砧木类型对甜樱桃花芽多胺代谢相关基因表达和休眠的影响

Effects of Rootstock Types of Sweet Cherry on the Expression of Polyamine Metabolism Related Genes and Dormancy in Floral Buds

为验证砧木类型对甜樱桃多胺代谢和花芽休眠的影响,该研究以嫁接于不同砧木——矮化砧木‘吉塞拉6号’[Gisela 6,(G6)]和乔化砧木‘马扎德’(Mazzard)的甜樱桃品种‘罗亚理’为试材,通过田间观察确定嫁接于两种砧木的‘罗亚理’休眠期和花期,利用生物信息学、基因克隆、实时荧光定量、亚细胞定位和双分子荧光互补等手段,对甜樱桃花芽内多胺代谢中关键基因[多胺氧化酶基因(PavPAOs)和精氨酸脱羧酶基因(PavADC)]进行克隆和功能分析,对可能与PavADC存在互作关系的蛋白进行预测和验证,并利用高效液相色谱法对花芽内游离多胺的含量进行测定。结果表明:(1)‘Mazzard-罗亚理’的休眠期(75 d)长于‘G6-罗亚理’(69 d),且初花期晚于‘G6-罗亚理’。(2)‘G6-罗亚理’花芽内游离多胺含量高于‘Mazzard-罗亚理’,其中Put含量高1.2%~163.4%,Spd含量高8.8%~261.1%,Spm仅在1月和3月的‘G6-罗亚理’花芽中检测到。(3)成功克隆获得甜樱桃多胺代谢关键基因PavPAO2、PavPAO4、PavPAO5和PavADC,其长度分别为1485、1611、1704和2307 bp;系统进化分析表明,甜樱桃PavPAOs和PavADC均与桃、扁桃和苹果等物种的亲缘关系较近;亚细胞定位显示,PavPAO2和PavPAO4定位在细胞膜,而PavPAO5和PavADC则定位在细胞核。(4)qRT-PCR分析显示,休眠期间‘G6-罗亚理’花芽中PavPAO5的表达量低于‘Mazzard-罗亚理’,而PavADC的表达量高于‘Mazzard-罗亚理’;在整个休眠期,PavPAO2和PavPAO4的表达量整体水平远远低于PavPAO5,推测PavPAO5可能是在休眠期起主导作用的家族成员。(5)BiFC验证结果表明,在多胺代谢过程中PavADC与PavSAMDC存在互作关系,推测两者相互作用参与调控花芽内多胺水平。研究认为,甜樱桃砧木类型能够通过影响多胺代谢进程而影响休眠以及开花的进程,关键基因PavPAOs和PavADC的高表达能够影响花芽内多胺水平,PavADC能够使多胺含量升高从而促进休眠解除以及提早开花;多胺代谢过程中关键基因的表达和多胺含量变化能够影响休眠和花期,多胺含量的升高能够促进休眠解除以及提早开花。

In order to verify the effect of rootstock types on polyamine metabolism and floral bud dormancy of sweet cherry,this study collected the floral buds of sweet cherry variety‘Royal Lee’grafted in the dwarf rootstock Gisela 6(G6)and the vigorous rootstock Mazzard as materials.Field investigations found that the dormancy process and florescence of the same sweet cherry variety grafted in dwarf and vigorous rootstocks are significantly different,and then we used the bioinformatics,gene cloning technique,real-time fluorescence quantification,subcellular localization methods to clone and do functional analysis of the key genes[polyamine oxidase gene(PavPAOs)and arginine decarboxylase gene(PavADC)]in the polyamine metabolism in sweet cherry floral buds.With online websites and bi-molecular fluorescence complementation we predicted and verified proteins that may interact with PavADC.In addition,the content of free polyamines in floral buds was determined by high performance liquid chromatography.Results showed that:(1)the dormancy period of‘Mazzard-Royal Lee’(75 d)is longer than that of‘G6-Royal Lee’(69 d).And the flowering period of‘Mazzard-Royal Lee’is later than‘G6-Royal Lee’.(2)The content of polyamines in floral bud of‘G6-Royal Lee’is higher than that of‘Mazzard-Royal Lee’.In floral buds of‘G6-Royal Lee’,Put content was 1.2%-163.4%higher than that of‘Mazzard-Royal Lee’and Spd content was 8.8%-261.1%higher than that of‘Mazzard-Royal Lee’.Spm was not detected in the floral buds of‘Mazzard-Royal Lee’,while was synthesized in‘G6-Royal Lee’during January and March.(3)The key genes of sweet cherry polyamine metabolism PavPAO2,PavPAO4,PavPAO5 and PavADC were successfully cloned,and their lengths were 1485,1611,1704,and 2307 bp,respectively.Phylogenetic analysis showed that sweet cherry PavPAOs and PavADC are closely related to species such as Prunus persica,Prunus dulcis,and Malus domestica;subcellular localization analysis shows that PavPAO2 and PavPAO4 are located in the cell membrane,while PavPAO5 and PavADC are located in the nucleus.(4)qRT-PCR analysis shows that the expression of PavPAO5 in floral buds of‘G6-Royal Lee’is lower than that of‘Mazzard-Royal Lee’,while the expression of PavADC is higher than that in‘Mazzard-Royal Lee’during dormancy.Throughout the dormant period,the overall expression of PavPAO2 and PavPAO4 were much lower than PavPAO5.It is speculated that PavPAO5 may be the dominant family member during the dormant period.(5)BiFC shows that PavADC could interact with PavSAMDC during the polyamine metabolism and we speculated that the two are involved in regulating the levels of polyamines in floral buds.Studies have shown that the types of sweet cherry rootstocks can affect the process of dormancy and flowering by affecting the process of polyamine metabolism.The high expression of key genes PavPAOs and PavADC can affect the level of polyamines in floral buds.PavADC can increase the content of polyamines to promote the release of dormancy and early flowering.The expression of key genes and changes in polyamine content during the process of polyamine metabolism can affect dormancy and florescence,and the increase in polyamine content can promote the release of dormancy and early flowering.