| Cinnamomum camphora,a species of evergreen tree,is an important evergreen tree species in China.It produces clusters of black,round,berry-like fruit.The C.camphora fruits fall after ripening,and fallen fruits not only affect the environment,but also cause waste of resources.Current research on C.camphora has mainly focused on the traits in seeds of camphor tree,reproductive characteristics,extraction of volatile oil from fruits and seeds,the biological activity of volatile oil,extraction of fruit pigment,anthocyanin stability,anthocyanin bacteriostasis and antioxidant capacity of anthocyanin.However,the phenotypic characters and anthocyanin biosynthesis pathway of C.camphora fruits have been rarely reported.The present study used C.camphora fruits as research object reveals the effects of environmental factors on phenotypic characters of C.camphora fruits from different provenances.Moreover,comparative transcriptome analysis was applied for fruit of C.camphora at three stages of maturity,including the green fruit stage,turning color stage and ripe red stages,to discover the hub genes involved in the regulation of developmental processes and the metabolic process of anthocyanins biosynthesis.Meanwhile,Investigating the inhibition mechanism of anthocyanin against alpha-glucosidase,we applied enzyme inhibition kinetic,molecular docking and simulation to reveal anthocyanin attenuated the rise in blood glucose.This research provides a timely and necessary study of C.camphora fruit resources.The main content of this paper is as follows:1 Digital image processing techniques used for shape analysis of C.camphora fruitsTo effectively avoid the effect of the shadow of the object,we designed a shadowless device.The correlation between Image J measurement results and vernier caliper measurement results is extremely significant.This proposed method offered a low-cost,simple,accuratzse and efficient measurement approach.Meanwhile,the shape parameters（area,perimeter,circularity and Feret’s diameter）was measured using Image J software,with the results saved as Excel file format.2 Study on fruit characters of C.camphora from different provenancesWe investigated the seed and fruit traits of C.camphora from 8 provenances.The anthocyanins content was determined through the differential p H method.The result revealed that the anthocyanin content of C.camphora fruit was not affected by different provenance.Principal component analysis and analysis of variance were applied to analyze the variations of seed and fruit traits of C.camphora from different provenances to reveal the effects of spatial geography and topography on the seed and fruit traits.It was found that there were significant differences in the characters of C.camphora in different areas.In Huangshan area of Anhui Province,C.camphora fruit is the biggest.100-kernel weight and the peel rate is 55.394g and 65.22%,respectively.The circularity of C.camphora fruit was negatively correlated with other phenotypic traits（except seed circularity）.The correlation coefficients of peel rate with circularity,seed diameter and 100-seed weight were-0.082,-0.101 and-0.074,respectively;The peel rate was positively correlated with100-kernel weight and diameter of fruit,and the correlation coefficients were 0.709 and0.521,respectively.Study on the correlation between the phenotypic characters and environmental factors of C.camphora fruit find that the peel rate had the highest negative correlation with the altitude（Spearman’s rank correlationρ?=?-0.351）.The fruit circularity is negatively correlated with other traits except for seed circularity.The correlation coefficient of 100-kernel weight with longitude and latitude reached significant level,which pearson correlation coefficient is 0.427（p<0.01）and 0.275（p<0.05）,respectively.However,There is no significant correlation between 100-kernel weight and altitude,average temperature and annual precipitation.Furthermore,the cluster analysis indicated that the phenotypic characters of fruits and seeds of Huangshan Mountain in Anhui Province were significantly different from those of other provenances.Moreover,they were classified into one group,and the other 7 provenances are the second group.3 Transcriptome analysis was performed using RNA-seq and hub genes of anthocyanin biosynthesis in camphora fruit peel.The amplified c DNA fragments of the green fruit stage,turning color stage and ripe red stages were sequenced using Illumina Hi Seq TM platform.222344 unigenes were assembled by De novo assembly with Trinity software.124201 Unigenes were annotated by at NR database.Sequence homology revealed that 93912 of the assembled unigenes were assigned at least one GO term and 48912 unigenes were assigned at KEGG Database.Combining with KEGG metabolic pathway according to transcriptome annotation results,We analyzed the synthetic pathways involved in anthocyanin biosynthesis:Phenylpropanoid biosynthesis（ko00940）,Flavonoid biosynthesis（ko00941）and Anthocyanin biosynthesis（ko00942）,finding that the hub genes including Cluster-23301.86991、Cluster-23301.86265、Cluster-23301.87325、Cluster-23301.87656belong to Phenylalanine Ammonia-Lyase（PAL）Gene Family;Cluster-23301.85520 is part of F3’5’H Gene Family;Cluster-23301.86036 and cluster-23301.21176 belong to UFGT Gene Family;cluster-23301.84973 belongs to UGAT Gene Family.These hub genes provide basic data for the study of anthocyanin accumulation and metabolism related genes expression in camphora peel.Additionally,we found 11 MYB transcription factors and 22b HLH transcription factors,which may regulate anthocyanin metabolism.4α-Glucosidase inhibitory effect of anthocyanins from C.camphorafruit:Inhibition kinetics and mechanistic insights through in vitro and insilicoThe present study evaluated the in vitroα-glucosidase inhibitory activity of three major pigment constituents of C.camphora fruit,namely cyanidin,cyanidin 3-rutinoside,and cyanidin-3-O-glucoside.We found that cyanidin exerted strong inhibitory activity onα-glucosidase,with IC50 of 5.293×10-3m M,whereas cyanidin 3-rutinoside and cyanidin-3-O-glucoside did not show inhibitory activity onα-glucosidase.The inhibitory activity of cyanidin was stronger than that of acarbose（IC50 1.644 m M）,the current most commonly used drug for postprandial glycemia.Kinetic analysis indicated that cyanidin inhibitedα-glucosidase through competition,with a Ki value of 0.0183 m M.Fluorescence spectrum titration showed only one binding site between cyanidin andα-glucosidase,and the binding constant was calculated.Further,molecular docking was conducted to simulate the binding interactions between cyanidin andα-glucosidase.Cyanidin was found to interact with several residues close to the catalytic site ofα-glucosidase throughπ-πstack interaction and hydrogen bonds.The calculated binding energy of the cyanidin and enzyme complex was-105.031 k J/mol.Molecular simulation and calculation showed that the van der Waals force played an essential role in the binding ofα-glucosidase and cyanidin. |
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