| Fruit softening is a complicated and genetically determined process,it affects the' fruit firmness,the other fruit qualities.At present,the process of flesh softening during ripening has rarely been known in hawthorn.The hard-fleshed hawthorn Qiu JinXing and the soft-fleshed hawthorn RuanrouShanlihong 3 Hao were maintained in the National Hawthorn Germplasm Repository of China at Shenyang.Ruanrou Shanlihong 3 Hao fruits became softer during ripening,whereas Qiu JinXing fruits remained hard.And insect resistance of soft-fleshed fruit in maturity was far higher than that of hard-fleshed fruit.Thus,Ruanrou Shanlihong 3 Hao and Qiu JinXing fruits were used as the experimental materials.In this study,We studied in TEM observation,related hydrolytic enzymes activities(PG,Gns,P-GAL,etc.),the RNA-Seq and analysis aspects in the fruit developmental stages,providing strong evidence for the formation of different texture hawthorn fruits.The main results were as follows:1.Observation the ultramicroscopic structure of hawthorn fruit flesh cells,finding that at the middle developmental stages II of Ruanrou Shanlihong 3 Hao and Qiu JinXing,the structures of the cell walls in the fruit flesh were complete,presenting a light-dark-light structure and a plasma membrane located next to the cell wall.Moreover,deep-colored and closely arranged microfilaments and dark middle lamella were observed;when fruits ripening,the middle lamella of Ruanrou Shanlihong 3 Hao and Qiu JinXing fruit flesh tissue was nearly all degraded,the light-dark-light structure disappeared.However,the micro filaments in the Qiu JinXing fruit tissue were arranged closely and colored deeply,whereas the microfilaments in the Ruanrou Shanlihong 3 Hao fruit flesh tissue were arranged loosely,partially degraded and became lighter in color.Indicating the different texture hawthorn fruits ripening was caused by the different microfilaments degradation degree.2.Through the analysis of hawthorn fruit RNA-Seq,46.72 Gb clean data,2182914 contigs,199204 transcripts and 72837 unigenes were obtained.12,143,27,411,8,280,20,603,22,628,22,240 and 39,248 were respectively annotated to the NR,Swiss-Prot,GO,COG,KOG,KEGG and Pfam databases.We identified 85 unigenes related to the cell wall between hard-and soft-fleshed hawthorn fruits,and galactose metabolisms were involved in fruit softening.Data obtained by the RNA-Seq analysis could be used to provide data support for the further in-depth research of hawthorn fruit softening.3.This study cloned the full-length of ?-GAL(CpBGALl)of Ruanrou Shanlihong 3 Hao and Qiu JinXing,and analyzed its bioinfomatics,obtained that CpBGALl gene CDS length of two resources were 2196 bp,which could code 731 amino acids,amino acids sequences had signal peptides,it belonged to glycosyl hydrolases 35 family,contained two conservative structure domains—galectin and glycosyl hydrolase 35;founding that the 52 and the 302 amino acid of CpBGALl of Qiu JinXing mutated.We speculated the mutation of two amino acids of CpBGALl conservative structure domains caused the change of structure and function of protein.4.Enzyme activity determination in hawthorn fruit found that at the beginning of hawthorn fruit softening,?-Gal activity in the soft-fleshed fruit was well above its activity in the hard-fleshed fruit,and in the process of fruit softening,its activity in the soft-fleshed fruits was higher than its activity in the hard-fleshed fruit,this was consistent with real-time PCR results,which means ?-Gal had an important function in the early softening of different textures.In the process of hawthorn fruit softening,PG activity in hard-fleshed fruits was higher than its activity in the soft-fleshed fruits,PL activity in the soft-fleshed fruit was similar to its activity in hard-fleshed fruit,and its activity was on the decline in the soft-fleshed hawthorn fruit,they were consistent with real-time PCR results,which signified PG and PL has nothing to do with the reasons for the formation of different texture of hawthorn fruit;in the process of hawthorn fruit softening,Ffase and Gns' activity in the soft-fleshed fruit was well above their activity in the hard-fleshed fruit,and their activity rapidly rised in the process of soft-fleshed hawthorn fruit softening,they were consistent with real-time PCR results,which signified Ffase and Gns play an important role in the process of hawthorn softening.Real-time PCR analyzed that at the beginning of hawthorn fruit softening,the expression levels of PE4 in the soft-fleshed fruit was well above its expression levels in the hard-fleshed fruit,and in the process of fruit softening,its expression levels in the soft-fleshed fruits was higher than its expression levels in the hard-fleshed fruit,which means PE4 had an important function in the early softening of different textures;in the process of hawthorn fruit softening,the expression levels of a-GAL,PE63 and XTH in the soft-fleshed fruit were higher than that in the hard-fleshed fruit,and their expression levels rised in the process of soft-fleshed hawthorn fruit softening,which signified a-GAL,PE63 and XTH play an important role in the process of hawthorn softening. |
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