苹果芽变品种珍富果实品质和花青苷合成基因表达分析

Analysis of fruit quality and expression of genes related to anthocyanins synthesis in apple bud sport Zhenfu

【目的】测定2001富士及其芽变品种珍富之间果实品质及花青苷合成相关基因表达水平差异,明确珍富品种特性,为其示范推广提供参考依据,也为研究果皮颜色芽变机制提供理论支撑。【方法】以芽变品种珍富及2001富士果实为试材,对其采收期及贮藏14 d后果实外观、内在品质、花青苷含量和合成相关基因表达水平进行测定。【结果】与2001富士相比,芽变品种珍富果实采收日期可以提前6 d,盛花期至采收期提前6 d;采收期及贮藏14 d后,外在品质方面,珍富果皮亮度、饱和度、花青苷总量及3种不同花青苷的含量均显著高于2001富士,但二者单果质量及果形指数无显著差异;果实内在品质方面,采收期及贮藏14 d后,珍富果实的可溶性固形物、维生素C含量及固酸比都显著高于2001富士,但珍富的可滴定酸含量显著低于2001富士;二者果肉硬度、破裂力在采收期及贮藏期均无显著差异;10个花青苷合成关键结构基因及5个转录因子类调控基因都有不同程度的提高,关键结构基因MdC4H、MdANS、MdUFGT及转录因子基因MdMYB10、MdMYB11、MdERF3表达量显著提高。【结论】采收期及贮藏14 d后,红色芽变品种珍富果皮亮度L*值及果皮颜色饱和度C*值均显著高于2001富士;珍富果实可溶性固形物、维生素C含量及固酸比显著提高,可滴定酸含量显著降低;珍富果皮中花青苷总量及3种不同花青苷含量都显著提高,花青苷合成相关结构基因和转录调控基因也显著上调表达。

【Objective】Fuji 2001 apple has been widely planted since it was introduced into China from Japan in 1993.Zhenfu is a red bud sport of Fuji 2001,and it passed variety registration on August 24,2021.The peel of Zhenfu shows more obvious red color than that of Fuji 2001.This mirable advantage in peel color has attracted the attention of apple growers.However,whether there are differences in fruit quality,anthocyanin compositions and contents,and expression of anthocyanin synthesis-related genes need to be further clarified.These results will provide reference for production extension and a research basis for disclosing the peel pigment mechanism of Zhenfu.【Methods】Traits of fruits,internal quality,composition of anthocyanin compounds and expression level of the genes related to pigment synthesis of Fuji 2001 apple and its bud sport Zhenfu fruits were measured and assessed.The maturity of apple was determined by the starch-iodine staining method.The vertical and horizontal diameters of each fruit were measured by the syntek electronic digital vernier caliper.The single fruit weight of each fruit was measured by an electronic balance.The flesh firmness and rupture force were measured by TMS-PRO texture analyzer.The content of soluble solids was measured by PR-101αrefractometer,and peel brightness(L*)and saturation(C*)were measured by CR-400 colorimeter.Titratable acid was determined by acid-base titration.The contents of vitamin C and soluble solids were determined by 2,6-dichloroindophenol titration and PAL^(-1) digital refractometer,respectively.Solid/acid ratio was expressed as the ratio of soluble solids to total acid.Peel sample of 1.00 g was grinded into homogenate in liquid nitrogen,dissolved in 5 mL of HCl-methanol(0.5∶99.5,v/v)solution,extracted for 24 h at 4℃under dark conditions,centrifuged at 12000 r·min-1 for 10 min at 4℃,and filtered through 0.22μm organic phase membrane.1.5 mL supernatant was transferred to an automatic injection bottle and detected by Waters HPLC high performance liquid chromatography to determine anthocyanin contents.The total RNA of fruit peel was extracted by trizol extraction kit.RNA was reverse transcribed into cDNA using the firststrand cDNA synthesis kit.The first-strand cDNA was applied to analyze gene expression levels by stepone fluorescence quantitative PCR.【Results】The fruit harvest date of Zhenfu was 6 days earlier than Fuji 2001.In terms of external qualities,both apple peels set red stripes,but Zhenfu peel showed more obvious red color.At the day of harvest and 14th day after storage,L*value of peel brightness and C*value of peel color saturation of Zhenfu were significantly higher than those of Fuji 2001.Three kinds of anthocyanin compounds,including cyanidin-3-galactoside,cyanidin-3-O-glucoside and anthocyanin rhamnoside,were detected in the peels of both Fuji 2001 and Zhenfu.Among them,the content of cyanidin-3-galactoside was the highest,and was the main anthocyanin component in the peel of Fuji 2001 and Zhenfu.At the day of harvest,the contents of cyanidin-3-galactoside,cyanidin-3-O-glucoside and anthocyanin rhamnoside in Zhenfu peel were 2.21,1.98 and 1.60 times higher than those of Fuji 2001 peel.The total contents of anthocyanins in Zhenfu were 2.18 times higher than those of Fuji 2001.After 14-day storage,the contents of 3 kinds of anthocyanins and the total amount of anthocyanins in Zhenfu were still higher than those in Fuji 2001.There was no significant difference in fruit quality and fruit shape index between them.In terms of internal quality,the contents of soluble solids,vitamin C and solid acid of Zhenfu were significantly higher than those of Fuji 2001.The titratable acid content of Zhenfu was significantly lower than that of Fuji 2001.However,there were no significant differences in flesh firmness and rupture force between the two varieties at the day of harvest and 14th day after storage.Some key structural genes and positively regulating transcription factor genes in anthocyanin synthesis pathway showed higher expression levels in Zhenfu than those in Fuji 2001,especially MdC4H,MdANS,MdUFGT,MdMYB10,MdMYB11 and MdERF3.【Conclusion】At harvest and 14th day after storage,peel brightness L*value and peel color saturation C*value of Zhenfu were significantly higher than those of Fuji 2001.The titratable acid content of Zhenfu was significantly lower than that of Fuji 2001,and the soluble solids content,vitamin C content and solid/acid ratio were significantly higher than those of Fuji 2001.In addition,the total amount of anthocyanins in the peel of Zhenfu was significantly higher than that of Fuji 2001.The key structural genes in the anthocyanin synthesis pathway of Zhenfu and the related transcriptional regulatory factors were significantly up-regulated in the peel of Zhenfu.