Molecular Cloning And Functional Analysis Of Key Genes Involved In Sugar Accumulation Of Chinese Dwarf Cherry[Cerasus Humilis(Bge.)Sok.]

Cerasus humilis,as a member of the Rosaceae family,is a new fruit resource with high comprehensive utilization value.Sugar not only plays a major role in flavor characteristics of the Cerasus humilis fruit,but also is one of the commercial indicators of fresh fruit quality.Sugar is the leading factor affecting fruit quality and an important quality index in breeding work.Therefore,study on physiological and molecular biology mechanism of sugar accumulation in Cerasus humilis will provide a theoretical basis in improving fruit quality and speeding up the breeding process.Here,expressions of the key genes involved in sugar metabolism were studied by RNA sequencing of the fruit at five different developmental stages,and the relationships of their relative transcript abundance and activities of enzymes with sugar accumulation were analyzed during fruit development;Twelve coding sequences of the key genes involved in sugar metabolism were cloned,and their spatio-temporal expression patterns were also analyzed;Over-expression of ChSPS1,ChSS1 and ChSS3 in tobacco by Agrobacterium-mediated transformation were carried out to determine their roles in sucrose synthesis.The main results are as follows:1.The contents of soluble sugar for cultivar 'Nongda No.4'at five different developmental stages(80d,110d,117d,124d,and 131d after anthesis)were determined.The result showed:The sucrose content,glucose content,fructose content,sorbitol content and total sugar content were increased steadily along with the development of the fruit;In the mature period of fruit,sucrose content,glucose content,fructose content,sorbitol content were 19.882 mg·g-1FW(about 30%of the total sugar content),14.681 mg·g-1FW(about 22%),28.884 mg·g-1FW(about 44%),2.364 mg·g-1FW(about 4%)respectively,indicated that fructose was the main form of sugar accumulation.2.Fruits of five different developmental stages from cutivar 'Nongda No.4' were used at the materials for the RNA sequencing through Illumina Hi-Seq2000 platform.In total,26870,32705,32745,32247 and 31228 unigene sequences were annotated respectively from the sequencing output of '4-1A','4,2A','4-3A','4-4A','4-5A'.The RPKM value of 27 genes involved in sugar metabolism was found in gene expression profile,and activities of enzymes were determined.At the stage of 110d after anthesis during fruit development,the enzyme activities of SC?vAINV?NINV?SDH and FK were relatively high,and the high transcript levels were responsible,which caused low levels of sugar accumulation.As the fruit continues to grow,the transcript levels and activities of these enzymes were down-regulated.The concomitant sugar started to accumulate in vacuole.At the last two weeks of fruit development,the transcript level of HT was up-regulated result in the accumulation of fructose and glucose;The sucrose accumulation was enhanced which was consistent with the elevated enzyme activities of SPS and SS,and an increase in their transcript levels;As a result,fruit sweetness was improved due to high contents of total soluble sugars.3.Based on the RPKM value of 27 genes related to sugar metabolism,12 highly expressed genes were selected which were ChSPS1,ChSPS2,ChSSl,ChSS3,ChvAINV,ChNINV3,ChCWINV,ChHKl,ChFK2,ChSDH2,ChSUT4 and ChSOT2 respectively.Primers were designed for these 12 genes according to their full length sequences from the RNA sequencing data,and these genes were cloned using the fruit of the ripening stage(124d after anthesis)from cultivar 'Nongda No.4'.These genes encode 1057,1059,806,640,686,680,557,498,555,367,499 and 538 amino acids,respectively.The phylogenetic analysis revealed that Cerasus humilis shared a close evolutionary relationship with other plants belonging in Rosaceae family,such as Prunus cerasus,Prunus armeniaca,Prunus mume,Prunus persica,Malus domestica and Pyrus bretschneideri.4.The spatio-temporal expression patterns of 12 genes involved in sugar metabolism at different organs(root,stem,leaf,flower and fruit)and different developmental stages after anthesis(80d,110d,117d,124d,13 1d)of cultivar 'Nongda No.4' were analyzed by real-time PCR,and the results showed that these gene were expressed in all tissues but their expression profiles were specific.The real-time PCR analysis results were consistent with the expression profiles which showed high quality RNA-sequencing data were generated in this study.5.Three ChSPS genes ChSPSl,ChSPS2 and ChSPS3 were found in the RNA sequencing data.ChSPSl had the highest RPKM value and it was concluded to play a crucial role in sucrose synthesis in Cerasus humilis.The RPKM value changes of ChSS1 and ChSS3 were completely opposite during fruit development.In order to further validate the roles of ChSPSl,ChSSl and ChSS3 in sucrose synthesis,three of over-expression vectors pBI-ChSPS1,pBI-ChSSl and pBI-ChSS3 were constructed,and two RNAi vectors pBI-ChSSI-RNAi and pBI-ChSS3-RNAi were also constructed.6.Agrobacterium-mediated transformation was carried out and transgenic tobacco lines were obtained by resistance screening,PCR identification and GUS staining after transforming with three of over-expression vectors.The relative expression level of ChSPSl and the enzyme activity of SPS in leaves of transgenic tobacco were 1624-4626 and 1.15-1.55 times as much as in leaves of the wild type tobacco.The sucrose content and total sugar content in transgenic tobacco were increased 0.353-0.876 mg·g-1FW and 2.169-2.577 mg·g-1 FW as much as the wild type,respectively,indicating that the ChSPSl boosted the sucrose accumulation in the leaves of transgenic tobacco.The relative expression level of ChSS1 and the enzyme activity of SS in leaves of transgenic tobacco were 2692-4829 and 1.10-1.26 times as much as in leaves of the wild type tobacco.The sucrose content and total sugar content in transgenic tobacco were increased 0.489-0.768 mg·g-1FW and 1.092-2.082 mg·g-1FW as much as wild type,respectively,indicating that the ChSSl boosted the sucrose accumulation in the leaves of transgenic tobacco.The relative expression level of ChSS3 and the enzyme activity of SC in leaves of transgenic tobacco were 890-1979 and 1.37-1.94 times as much as in leaves of the wild type tobacco.The sucrose content and total sugar content were decreased 0.625-0.958 mg·g-1FW and 0.907-1.590 mg·g-1FW.The result indicated that the ChSS3 inhabited the sucrose accumulation in the leaves of transgenic tobacco.Above all,this study showed that the ChSS1 and ChSS3 might have opposite effects on sucrose accumulation in Cerasus humilis fruits.