外源脱落酸抑制蓝莓早花及相关基因表达特性研究

Research on application exogenous abscisic acid in inhibiting early flowering and associated genes expression characteristics in blueberry

【目的】为抑制蓝莓暖冬早花,对南高丛蓝莓品种‘Emerald’进行外源脱落酸处理,了解脱落酸对蓝莓花芽内休眠及相关基因表达的影响。【方法】通过统计冬季低温积累量,以萌芽率为指标,确定蓝莓南高丛品种‘Emerald’花芽的不同休眠状态和打破休眠的需冷量;以不同休眠状态下的花芽为试验材料,通过外源施用不同浓度的ABA(50、100、150μmol·L^(-1))及ABA处理后给予不同的冷量,探究外源ABA及低温对蓝莓花芽内休眠促进及解除的影响。利用实时荧光定量PCR技术,探究外源ABA和低温处理对蓝莓花芽休眠相关基因表达的影响。【结果】打破蓝莓花芽内休眠时间为2019年12月4—11日,此后花芽萌芽能力强,进入生态休眠阶段。外源ABA处理具有促进花芽内休眠作用,在进入生态休眠之前外源施用ABA能显著抑制花芽萌发,8 d以上冷处理能抵消外源ABA对萌芽的抑制,并显著提高开花的整齐度,而在生态休眠阶段外源施用ABA,抑制效果不显著。花芽休眠相关基因中VcNCED3、VcPYL1的表达量在ABA处理后显著上调,随低温打破休眠显著下调。VcPYL2、VcCBF、VcGA20OX-1、VcFT的表达规律与其相反,说明其参与蓝莓花芽内休眠解除调控。【结论】生态休眠之前外源施用ABA能够调控相关基因的表达,加深蓝莓内休眠并抑制蓝莓早花。

【Objective】The study aimed at investigating the effect of abscisic acid on flower bud dormancy and related genes expression in blueberry(Vaccinium spp.).【Methods】The chilling accumulation of blueberry in winter was calculated using the Utah model in Jinhua.Annual branches of 20 cm length with flower buds of Southern highbush blueberry cultivar‘Emerald’were collected at the blueberry base for the study of sprouting rate estimation,hormone treatment,and cold treatment following hormone treatment.The branches with flower buds were randomly divided into three replicates.Sprouting rate was used as an indicator to determine dormant states of the flower buds and chilling requirement for breaking dormancy.‘Emerald’blueberry branches with flower buds were sampled on November 20,2019,December 11,2019 and January 8,2020.Flower buds in different dormancy states were used as experimental materials which were treated with exogenous ABA(50,100,150μmol·L^(-1))and different chilling times following ABA treatment.We aimed at investigating the effects of ABA and chilling time on dormancy promotion or release on blueberry floral buds.The qRT-PCR was employed to explore the expression of dormancy related genes in blueberry flower buds with treatments of exoge nous ABA and different chilling times.The total RNA of‘Emerald’flower buds was extracted by modified CTAB method.Reverse transcription of the total RNA into cDNA was performed by HiScriptⅢRT reagent Kit.Eight genes,VcNCED3,VcPYL1,VcPYL2,VcSVP,VcCBF,VcICE,VcGA20OX-1 and VcFT were filtered from differently expressed genes of a transcriptome profile of flower bud dormancyrelease.The specific primers of those genes in blueberry were designed by Primer 5.0 software.The two-steps qRT-PCR was performed with four biological replications per sample using a 2×Sybr Green qPCR Mix(High ROX)kit in the following system including2×SybrGreenqPCRMix of 10μL,5μmol forward primer and reverse primer respectively,cDNA(200 ng·μL^(-1))1μL,ddH2O 8μL,composing a 20μL total volume.The relative expressions of genes were estimated using 2^(-ΔΔCt)method.The significant differences were detected using SPSS Statistics 21 software.The data results were analyzed using single factor analysis of variance,such as LSD,Duncan,etc.,and using prism software for plotting.【Results】The endo-dormancy of flower buds was broken on between December 4 and December 11 in 2019.The sprouting rate of flower buds on December 11 in 2019 reached 83.14%,which indicated the endo-dormancy was broken based on an arbitrary release standard of 50%.Chilling requirement of‘Emerald’was 177.33 C·U.After that point,flower buds of blueberry had strong sprouting potential and high germination rate before entering eco-dormancy phase.Accordingly,we defined an endo-dormancy state,endo-dormancy release state and eco-dormancy state for blueberry variety‘Emerald’flower buds on Nov.20,Dec.2019 and Jan.8,2020.Exogenous application of ABA treatment before ecodormancy state could significantly inhibit sprouting of flower bud and promote flower bud into endodormancy.The germination rates of flower buds gradually decreased with the increase of ABA concentration.The exogenous application of 150μmol·L^(-1)ABA during the endodormancy state could significantly inhibit the germination of flower buds.The results showed that with the increase of 4℃cold treatment time,the germination rate of flower buds increased significantly,the accumulation of 8 days low temperature could counteract the inhibition of ABA on germination.At the same time,cold treatment could also promote the uniformity of flowering.According to the Utah model,chilling accumulation at 4℃for 4,8 and 12 days is equivalent to 96,192,288 C·U which could achieved in the field.However,no inhibitory effect was found when exogenous application of ABA was done in ecodormancy state.The expression level of the VcNCED3 in dormancy flower buds was significantly up-regulated after ABA treatment,and the expression level of the VcNCED3 was promoted by 3.67 fold.It was significantly down-regulated with low temperature breaking dormancy,the expression reached 0.15 when treated at 4℃for 8 days.The expression profile of the VcPYL1 showed similar pattern with the VcNCED3.By contrast,the genes VcPYL2,VcCBF,VcGA20OX-1 and VcFT were down-regulated with exogenous ABA treatment probably due to promoting flower buds into dormancy and being up-regulated by low temperature to break dormancy.Our results suggested that the genes might be involved in the regulation of blueberry flower bud dormancy.【Conclusion】The exogenous application of ABA could promote blueberry flower buds into deep endo-dormancy state through regulating the expression of flower buds dormancy related genes and inhibit blueberry early flowering.