Cloning And Expression Of NCED And CYP707A During The Dormancy Of Flower Buds In Pear

Pear?Pyrus L.?belongs to Rosaceae,and pear flower buds will take place dormancy to adapt to discomfort environment in the winter,and if they want to break dormancy need to experience the low temperature period.It was known that a change in ABA abundance may enhance or break the dormancy of flower buds.The variations of ABA abundance responding to external environmental changes were dependent on the comprehensive effect of the biosynthetic pathway and main metabolic pathway.In this study two kinds of pear?'Huanghua','Mixue'?were as the research objects,and the changes of ABA abundance during the pear flower bud dormancy and the characters of the genes?PpNCEDs,PpCYP707As?that play an important role in the biosynthetic pathway and main metabolic pathway of ABA were studied.1.Change of ABA abundance during the dormancy of pear flower budsChanges of ABA abundance during dormancy in 'Huanghua' and 'Mixue' pear were investigated by High Performance Liquid Chromatography?HPLC?.It was found that the change trend of ABA abundance during flower bud dormancy in'Huanghua' was similar as 'Mixue' that liked wave.There were four peaks in the change trend of ABA abundance in 'Huanghua',but there were three peaks in 'Mixue'.The ABA content in dormant flower buds of 'Mixue' was higher than 'Huanghua'.The ABA contents of 'Huanghua' and 'Mixue' decreased before flower buds dormancy release,respectively increased and decreased after the first increased in the process of flower buds dormancy release,and both increased after dormancy released.The experimental results showed the reason why the change of ABA abundance in'Mixue' pear flower buds during dormancy was different from 'Huanghua' may be that their chilling requires was different.A decrease in ABA abundance was necessary for pear bud dormancy break.Moreover,the ABA abundance was not only related to the regulation of pear flower buds dormancy,but also associated with response to external temperature variation.The change trend of single ABA content can not be as the judgment of pear flower bud dormancy process.2.The characteristics of bioinformatics of PpNCEDs and PpCYP707AsThe sequences of NCED and CYP707A were retrieved from the dataset of Pear Genome project by homologues search using local blasts,and the specific primers of NCEDs and CYP707As were designed by DNAMAN.Three NCEDs and five CYP707As were cloned from 'Huanghua' and 'Mixue' pear respectively.The genes were named as PpNCED1,PpNCED2,PpNCED3,PpCYP707A1,PpCYP707A2,PpCYP707A3,PpCYP707A4 and PpCYP707A5.The genes had been logined in NCBI.The phylogenetic tree analysis showed that each gene in the families within dispersed to several big branch cluster,in addition PpCYP707A4 and PpCYP707A5 may be similar.PpCYP707A4 from pear and the gene?XP₀08346457.1?from apple priority clustering,PpCYP707A5 from pear and the gene?XP₀08382035.1?from apple priority clustering,they may have originated from the common ancestor of apples and pears.According to the analysis of bioinformatics,the number of amino acids of PpNCED1,PpNCED2 and PpNCED3 proteins from 'Huanghua' was 617,613 and 607 respectively.The PpNCEDs proteins were hydrophilic protein and contained a PLNO2258 putative conserve domains.The number of amino acids of PpCYP707A1,PpCYP707A2,PpCYP707A3,PpCYP707A4 and PpCYP707A5 from 'Huanghua'was 452,451,479,476 and 474 respectively.The PpCYP707As proteins were hydrophilic protein and contained a PLN02196 putative conserve domains.The amino acid compositions within the family proteins were different,so the bioinformatics of the genes were different.3.Expression of PpNCEDs and PpCYP707As during the dormancy of flower buds in pearThe relative expression levels of PpNCED1,PpNCED2,PpNCED3,PpCYP707A1,PpCYP707A2 and PpCYP707A3 during the dormancy of 'Huanghua'pear and 'Mixue' pear flower buds were analyzed by qPCR.The results showed that the change trend of PpNCEDs and PpCYP707As express quantity during flower bud dormancy in 'Huanghua' and 'Mixue' liked wave.They were similar as the change trend of ABA abundance in 2012 and 2013.During 'Mixue' flower bud dormancy the change trend of PpNCED1 and PpCYP707A3 express quantity were similar as the change trend of ABA abundance,too.The expression patterns of each gene of PpNCEDs and PpCYP707As during the pear flower buds dormancy were different from each other,but they may play a role together in the ABA abundance.The experimental results showed that there was a dynamic balance about the regulation of ABA abundance by PpNCEDs and PpCYP707As.The expressions of PpCYP707As were involved in negative feedback regulation of ABA abundance,and the function performances of PpCYP707As were different in specificity of time and space between different varieties.But the expressions of PpCYP707As were not completely under the thumb of ABA abundance.In cold days PpNCEDs express quantity increased when ABA in pear flower buds deposited,so PpNCEDs may involve in the response to low temperature during pear flower buds dormancy.PpCYP707A2 may play an important role in the ABA decrease before the flower buds dormancy release of 'Huanghua',and PpNCEDs may play the same role in 'Mixue'.PpCYP707A1 and PpCYP707A2 may play an important role in the ABA decrease in the dormancy entirely release time of‘Mixue'pear.The relationship between the dormancy mechanism and the ABA regulation mechanism in low-chilling pear may be different from other chilling varieties.4.Analysis of elements on the promoter regions of PpNCEDs and PpCYP707AsThe promoters of PpNCED1,PpNCED2,PpNCED3,PpCYP707A1,PpCYP707A2 and PpCYP707A3 were cloned.The cis-elements on the six gene promoter were analyzed 21 kinds of elements involved in plant response to light were found,such as ACE,G-Box and AE-box.And 6 kinds of elements involved in plant response to hormone were found,like ABRE,GARE and TGA-elemen.Expressions of PpNCEDs and PpCYP707As were related to photoperiod changes and hormone balance,moreover they were adjusted by bZIP.5.Prokaryotic expression of PpNCEDl,PpCYP707A1 and PpCYP707A2PpNCED1,PpCYP707A1 and PpCYP707A2 from 'Huanghua' pear were expressed in E.coli in order to get recombinant protein.The open reading frames of the gene were cloned in pEASY-BluntE1 vector to construct the expression recombinant plasmid pEASY-PpNCED1,pEASY-PpCYP707A1,pEASY-PpCYP707 A2.The recombinant vectors were transferred into E.coli strain BL21?DE3?,and the expression strains were named as BL21?DE3?-pEASY-PpNCED1,BL21?DE3?-pEAS Y-PpCYP707A1 and BL21?DE3?-pEASY-PpCYP707A2.Total proteins of E.coli were analyzed by SDS-PAGE.After 6 hours induced by lmol/l IPTG,heterogenous PpCYP707A1 and PpCYP707A2 were expressed in the E.coli expression strains,but heterogenous PpNCED1 was not expressed.