results.(3)For the first time,a full-length cDNA library was constructed using loquat peel,pulp and leaf as materials.The library identification results showed that the extracted total RNA and treated dscDNA were of good quality,the library capacity reached 3.72×107 cfu/mL,and the average length of the insert was 1000 bp.The EjCYP716A gene and promoter were cloned and constructed into pHIS2 vector for subsequent yeast single-hybrid library assays. |
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