Effects Of Different Temperature On Quality Of Post-harvest Pomegranate And Expression Of Related Genes

Pomegranate(Punica granatum L.),a species belonging to the family Punicaceae,small deciduous trees or shrubs.was a multifunction species with a wide range of economic value,such as fruit for eating,flower for ornamental,tree for virescence and exocarp for Chinese medicines.Currently,there was a little research on quality changes of post-harvest pomegranate.Storage has become a bottleneck affecting the development of pomegranate industry.Taking pomegranate cultivar ‘Yushizi’ as test materials,we researched the influence of different temperature(0℃,5℃,10℃,15℃)treatments on the content changes of souble sugar,titratable acid,Vitamin C,anthocyanin,total phenolic and total flavonoid in pomegranate seeds of post-harvest different storage period(0-56 d).At the same time the anthocyanin biosynthesis-related genes(F3’5’H,CHI,CHS)and ethylene signal transduction pathway related genes(ERF,ETR)were cloned from pomegranate seeds by RACE.The gene expression levels in pomegranate seeds during different temperature storage periods were analyzed by RT-PCR.Exploring the physiological and molecular mechanism of pomegranate post-harvest quality changes provided the theoretical basis in order to pomegranate post-harvest storage and preservation.The main results as follows:1.We analyzed the quality changes of pomegranate seeds of different storage time under different temperature treatments.(1)The contents of souble sugar rose at first,and then declined,the peaks of 0℃ and 5℃ were at about 28 d,10℃ and 15℃ were at around14 d.(2)Under the condition of 0℃ treatment,titratable acid contents declined significantly after peaking at 28 d;The contents were at a stable level under 5℃ and 10℃,and that showed a trend of rising under 15℃.(3)VC contents had no obvious change under0℃;VC accumulated fast before 14 d,then being at the stable stage under 5℃,10℃ and15℃.(4)Under different temperature(0℃,5℃,10℃,15℃)treatments,anthocyanin contents kept rising to maximums,which was 2.03 times,2.88 times,3.25 times and 4.17 times than 0 d,respectively.(5)Total phenolic contents had no obvious changes under 0℃and 10℃;The contents significantly reduced before 14 d,then had been in a stable level under 5℃;The contents showed a trend of rising under 15℃.(6)Under different temperature(0℃,5℃,10℃,15℃)treatments,total flavonoid contents rose at first,and then declined,all the the peaks were at 42 d,respectively 1.25 times,1.06 times,1.69 times and 1.56 times than 0 d.2.The full-length cDNA of anthocyanin biosynthesis-related genes(PgF3’5’H,PgCHI,PgCHS)were cloned by RACE.(1)The full-length cDNA of PgF3’5’H(Gen Bank accession No.: KU058892)was 1199 bp with an open reading frame of 918 bp encoding 305 amino acids.The 36 th to 39 th amino acids of the N-terminus of PgF3’5’H contained the characteristic conserved sequences(CYP motif "PPGP")of the cytochrome P450 family genes;Bioinformatics showed that the amino acid sequences of PgF3’5’H had high consistency with Eucalyptus grandis Eg F3’5’H2,Jatropha curcas Jc F3’5’H2 and Nelumbo nucifera Nn F3’5’H2 as 86%,82% and 81%,respectively.(2)The full-length cDNA of PgCHI(Gen Bank accession No.: KU058887)was 1154 bp with an open reading frame of693 bp encoding 230 amino acids.Bioinformatics showed that the identity of PgCHI with Paeonia lactiflora Pl CHI,Camellia nitidissima Cn CHI,Camellia sinensis Cs CHI were over 80%.(3)The full-length cDNA of PgCHS(Gen Bank accession No.: KU058888)was1676bp with an open reading frame of 1197 bp encoding 398 amino acids.PgCHS has two conserved domains of other CHS containing,namely cd00831 and PLN03173,also has the characteristics peptide sequence of CHS family: RLMMYQQGCFAGGTVLR;Bioinformatics showed that PgCHS had high consistency with Dimocarpus longan Dl CHS,Nelumbo nucifera Nn CHS,Populus euphratica Pe CHS as 93%.3.The full-length cDNA of ethylene signal transduction pathway related genes(ERF1,ERF2,ETR)were cloned by RACE.(1)The full-length cDNA of PgERF1(Gen Bank accession No.: KU058889)was 1612 bp with an open reading frame of 1185 bp encoding394 amino acids;The full-length cDNA of PgERF2(Gen Bank accession No.: KU058890)was 1611 bp with an open reading frame of 1185 bp encoding 394 amino acids.Both of PgERF1 and PgERF2 contained the DNA-binding-site at the 120 th to 177 th amino acids of the N-terminus,is a typical AP2/ERF center structure domain;Bioinformatics showed that the identity of PgERF1 and PgERF2 with Eucalyptus grandis Eg ERF12,Theobroma cacao Tc ERF1,Malus domestica Md ERF12 were over 64%.(2)The full-length cDNA of Pgetr(Gen Bank accession No.: KU058891)was 843 bp with an open reading frame of582 bp encoding 193 amino acids.Bioinformatics showed that Pg ETR had high consistency with Carica papaya Cp ETR,Momordica charantia Mc ETR,Pisum sativum Ps ETR as92%.4.Real time-PCR analyzed the expression of anthocyanin biosynthesis-related genes(PgF3’5’H,PgCHI,PgCHS).(1)The expression levels of PgF3’5’H showed a rising trend under 0℃,5℃ and 10℃,being unstable under 15℃;The expression of PgF3’5’H was significantly positively correlated with anthocyanin contents of pomegranate seeds under different temperature treatments.(2)After 14 d,the expression levels of PgCHI rosesignificantly under 0℃,keeping declining under 5℃,10℃ and 15℃;The expression of PgCHI was significantly positively correlated with anthocyanin contents under 0℃.(3)The expression levels of PgCHS showed a rising trend under 0℃;That of 5℃,10℃ and 15℃were significantly lower than 0℃;The expression of PgCHS was significantly positively correlated with anthocyanin contents under 0℃.5.Real time-PCR analyzed the expression of ethylene signal transduction pathway related genes(ERF1,ERF2,ETR).The consistency of PgERF1 and PgERF2 expression was as high as 0.954**;Under 0℃ treatments,the expression levels of the two genes showed a rising trend as the storage time(0-56 d)went on;Under 5℃,10℃ and 15℃treatments,the expression of the two genes rose at first,and then declined,at 28 d,14d and14 d respectively reached maximum value.(2)Under 0℃,5℃ and 10℃,the expression of Pg ETR rose to the maximum at 14 d,then suffered a drop though to various degrees;The expression showed a trend of declining after rising,peaked at 42 d.