| Pear,originated in China,is known as one of the most popular fruits,and is widely planted all over the world.It has been used as folk medicine and health food in daily life for more than two thousand years.It is not only an important source of many essential nutrients such as water,dietary fiber,vitamins and potassium,but also a source of phytochemistry.Arbutin,a phytochemical component with the functions of whitening,relieving cough,eliminating phlegm and diuresis,is regarded as a characteristic metabolite in pear.It is of great importance to carry out in-depth and systematic research on arbutin for the acquisition and innovative utilization of arbutin resources in pear.At present,the arbutin in pear has not been systematically reported,there are very few studies on the biosynthesis of arbutin to date.Firstly,we analyzed the antioxidant capacity and the distribution characteristics of phytochemical components in pear fruits,and investigated the role of arbutin in the antioxidant capacity of pear in this study.Moreover,focusing on arbutin biosynthesis,we screened for genes potentially related to arbutin biosynthesis using BSA-resequencing technology for the first time.In addition,we mined the key genes involve in arbutin synthesis using transcriptome sequencing technology.Furthermore,we systematically identified the members of UDP-glycosyltransferase(UGT)family that involved in arbutin glycosylation based on bioinformatics methods.And based on the above research,and one of them was cloned and its function was verified.It is expected to provide a theoretical basis for further elucidating the mechanism of arbutin biosynthesis in pear.The main results are shown as follow:1.In this study,the antioxidant activities of 58 pear fruits were analyzed through antioxidant potency composite index,and the correlations between total phenols,total flavonoids,arbutin and antioxidant capacity were investigated.The results showed that the antioxidant capacity of different pear fruits varied significantly,of which ‘Balixiang’ had the strongest comprehensive antioxidant capacity.The variation of total phenol contents was ranged from 266.73 μg/g to 1707.03 μg/g,and total flavonoid contents ranged from 64.81μg/g to 375.69 μg/g.The sum of total phenol contents and total flavonoids of ‘Jianba’ and ‘Balixiang’ was the highest.Arbutin was commonly detected in different pear varieties,and the content ranged from 22.43 μg/g(‘Dashuihe’)to 145.13 μg/g(‘Jianba’).Correlation analysis showed that the antioxidant capacity of arbutin and total phenol contents showed similar patterns,both of them had the highest Pearson correlation coefficients with ABTS free radical scavenging capacity.These results indicated that arbutin was an important antioxidant phenolic component in the pear fruits.2.The arbutin content of ‘Xuehua pear’ and ‘Korla fragrant pear’ at developmental stage as well as 143 individuals of their hybrid F1 offspring was determined using high performance liquid chromatography.The results showed that the fruit arbutin content of ‘Xuehua pear’ was significantly higher than that of ‘Korla fragrant pear’.And among 143 individuals of the F1 offspring,plant line-62 had the highest arbutin content.15 strains were selected respectively based on arbutin content to construct two extreme pools,and genes related to arbutin synthesis were mined by BSA genome resequencing.The candidate intervals of 3.78 Mb were obtained,including 7 subintervals on Chr1,11 and 15.And the results showed that the candidate intervals of 3.78 Mb,including 7 subintervals on Chr1,11 and 15 were obtained.360 non synonymous mutant genes were located in the candidate region,and 29 genes were related to metabolism,mainly involved in carbon metabolism,amino acid metabolism,sucrose and starch metabolism.Among them,Pbr008761.1 and Pbr017545.1 were closely related to the glycolytic process and encoded ATP-phosphofructokinase and enolase,respectively.In the promoter region of these genes,light-responsive elements as well as hormonerelated cis-elements such as salicylic acid(SA)and methyl jasmonate(Me JA)were predicted.3.It has been considered that arbutin was an upstream product of secondary metabolism,therefore pear varieties with highest arbutin and with lowest arbutin were selected to analyse the expression characteristics of genes encoded phenylalanine ammonia lyase(PAL),cinnamate 4-hydroxylase(C4H)and 4-coumarinyl coenzyme A ligase(4CL).The results showed that 14 genes were significantly correlated with arbutin content.In the promoter region of the above genes,lightresponsive elements and hormone-related cis-elements such as SA and Me JA were predicted.Therefore,the mechanisms of arbutin synthesis in response to light quality and SA were explored further.On the one hand,the different light quality environments simulated by the filter film were used for treatment.The results showed that,the content of arbutin in the fruit was the highest under the treatment of red filter film.The callus under the green filter film treatment had the lowest arbutin content,while had the highest content under the blue and purple filter film treatments.Transcriptome sequencing was performed,and 4814 differentially expressed genes(DEGs)were screened,of which 2020 were up-regulated and 2794 were down-regulated.The KEGG annotation results showed that DEGs were annotated on carbon metabolism,amino acid synthesis,sucrose and starch metabolism,and phenylpropane synthesis-related pathways.The metabolism-related genes Pbr008761.1 and Pbr017545.1 selected in the third chapter were significantly up-regulated after treated with purple filter.And the expression of C4 H and 4CL genes in the upstream of phenylpropane metabolism were up-regulated.On the other hand,different concentrations of SA were applied to pear callus,the results showed that the accumulation of arbutin were promoted by treatment with all concentrations of SA for 24 hours,and the effect of 100 μmol/L SA treatment was most best.Transcriptome sequencing was used to analyse the gene transcription levels in callus under SA treatment,and 380 DEGs were screened,of which 335 were up-regulated and 45 were down-regulated.The KEGG annotation results showed that the number of DEGs annotated to phenylpropane anabolism was the largest.Among all DEGs,transcription level of Pbr008363.1 was induced by SA,and it was also significantly correlated with arbutin content in different pear genotypes.In addition,the upstream genes of phenylpropane metabolism including Pbr017290.1,Pbr036272.1 and Pbr039972.1 were co-expressed genes after treatment by light and SA.And it is worth noting that three genes including Pbr006844.1,Pbr021064.1 and Pbr021069.1,annotated as hydroquinone glycosyltransferase activity,were significantly up-regulated induced by SA.The above genes may be the key genes involved in the arbutin synthesis.4.Glycosidation is the last step in arbutin synthesis.Therefore this study systematically analysed the members of the UDP-glycosyltransferase family in the white pear genome.178 UGT genes were identified,all of which had the motif of plant secondary product glycosyltransferase(PSPG).These genes were divided into 16 groups(A-P)through phylogenetic analysis.And the results showed that Pbr006844.1,Pbr014154.1,Pbr014155.1,Pbr021064.1 and Pbr021069.1 were closest with arbutin synthase of Rauvolfia verticillata.In addition,based on the correlation analysis between the transcription levels of UGT genes and the content of arbutin,11 UGT genes were screened which closely related to arbutin content.Furthermore,the arbutin content in the callus increased significantly treated with hydroquinone as substrate,and except Pbr039674.1,the transcriptional abundances of other genes were increased.These results provided important insights into the glycosylation process of arbutin in pear.5.Based on the above results,Pbr021069.1 was cloned in this study and named as Pb UGT72B1.Through the methods of gene transient transformation system and heterologous transformation Arabidopsis system,the results suggested that the Pb UGT72B1 can participate in the synthesis of arbutin. |
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