Characteristics And Expression Of Dormancy-associated MADS-box In Pyrus Pyrifolia

Pyrus pyrifolia is a perennial deciduous fruit tree which belongs to Rosaceae,Maloideae,pear(Pyrus L)plants.It is one of the important economic fruits in China,and was mainly distributed in southern’China region,which bring huge economic benefits for the south growers.However,pear flower buds have the characteristics of dormancy in winter.So it’s very important to explore the related mechanism of pear bud dormancy.In this study,the buds from ’huanghua’ and ’mixue’,two cultivars of Pyrus pyrifolia,were used as the experiment to explore the expression of dormancy associated transcription factor genes.Moreover,we studied the sequence characteristics of DAM genes and analyzed their expression patterns under different treatment.Main results are as follows.1.We got 18 screening involves five types of transcription factors(WRKY、MYB、MADS-box、bZIP and HD-ZIP).Among them,WRKY71,MYB108-like-2,MYB36,PpbZIP,PpMADS13-1,PpMADS13-3,PpMADS21 and PpMADS35 genes’ expression of peaks distribution in bud dormancy period,bud dormancy breaking critical period and dormancy breaking period,which shows that they may participate in the regulation of flower bud dormancy process conversion mechanism.WRKY33,WRKY7 are significantly expressed mainly in deep dormancy period,and they may participate in some physiological mechanism of regulation in the depth of the flower bud dormancy stage.MYB108-like-1,bZIP60,PpFT2a genes are expressed in the stage of flower bud sprouting,shows that they are mainly involved in the process of flower bud to blossom.PpFLC-like-1,PpFLC-like-2 are expressed in January 16 before the stage of dormancy breaking,suggests they may be a key regulation gene for bud dormancy.MYB21-like,APL-like,ATHB-12-like genes accumulated during the ecodormancy,suspected that they were raised in response to the stimulation in low temperature.2.Four MADS-box genes were cloned from ’huanghua’ and ’mixue’ respectively.Both of them had been login in GenBank.PpMADS13-1,PpMADS13-3,PpMADS21 and PpMADS35 amino acid sequence similarity was 100%,91.45%,100%and 81.7%respectively.The nucleotide sequence similarity was 81.7%for PpMADS35mx and PpMADS35hh which means the less conservation.The analysis of evolutionary relationship shows that they are clustering together with PpDAMs and MdDAMs which means a close genetic relationship.Among them,PpMADS21 close relationship with PpMADS13-3 and PpDAM1;PpMADS21 close relationship with PpMADS13-1 and PpDAM3.Subcelluar localization analysis show that thay were both located within the nucleus.3.We analysis the four DAM genes’ expression from ’huanghua’ for two consecutive years during the period of flower bud dormancy.Different from four DAM genes to fluctuations from’mixue’,four genes from ’huanghua’ showed reduction concomitant with dormancy release.And we also observed that the accumulation of DAM transcript has a little increase before or after the period of endodormancy breaking.The qPCR analysis in 2015-2016 shows that four DAM genes from ’huanghua’ expressed in Dec which consistent with the hypothesis of expression pattern of the DAM genes:Then,we accelerate endodormancy release by HC and GA4+7 treatments for ’huanghua’ in field.The results shows that four DAM genes tend to down-regulated both control group and treatment group.Compare with control group,the expression level of PpMADS13-1hh,PpMADS13-3hh and PpMADS21hh in HC treatment group were higher before Jan 14.But with the deepening of ecodormancy,their expression in HC treatment group lower than in control group.We suggested that Pyrus pyrifolia DAM genes could be in induced by low temperature and their own dormant state.Next,we treated ’huanghua’ branch with HC and low temperature in manual climatic box.The result shows that four DAM genes in HC treatment group down-regulated suddenly as well as in control group under the condition of 25℃.And the expression level of DAM genes in HC treatment group were lower than in control group.However DAM genes maintain the high level of expression in first 5 days after cooling treatment,and then reduced gradually.We also treated in ’mixue’ branch and the same result was obtained.Moreover,the accumulations of DAM genes were also found in the phloem.The above results suggested that DAM genes will be inducted during the endodormancy both in ’huanghua’ and ’mixue’.DAM genes were inducted by cooling and inhibited by heat.Dormant state is one of the important regulate factor for DAM genes.There is a possible relationship between chilling requirement and DAM expression.We suggested that DAM may need a continuous expression to maintain dormancy state in high chilling requirement pear,however DAM in low chilling requirement pear may just need expressed in some specific period to maintain dormancy state.4.Analyzed the expression of PpCBF and PpFT2a from ’mixue’ and ’huanghua’,which are suspected to respond to DAM.The result shows that PpCBF from ’huanghua’ was inducted in Jan 28 and PpCBF from ’mixue’ was inducted in Dec 12.We did not find marked relationship between PpCBF and DAM.PpFT2a from ’huanghua’ and ’mixue’ were up-regulated concomitant with dormancy release which were contrast to DAM.5.The promoters of PpMADS13-1,PpMADS13-3 and PpMADS21 from ’huanghua’ and’mixue’ were obtained.They contained the same elements such as light responsive elements,hormone-responsive elements and stress responsive elements.We also found the C-repeated/DRE from the promoters of PpMADS13-1mx.So we could suspect that DAMs were involved in the response of stress such as cooling,drought and heat and then regulated bud dormancy.6.The protein of PpMADS13-1mx was expressed in prokaryotic expression system.We succeeded in building pEASY-PpMADS13-1 expression vector which was transferred into E.coli strain BL21(ED3).During the 6 and 24 hours induced by IPTG(0.5mmol/L),the target band was found through the SDS-PAGE analysis.