野酿2号毛葡萄果实发育进程中苹果酸代谢规律及相关基因表达分析

Malic acid metabolism regulation and related genes expression during berry development in Vitis heyneana cv.Yeniang No.2

【目的】分析野酿2号毛葡萄果实发育进程中苹果酸代谢规律及相关基因表达情况,为提高野酿2号毛葡萄的酿造品质及优质葡萄酒生产提供参考依据。【方法】以5年生野酿2号毛葡萄为试验材料,于盛花后5~17周采集不同生长发育阶段果实,测定果实单粒重、pH、可溶性固形物和可滴定酸含量,采用高效液相色谱法测定果实中苹果酸、柠檬酸和酒石酸含量;使用酶标仪测定苹果酸代谢关键酶[苹果酸脱氢酶(NAD-MDH)、NADP-苹果酸酶(NADP-ME)和磷酸烯醇式丙酮酸羧化酶(PEPC)]活性;采用实时荧光定量PCR检测苹果酸代谢相关基因PEPC、MDH和ME的相对表达量,并分析3个基因与苹果酸含量的相关性。【结果】野酿2号毛葡萄果实单粒重在转色期前增长较快,转色期的果实pH、可溶性固形物、苹果酸含量分别是盛花后5周的1.03、1.36和2.38倍,盛花后11周可滴定酸含量达峰值,为35.68 mg/g。盛花后5~17周,野酿2号毛葡萄果实中酒石酸和柠檬酸含量变化不明显,而苹果酸含量变化幅度较大。盛花后8周,NAD-MDH和NADP-ME活性最高,分别为736.06和453.50 nmol/(g·min),幼果期PEPC活性较高。转色期果实中MDH基因的相对表达量是盛花后5周的1.62倍,而PEPC和ME基因的相对表达量均低于盛花后5周。相关分析结果表明,苹果酸含量与MDH基因呈极显著正相关(P<0.01),与PEPC基因呈显著正相关(P<0.05,下同),与ME基因呈显著负相关。【结论】野酿2号毛葡萄苹果酸含量变化是影响总酸变化趋势的主要因素,转色期是苹果酸含量由积累到降低的转折期,苹果酸含量受相关基因MDH、PEPC和ME表达调控。

【Objective】This study aimed to analyze the dynamics of malic acid metabolism and the expression patterns of related genes during berry development in Vitis heyneana cv.Yeniang No.2,provided reference for improving the brewing quality of V.heyneana cv.Yeniang No.2 and facilitating the production of high-quality wines.【Method】Fiveyear-old V.heyneana cv.Yeniang No.2 grapes were used as experimental materials,berries at different developmental stages were collected at 5-17 weeks after full bloom.The single berry weight,pH,soluble solids content(SSC)and titratable acidity(TA)content were measured.High-performance liquid chromatography(HPLC)was employed to determine the contents of malic acid,citrate,and tartrate in the berries.Enzyme-linked immunosorbent assay(ELISA)was used to assess the activities of key enzymes in malic acid metabolism:activities of NAD-dependent malate dehydrogenase(NAD-MDH),NADP-malic enzyme(NADP-ME)and phosphoenolpyruvate carboxylase(PEPC).Real-time fluorescence quantitative PCR was conducted to quantify the relative expression levels of genes related to malic acid metabolism(PEPC,MDH and ME),and the correlations between these genes and malic acid content were analyzed.【Result】The single berry weight of V.heyneana cv.Yeniang No.2 grapes increased rapidly before the veraison stage.During veraison,pH,SSC and malic acid content of the berries were as 1.03,1.36,and 2.38 times as those of 5 week after full bloom respectively.The TA peaked at 11 weeks after full bloom,reaching 35.68 mg/g.From 5 to 17 weeks after full bloom,the contents of tartrate and citrate did not change greatly,while the malic acid content fluctua-ted greatly.The activities of NAD-MDH and NADP-ME peaked at 8 weeks after full bloom,with values of 736.06 and 453.50 nmol/(g·min)respectively,while PEPC activity was high during the early berry development stage.During veraison,the relative expression of MDH gene was as 1.62 times as that at 5 weeks after full bloom,while the relative expression levels of PEPC and ME genes were lower than that at 5 weeks after full bloom.Correlation analysis revealed extremely significant positive correlation between malic acid content and MDH gene expression(P<0.01),significant positive correlation with PEPC gene expression(P<0.05,the same below),and significant negative correlation with ME gene expression.【Conclusion】Malic acid content variation is the primary factor influencing the trend of total acidity in V.heyneana cv.Yeniang No.2 grapes.The veraison stage marks a transition from malic acid accumulation to degradation.The expression of related genes MDH,PEPC and ME regulates malic acid content during berry development.