‘靖安椪柑’果实发育阶段柠檬酸含量变化及其代谢相关基因的表达分析
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摘要
【目的】明确‘靖安椪柑’果实发育过程中柠檬酸含量的变化规律及其与相关基因的表达关系。【方法】以‘靖安椪柑’发育阶段的果实为材料,采用高效液相色谱法(HPLC)测定了盛花后60~200 d果实有机酸含量变化,运用实时荧光定量PCR(q RT-PCR)分析柠檬酸代谢相关基因相对表达量,同时分析有机酸含量变化与相关基因表达的关系。【结果】柠檬酸为‘靖安椪柑’果实主要有机酸,在果实中含量最高;在椪柑果实发育期间,柠檬酸含量呈现出先上升后下降的变化规律,奎宁酸、酒石酸和苹果酸含量呈递减的趋势,总有机酸和可滴定酸含量变化与柠檬酸含量变化一致;柠檬酸代谢相关基因表达及相关性分析结果显示,在发育期间,‘靖安椪柑’果实Cit CS1相对表达略有下降,Cit CS2和CitPEPC1相对表达先增加后减少,Cit PEPC2和Cit PEPC4的表达量在果实发育后期有所升高,这些柠檬酸合成基因表达均与柠檬酸含量无显著性相关;柠檬酸降解相关基因中,Cit PEPCKs、Cit Aco1和Cit FBPases相对表达整体呈缓慢下降的趋势,与柠檬酸含量无显著性相关,Cit Aco2/3和Cit ACLα1相对表达整体呈增长的趋势,Cit IDH1、Cit ACLα1和Cit ACLβ相对表达呈先下降后上升的趋势,Cit IDH2/3、Cit GADs和Cit GS2相对表达均呈先上升后下降的趋势,其中Cit Aco2/3和Cit ACLα1相对表达与柠檬酸含量呈显著负相关,Cit IDH1/2相对表达与柠檬酸含量呈极显著负相关。【结论】‘靖安椪柑’果实发育阶段有机酸含量主要由柠檬酸含量决定,随着果实发育成熟,柠檬酸和有机酸含量呈先升高后降低的变化,这种变化与柠檬酸合成相关基因表达无直接关联,主要受降解相关基因影响,尤其是Cit Aco2/3、Cit IDH1/2和Cit ACLα1的相对表达量的变化可能是调控‘靖安椪柑’发育阶段果实柠檬酸含量下降的主要原因之一。
【Objective】‘Jing'an ponkan'is an important citrus fruit in Jiangxi province. The fruits are rich in acids which are related to fruit quality. The study aimed to explore the changes of citric acid content and its relationship with the related gene expression in‘Jing'an ponkan'during fruit development.【Methods】The fruits of‘Jing'an ponkan'were picked up at 60, 80, 100, 120, 140, 180 and 200 days after full blossom(DAFB) respectively from 10 trees in an orchid of Jing'an county, Jiangxi province.A sample of 30 fruits were divided into three biological replicates, the flesh portions of the fruit were frozen with liquid nitrogen then stored at-80 ℃. The titratable acid(TA) was determined by acid-base neutralization titration, and the result was converted by the amount of citric acid. The organic acid content was determined by high performance liquid chromatography(HPLC). The extraction of RNA was carried out by CTAB method. And it was detected by agarose gel electrophoresis. Expressiones of genes involved in citric acid biosynthesis and degradation(Cit CSs, Cit PEPCs, Cit PEPCKs, Cit Acos, Cit IDHs,Cit GADs, Cit GS2, Cit ACLs and Cit FBPases) were analyzed by real-time fluorescence quantitative PCR(Q-PCR).【Results】Citric acid was the most important organic acid in‘Jing'an ponkan'fruits, followed by quinic acid and tartaric acid. The citric acid accounted for more than 71.15% of the total organic acid content in ponkan fruits. The quinic acid and the tartaric acid content accounted for 14.26%and 26.22% of the total organic acid content, while malic acid content was less than 0.2% of the total organic acid content. The citric acid increased from 60 DAFB to 120 DAFB, then decreased. This was consistent with the variation of TA and total organic acids content. At 120 DAFB, the citric acid content reached the maximum value of 32.91 mg· g-1. At 200 DAFB, the citric acid, the total organic acids and the titratable acid content were 17.20 mg· g-1, 22.49 mg· g-1 and 1.36%. The content of the quinic acid and the tartaric acid gradually decreased during fruit development and maturation. At 200 DAFB, the quinic acid and the tartaric acid content were 4.68 mg· g-1 and 0.56 mg· g-1. The malic acid content was very low in the whole developmental stage, and the total content decreased slightly. The results of the gene expressiones related to the citric acid metabolism and correlation analysis revealed that the relative expression of Cit CS1 decreased slightly, and the relative expression of Cit CS2 and Cit PEPC1 increased first and then decreased. The relative expression of the Cit PEPC2 and the Cit PEPC4 increased at the late stage in‘Jing'an ponkan'fruit, both of them had no significant correlation with the citric acid content. The relative expression of Cit PEPCKs, Cit Aco1 and Cit FBPases showed a slow decline, which also had no significant correlation with the citric acid content. The relative expression of Cit Aco2/3 and Cit ACLα 1 rose slowly at early developmental stage then increases rapidly during fruit maturation,which showed a significant negative correlation with the citric acid content. The relative expression of Cit IDH1 and Cit ACLα 1/β decreased firstly then increased, but the relative expression of Cit IDH2/3,Cit GADs and Cit GS2 was reverse, which increased firstlyand then decreased. The relative expression of Cit IDH1/2 and the citric acid content showed a significant negative correlation.【Conclusion】The citric acid was the most important acid in the organic acid of‘Jing'an ponkan'fruits during fruit development. With fruit ripening, the citric acid and the organic acid content increased firstly and then decreased. The change of the citric acid content in the fruit was not directly related to the gene expression related to the citric acid synthesis it is mainly influenced by the genes related to degradation. Especially the increase of the relative expression of the Cit Aco2/3,the Cit IDH1/2 and the Cit ACLα 1 in the late mid period of fruit development might be an important factor in regulation of of organic acid in‘Jing'an ponkan'fruit developmental period in the late fall.
【Objective】‘Jing'an ponkan'is an important citrus fruit in Jiangxi province. The fruits are rich in acids which are related to fruit quality. The study aimed to explore the changes of citric acid content and its relationship with the related gene expression in‘Jing'an ponkan'during fruit development.【Methods】The fruits of‘Jing'an ponkan'were picked up at 60, 80, 100, 120, 140, 180 and 200 days after full blossom(DAFB) respectively from 10 trees in an orchid of Jing'an county, Jiangxi province.A sample of 30 fruits were divided into three biological replicates, the flesh portions of the fruit were frozen with liquid nitrogen then stored at-80 ℃. The titratable acid(TA) was determined by acid-base neutralization titration, and the result was converted by the amount of citric acid. The organic acid content was determined by high performance liquid chromatography(HPLC). The extraction of RNA was carried out by CTAB method. And it was detected by agarose gel electrophoresis. Expressiones of genes involved in citric acid biosynthesis and degradation(Cit CSs, Cit PEPCs, Cit PEPCKs, Cit Acos, Cit IDHs,Cit GADs, Cit GS2, Cit ACLs and Cit FBPases) were analyzed by real-time fluorescence quantitative PCR(Q-PCR).【Results】Citric acid was the most important organic acid in‘Jing'an ponkan'fruits, followed by quinic acid and tartaric acid. The citric acid accounted for more than 71.15% of the total organic acid content in ponkan fruits. The quinic acid and the tartaric acid content accounted for 14.26%and 26.22% of the total organic acid content, while malic acid content was less than 0.2% of the total organic acid content. The citric acid increased from 60 DAFB to 120 DAFB, then decreased. This was consistent with the variation of TA and total organic acids content. At 120 DAFB, the citric acid content reached the maximum value of 32.91 mg· g-1. At 200 DAFB, the citric acid, the total organic acids and the titratable acid content were 17.20 mg· g-1, 22.49 mg· g-1 and 1.36%. The content of the quinic acid and the tartaric acid gradually decreased during fruit development and maturation. At 200 DAFB, the quinic acid and the tartaric acid content were 4.68 mg· g-1 and 0.56 mg· g-1. The malic acid content was very low in the whole developmental stage, and the total content decreased slightly. The results of the gene expressiones related to the citric acid metabolism and correlation analysis revealed that the relative expression of Cit CS1 decreased slightly, and the relative expression of Cit CS2 and Cit PEPC1 increased first and then decreased. The relative expression of the Cit PEPC2 and the Cit PEPC4 increased at the late stage in‘Jing'an ponkan'fruit, both of them had no significant correlation with the citric acid content. The relative expression of Cit PEPCKs, Cit Aco1 and Cit FBPases showed a slow decline, which also had no significant correlation with the citric acid content. The relative expression of Cit Aco2/3 and Cit ACLα 1 rose slowly at early developmental stage then increases rapidly during fruit maturation,which showed a significant negative correlation with the citric acid content. The relative expression of Cit IDH1 and Cit ACLα 1/β decreased firstly then increased, but the relative expression of Cit IDH2/3,Cit GADs and Cit GS2 was reverse, which increased firstlyand then decreased. The relative expression of Cit IDH1/2 and the citric acid content showed a significant negative correlation.【Conclusion】The citric acid was the most important acid in the organic acid of‘Jing'an ponkan'fruits during fruit development. With fruit ripening, the citric acid and the organic acid content increased firstly and then decreased. The change of the citric acid content in the fruit was not directly related to the gene expression related to the citric acid synthesis it is mainly influenced by the genes related to degradation. Especially the increase of the relative expression of the Cit Aco2/3,the Cit IDH1/2 and the Cit ACLα 1 in the late mid period of fruit development might be an important factor in regulation of of organic acid in‘Jing'an ponkan'fruit developmental period in the late fall.
引文
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[2]舒桂南,熊尚知.靖安椪柑生物学特性观察及栽培技术要点[J].现代园艺,2008(4):11-12.SHU Guinan,XIONG Shangzhi.The observed biological characteristics and cultivation techniques of Jing’an Ponkan[J].Modern Horticulture,2008(4):11-12.
[3]卢晓鹏,李菲菲,谢深喜.柑橘果实柠檬酸积累调控基因研究进展[J].果树学报,2018,35(1):118-127.LU Xiaopeng,LI Feifei,XIE Shenxi.Citrate accumulation in citrus fruit:a molecular perspective[J].Journal of Fruit Science,2018,35(1):118-127.
[4]郑丽静,聂继云,闫震.糖酸组分及其对水果风味的影响研究进展[J].果树学报,2015,32(2):304-312.ZHENG Lijing,NIE Jiyun,YAN Zhen.Advances in research on sugars,organic acids and their effects on taste of fruits[J].Journal of Fruit Science,2015,32(2):304-312.
[5]LI S J,YIN X.R,XIE X L,ALLAN C,GE H,SHEN S L,CHEN K S.The citrus transcription factor,citerf13,regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump,citvha-c4[J].Scientific Reports,2016,6:20151.
[6]HUSSAIN S B,SHI C Y,GUO L X,KAMRAN H M,SADKA A,LIU Y Z.Recent advances in the regulation of citric acid metabolism in citrus fruit[J].Critical Reviews in Plant Sciences,2017,36(4):241-256.
[7]林琼.柠檬酸代谢及转运相关基因对柑橘果实酸度的调控机制[D].杭州:浙江大学,2015.LIN Qiong.Regulation of citrus fruit acidity by genes related to citrate metabolism and transportation[D].Hangzhou:Zhejiang University,2015.
[8]赵淼,吴延军,蒋桂华,谢鸣,林毅,蔡永萍.柑橘果实有机酸代谢研究进展[J].果树学报,2008,25(2):225-230.ZHAO Miao,WU Yanjun,JIANG Guihua,XIE Ming,LIN Yi,CAI Yongping.Advances in research on the metabolism of organic acids in Citrus fruits[J].Journal of Fruit Science,2008,25(2):225-230.
[9]KAYZ E,BOO K H,KIM H Y,EIGENHEER R A,PHINNEY B S,SHULAEV,V,NEGRE Z F,SADKA A,BLUNWALD E.Label-free shotgun proteomics and metabolite analysis reveal a significant metabolic shift during citrus fruit development[J].Journal of Experimental Botany,2014,62(15):5367-5384.
[10]杨滢滢,高阳,向妙莲,陈明,陈金印.‘纽荷尔’脐橙果实发育过程中几种柠檬酸代谢相关基因的表达特征分析[J].果树学报,2016,33(4):400-408.YANG Yingying,GAO Yang,XIANG Miaolian,CHEN Ming,CHEN Jinyin.Expression profile analysis of several citric acid metabolism related genes in the‘Newhall’navel orange during fruit development[J].Journal of Fruit Science,2016,33(4):400-408.
[11]NOUR V,TRANDAFIR I,IONICA M E.Hplc organic acid analysis in different citrus juices under reversed phase conditions[J].Notulae Botanicae Horti Agrobotanici Cluj-Napoca,2010,38(1):44-48.
[12]张规富,卢晓鹏,谢深喜.不同时期水分胁迫对椪柑果实柠檬酸代谢相关基因表达的影响[J].果树学报,2015,32(4):525-535.ZHANG Guifu,LU Xiaopeng,XIE Shenxi.Influence of water stress in different development stage on the citric acid metabolism-related genes expression in the ponkan fruits[J].Journal of Fruit Science,2015,32(4):525-535.
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