Quality Comparison Of Momordica Grosvenorii From Different Habitats And Cloning And Expression In This Species ?-Amyrin Synthase Gene

Siraitia grosvenorii is a unique traditional health medicinal material in China and one of the first batch of medicinal and edible plants published by the ministry of health.With the application of Siraitia grosvenorii in various fields expanding,the market demand at home and abroad is increasing,and the planting area is also growing.Therefore,the contents of total saponins,glycoside V and selenium of Siraitia grosvenorii from different producing areas in the same harvest period were compared to provide scientific basis for its development and utilization.Triterpenoid saponins are a group of cucurbits,which are considered to be the main active ingredients of Siraitia grosvenorii.The beta-carotene synthase plays an important drainage role in many medicinal plants with triterpenoid saponins as the key active ingredients.The study of this gene can not only reveal its mechanism in the synthesis of triterpenoid saponins of Siraitia grosvenorii,but also provide theoretical guidance for the regulation of secondary metabolism and genetic improvement,to provide scientific basis for better development and utilization of Siraitia grosvenorii.The total saponin content of each Siraitia grosvenorii fruit sample was measured by vanillin-concentrated sulfuric acid reagent spectrophotometric colorimetric method.high performance liquid chromatography was used to measure the content of glycoside V,hydride atomic fluorescence spectrometry was used to measure the content of selenium.Then significant difference analysis of each indicator.The significance of the difference was analyzed.Based on the transcriptome UniGene sequence and the rapid cloning technology of the end of the cDNA,the full-length cDNA of the key enzyme of triterpenoid saponin biosynthesis pathway,beta-amyrin synthase gene and minus strand beta-amyrin synthase-like gene,named Sg?AS?GenBank Registration No.:MK580640?and Sgm?AS?GenBank Registration No.:MK605687?.Real-time fluorescence quantitative PCR was used to detect the temporal and spatial expression of Sg?AS and Sgm?AS genes in different organs and tissues of Siraitia grosvenorii.The results are as follows:1.The total saponin content of each sample ranged from 2.42%to3.34%,the glycoside V content ranged from 0.68%to 1.41%,and the selenium content ranged from 5.634 to 41.462 ug.kg^-1.The total saponins content of Siraitia grosvenorii samples from different habitats did not differ significantly,but the contents of glycoside V and selenium showed significant differences.In Guangxi,Hunan and Sichuan,the contents of total saponins and glycoside V in Siraitia grosvenorii fruit can meet the requirements of pharmacopoeia.Therefore,Siraitia grosvenorii fruit has a wide planting area,but the content of glycoside V in different habitats is significantly different,suggesting that the conversion efficiency or maturity of fruit glycoside V have a certain difference,which may be related to the effective accumu Lated temperature of fruiting period in the habitats.It is appropriate to adjust it according to the habitats and annual climatic conditions.Harvest period to meet the needs of different processing and utilization.2.The full length sequence of Sg?AS gene was 2 547 bp,the longest ORF was 2 250 bp,encoding 749 amino acids.The theoretical molecular weight of the encoding protein was 85.94 kD,the isoelectric point was 6.17,the average total hydrophilicity was-0.330,the fat coefficient was 76.73,and the unstable coefficient of the encoding protein was 42.86.The protein was a non-transmembrane protein and belonged to non-secretory protein.The alpha helix accounted for 46.52%of the protein and the beta folding took up 3.74%with irregular crimp making up 49.73%.Molecular evolutionary analysis showed that Siraitia grosvenorii Sg?AS had the closest relationship with the corresponding proteins of the related homologous species Momordica charantia.The full length sequence of Sgm?AS gene was 2219 bp,the longest ORF was1926 bp,encoding 641 amino acids.The theoretical molecular weight of the encoding protein was 73.36 kD,the isoelectric point was 6.27,the average total hydrophilicity was-0.158,the fat coefficient was 87.21,and the unstable coefficient of the encoding protein was 49.84.It had a transmembrane helical structure with 7-29 aa,and the location coefficient of plasma membrane was 2?plas:2?.Location coefficient was 7?cyto:7?and nucleus was 4?nucl:4?.Molecular evolutionary analysis showed that Sgm?AS had the closest relationship with the corresponding proteins of pumpkin.3.Real-time fluorescence quantitative PCR analysis showed that both Sg?AS gene and Sgm?AS gene were expressed in stem,leaf and fruit.The relative expression of Sg?AS gene was highest in fruited leaves,followed by high expression in fruits on 50 days.The expression of Sg?AS gene in fruits increased continuously before 50 days,reached the highest value about 50 days,and then decreased gradually.The expression of Sgm?AS gene was highest in fruits at 40 days of fruit stage,followed by higher expression in leaves of main vines at greenhouse stage.The expression of Sgm?AS gene increased first and then decreased in fruits.The content of Sgm?AS gene was highest in leaves at greenhouse stage,and gradually decreased with the passage of time.Sg?AS and Sgm?AS may participate in the early terpene skeleton metabolism and triterpene synthesis shunt control process,respectively.It lays a foundation for research in-depth genetic transformation.