Screening of internal reference genes of cut flowers of Paeonia lactiflora and expression analysis of key genes of ethylene biosynthesis
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摘要:目的
筛选芍药切花开放至衰老过程中和盛开期不同组织中稳定表达的最适内参基因，深入解析ACS和ACO基因在乙烯介导的芍药切花开放至衰老过程中的功能。
方法（1）以芍药‘桃花飞雪’为材料，基于qRT-PCR技术分析13个候选内参基因在芍药切花开放至衰老不同时期及盛开期不同组织中表达水平的变化，应用geNorm、NormFinder、BestKeeper等软件和ΔCt法对其表达稳定性进行了评价；通过在线RefFinder网站分析获得候选内参基因表达稳定性的综合排名。（2）利用筛选出的双内参基因，对乙烯生物合成关键基因ACS和ACO在芍药切花开放至衰老不同时期及盛开期不同组织中的表达水平进行定量分析。
结果（1）在芍药切花开放至衰老的不同时期，geNorm分析表明GAPDH和RAN3表达稳定性最好；NormFinder分析显示RAN3表达最稳定；BestKeeper分析表明18S rRNA表达稳定性最好；RefFinder综合分析表明RAN3和GAPDH为最适内参基因。（2）在盛开期芍药切花花器官不同组织中，geNorm分析表明ARF和PP2A表达稳定性最好；NormFinder分析显示UBQ表达最稳定；BestKeeper分析表明GAPDH表达稳定性最好；RefFinder综合分析表明UBQ和ARF为最适内参基因。（3）在芍药切花开放至衰老过程中，ACS基因表达量显著低于ACO基因表达量，是调控乙烯合成的开关；ACO主要在切花开放前期（瓶插0 ~ 12 h）发挥作用；ACS表达量在绽口期和衰败期（瓶插6 h和144 h）均发挥重要作用，并在盛开期对乙烯合成起到限制作用；（4）在芍药切花盛开期不同组织中，ACS基因表达量显著低于ACO，ACS和ACO基因的表达存在组织差异性，且均在茎和花萼中表达较高。
结论本研究筛选出芍药切花开放至衰老不同时期（RAN3和GAPDH）以及盛开期不同组织中（UBQ和ARF）qPCR定量表达分析的双内参基因组合；乙烯生物合成关键基因ACO主要在芍药切花开放过程发挥作用，ACS基因在芍药切花开放和衰老过程均发挥重要的作用。研究结果将为切花寿命的改良提供参考。
Abstract:ObjectiveThis study aims to select the most suitable reference genes, which are stably expressed during the process from the opening to senescence and different tissues in blooming period of peony cut flowers, and to deeply analyze the function of ACS and ACO genes in ethylene-mediated process of peony cut flower opening to senescence.
Method(1) With cultivar ‘Taohuafeixue’ as materials, the variations of expression levels of 13 candidate internal reference genes at different stages from blooming to senescence and in different tissues of the full bloom stage in herbaceous peony cut flowers were analyzed by qRT-PCR. The expression stability of 13 candidate internal reference genes was assessed by ΔCt and 3 softwares including geNorm, NormFinder, and BestKeeper. The comprehensive rankings of expression stability of candidate internal reference genes were obtained by online RefFinder analysis. (2) Based on the selected double internal reference genes, the expression profile of ACS and ACO, key genes of ethylene biosynthesis were quantitatively analyzed at different stages from blooming to senescence and in different tissues of full bloom stage in herbaceous peony cut flowers.
Result(1) geNorm analysis showed that the expression stability of GAPDH and RAN3 at different stages was best. NormFinder analysis indicated that the expression stability of RAN3 was optimal. BestKeeper analysis illustrated that 18S rRNA expression was the most stable. RefFinder analysis indicated that RAN3 and GAPDH were the best suitable internal reference genes at different stages. (2) geNorm analysis indicated that the expression of ARF and PP2A in different tissues had the best expression stability. NormFinder analysis showed that the expression of UBQ was the most stable. BestKeeper analysis illustrated that GAPDH was best. RefFinder analysis indicated that UBQ and ARF were the best internal reference genes in different tissues in herbaceous peony cut flowers. (3) From opening to senescence process of peony cut flowers, the expression of ACS gene was significantly lower than that of ACO gene, and ACS was the ‘switch’ for the regulation of ethylene synthesis. ACO mainly played a role in pre-opening stage of cut flowers (0−12 h). ACS expression played an important role both in the bloom stage and in the senescence stage (6 h and 144 h), and played a limiting role in ethylene synthesis during the bloom stage. (4) ACS expression was significantly lower than ACO in different tissues of peony cut flowers during blooming period, there was tissue variability in the expression of ACS and ACO, and both of them were higher in stems and calyxes.
ConclusionThis study screens double internal reference gene combinations for qPCR quantitative expression analysis in different tissues of herbaceous peony cut flowers at varied periods from opening to senescence (RAN3, GAPDH) and at full bloom (UBQ, ARF). ACO gene, a key gene of ethylene biosynthesis mainly plays a role in the blooming process, and ACS plays an important role in both blooming and senescence process in herbaceous peony cut flowers. The finding will provide a reference for the improvement of cut flower longevity.
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图 1 13个候选内参基因PCR产物的熔解曲线
Figure 1. Melting curves of PCR products of 13 candidate internal reference genes
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图 3 13个内参基因表达稳定性的geNorm分析结果
Figure 3. geNorm analysis results of expression stability of 13 internal reference genes
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图 4 标准化的内参基因个数的geNorm分析结果
Figure 4. geNorm analysis results of standardized internal reference gene number
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图 5 13个内参基因表达稳定性BestKeeper分析结果
Figure 5. BestKeeper analysis result of expression stability of 13 internal genes
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图 6 芍药切花开放至衰老不同时期花瓣中ACS和ACO基因相对表达水平
不同大写字母表示同一基因不同瓶插时间之间差异显著（P < 0.05）；不同小写字母表示不同基因相同瓶插时间之间差异显著（P < 0.05）。
Figure 6. Relative expression level of ACS and ACO genes at different developmental stages from blossoming to senescence in herbaceous peony cut flowers
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图 7 ACS和ACO基因在芍药切花不同组织中的定量表达分析
不同大写字母表示同一基因不同组织之间差异显著（P < 0.05）；不同小写字母表示不同基因相同组织之间差异显著（P < 0.05）。
Figure 7. Quantitative expression analysis of ACS and ACO genes in different tissues during full bloom stages in herbaceous peony cut flowers
表 1 qRT-PCR引物信息
Table 1 Information for qRT-PCR primers
基因 引物序列 产物长度/bp 18S rRNA F：TCAGCCTTGCGACCATACTCC 113 R：AACCATAAACGATGCCGACCA Actin1 F：AAGCCCAGTCCAAGAGAGGTA 107 R：ATTGTAGAAGGTGTGATGCCA Actin2 F：TAACCCCAAAGCCAACAGAGAA 140 R：CACCAGAATCCAGCACAATACC RPL40 F：AATACCTCCTGACCAACA 133 R：CTCAATAATTCCACCTCG GAPDH F：TAAAGGGTGGTGCTAAGAAG 105 R：CAATGTGAAGATCAGGAGTG UBQ F：GACTCTCCATCTGGTCCTCA 116 R：CCTTCACATTATCAATCGTATC UBQ-L10 F：AAGGCCAAGATCCAGGATAA 137 R：CGCAAGACAAGGTGAAGAGT ARF F：AGACATTACTTCCAAAACACCC 122 R：GCATCCCTTAGTTCATCCTCGT EF1α F：GCCCTACTGGACTGACCACTGA 140 R：GGATGCTACATAACCACGCTT PP2A F：GCTGCTCAGTTCAACCACACC 113 R：GCACTAAATACCGTTACCACAT RAN3 F：AAGAACAGGCAGGTGAAGGCA 120 R：ACGGGCAAGGTAGAGAAAAGG RPL23 F：GAGCCAAGAACCTTTACATC 100 R：TTGACAGTTGCCATCACCAT TUA F：AGTCTACCCATCCCCACAAG 104 R：CAAGGAGAACTGCCACATCA ACS F：TCGGAGCCTGGATGGTTTAG 135 R：TTGCCAACTTTGTTTCTTCCTT ACO F：GTTCCTCCTATGCGTCATTCCA 118 R：TCCTATTACCATCGGTTTGAGC 
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表 2 内参基因PCR引物序列及相关信息
Table 2 PCR primer sequences and related information for internal reference genes
基因 扩增效率（E） 决定系数（R2） 18S rRNA 1.811 0.999 29 ACT1 1.978 0.999 39 ACT2 1.965 0.999 59 RPL40 1.903 0.999 21 GAPDH 1.954 0.999 27 UBQ 1.926 0.999 25 UBQ-L10 2.037 0.999 14 ARF 1.958 0.999 21 EF1α 1.989 0.999 37 PP2A 1.981 0.999 26 RAN3 1.994 0.999 27 RPL23 1.931 0.999 25 TUA 1.924 0.999 55
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表 3 13个内参基因表达稳定性的NormFinder分析结果
Table 3 NormFinder analysis result of expression stability of 13 internal reference genes
基因 切花开放至衰老不同时期 盛开期不同组织 稳定值（M） 排名 M 排名 18S rRNA 1.204 12 1.311 12 ACT1 0.357 4 0.328 4 ACT2 0.743 10 0.236 2 RPL40 0.959 11 0.862 8 GAPDH 0.454 6 0.978 9 UBQ 0.481 7 0.116 1 UBQ-L10 1.699 13 1.903 13 ARF 0.222 3 0.333 5 EF1α 0.391 5 0.760 7 PP2A 0.173 2 0.509 6 RAN3 0.160 1 0.274 3 RPL23 0.504 8 1.205 10 TUA 0.665 9 1.240 11
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表 4 13个内参基因在‘桃花飞雪’切花开放至衰老不同时期花瓣中的表达稳定性综合排名
Table 4 Overall ranking of expression stability values of 13 internal reference genes in petals at different times from opening to senescence
基因 ΔCt Bestkeeper NormFinder geNorm RefFinder 18S rRNA 12 1 12 11 9 ACT1 7 6 4 8 7 ACT2 10 11 10 10 11 RPL40 11 12 11 12 12 GAPDH 4 4 6 1 2 UBQ 6 3 7 3 4 UBQ-L10 13 13 13 13 13 ARF 2 9 3 5 3 EF1α 5 5 5 6 6 PP2A 3 10 2 7 5 RAN3 1 8 1 1 1 RPL23 8 7 8 4 10 TUA 9 8 9 9 8 注：表中数据为排名次序。下同。
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表 5 13个内参基因在‘桃花飞雪’不同组织中的表达稳定性综合排名
Table 5 Overall ranking of expression stability values of 13 internal reference genes in different tissues
基因 ΔCt Bestkeeper NormFinder geNorm RefFinder 18S rRNA 12 11 12 12 12 ACT1 5 2 4 5 5 ACT2 4 3 2 6 4 RPL40 8 12 8 7 9 GAPDH 9 1 9 9 7 UBQ 1 4 1 4 1 UBQ-L10 13 13 13 13 13 ARF 3 7 5 1 2 EF1α 7 8 7 8 8 PP2A 6 9 6 1 6 RAN3 2 6 3 3 3 RPL23 10 10 10 10 11 TUA 11 5 11 11 10
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[1] 李嘉珏. 中国牡丹与芍药[M]. 北京: 中国林业出版社, 1999. Li J J. Chinese tree peony and herbaceous peony[M]. Beijing: China Forestry Publishing House, 1999.
[2] 于晓南. 观赏芍药[M]. 北京: 中国林业出版社, 2019. Yu X N. Ornamental herbaceous peony[M]. Beijing: China Forestry Publishing House, 2019.
[3] 王哲, 史国安, 马雪情, 等. 芍药‘桃花飞雪’开花衰老期间乙烯代谢生理机制的研究[J]. 园艺学报, 2014, 41(11): 2268−2274. Wang Z, Shi G A, Ma X Q, et al. The ethylene metabolism in flowers of Chinese peony ‘Taohuafeixue’ during opening and senescence[J]. Acta Horticulturae Sinica, 2014, 41(11): 2268−2274.
[4] Lu J Y, Zhang G F, Ma C, et al. The F-box protein RhSAF destabilizes the gibberellic acid receptor RhGID1 to mediate ethylene-induced petal senescence in rose[J]. Plant Cell, 2024, 36(5): 1736−1754. doi: 10.1093/plcell/koae035
[5] Wang T, Sun Z, Wang S Q, et al. DcWRKY33 promotes petal senescence in carnation (Dianthus caryophyllus L.) by activating genes involved in the biosynthesis of ethylene, abscisic acid and accumulation of reactive oxygen species[J]. The Plant Journal: for Cell and Molecular Biology, 2023, 113(4): 698−715. doi: 10.1111/tpj.16075
[6] Wang M, Wang M, Ni C, et al. Differences in ethylene sensitivity, expression of ethylene biosynthetic genes and vase life among carnation varieties[J]. Ornamental Plant Research, 2024, 4: e004.
[7] Khan S, Alvi A F, Saify S, et al. The ethylene biosynthetic enzymes, 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) and ACC oxidase (ACO): the less explored players in abiotic stress tolerance[J]. Biomolecules, 2024, 14(1): 90. doi: 10.3390/biom14010090
[8] 李芳. 芍药花乙烯代谢及信号转导主要基因的克隆与分析[D]. 洛阳: 河南科技大学, 2013. Li F. Cloning and analysis of genes encoding ethylene biosynthesis and signal transduction elements from herbaceous peony (Paeonia lactiflora Pall.)[D]. Luoyang: Henan University of Science and Technology, 2013.
[9] 肖士奎, 李芳, 张文婷, 等. 芍药ACS基因的克隆及其蛋白质原核表达[J]. 华北农学报, 2022, 37(5): 36−44. Xiao S K, Li F, Zhang W T, et al. Cloning and prokaryotic expression of ACS gene from Paeonia lactiflora Pall. [J]. Acta Agriculturae Boreali-Sinica, 2022, 37(5): 36−44.
[10] Ashraf J, Zuo D, Wang Q, et al. Recent insights into cotton functional genomics: progress and future perspectives[J]. Plant Biotechnology Journal, 2018, 16(3): 699−713. doi: 10.1111/pbi.12856
[11] Wagner E M. Monitoring gene expression: quantitative real-time RT-PCR[J]. Methods in Molecular Biology, 2013, 1027: 19−45.
[12] Huggett J, Dheda K, Bustin S, et al. Real-time RT-PCR normalization; strategies and considerations[J]. Genes and Immunity, 2005, 6(4): 279−284. doi: 10.1038/sj.gene.6364190
[13] Peng S J, Ali S I, Hu X L, et al. Advancements in reference gene selection for fruit trees: a comprehensive review[J]. International Journal of Molecular Sciences, 2024, 25(2): 1142. doi: 10.3390/ijms25021142
[14] 李健. 芍药实时定量PCR内参基因的筛选和验证[J]. 分子植物育种, 2017, 15(7): 2544−2549. Li J. Selection and validation of reference genes for quantitative real-time PCR in herbaceous peony[J]. Molecular Plant Breeding, 2017, 15(7): 2544−2549.
[15] 梁蕤, 徐雷锋, 毕蒙蒙, 等. 柠檬色百合实时荧光定量PCR内参基因筛选[J]. 中国农业大学学报, 2023, 28(2): 59−73. doi: 10.11841/j.issn.1007-4333.2023.02.006 Liang R, Xu L F, Bi M M, et al. Validation of reference genes for quantitative real time-PCR in Lilium leichtlinii[J]. Journal of China Agricultural University, 2023, 28(2): 59−73. doi: 10.11841/j.issn.1007-4333.2023.02.006
[16] 周琳, 张永春, 蔡友铭, 等. 彩色马蹄莲不同品种和组织qRT-PCR内参基因筛选[J]. 分子植物育种, 2020, 18(12): 3971−3979. Zhou L, Zhang Y C, Cai Y M, et al. Screening of qRT-PCR reference genes in different varieties and tissues of Zantedeschia hybrida[J]. Molecular Plant Breeding, 2020, 18(12): 3971−3979.
[17] 李雪, 潘学军, 张文娥, 等. 核桃内参基因实时荧光定量PCR表达稳定性评价[J]. 植物生理学报, 2017, 53(9): 1795−1802. Li X, Pan X J, Zhang W E, et al. Stability evaluation of reference genes for quantitative real-time PCR analysis in walnut (Juglans spp.)[J]. Plant Physiology Journal, 2017, 53(9): 1795−1802.
[18] 王荣花, 刘雅莉, 李嘉瑞. 不同发育阶段牡丹和芍药切花开花生理特性的研究[J]. 园艺学报, 2005, 32(5): 861. doi: 10.3321/j.issn:0513-353X.2005.05.019 Wang R H, Liu Y L, Li J R. Studies on the blossom physiology in the different development stage of peony and Chinese peony flower[J]. Acta Horticulturae Sinica, 2005, 32(5): 861. doi: 10.3321/j.issn:0513-353X.2005.05.019
[19] Gasic K, Hernandez A, Korban S S. RNA extraction from different apple tissues rich in polyphenols and polysaccharides for cDNA library construction[J]. Plant Molecular Biology Reporter, 2004, 22(4): 437−438. doi: 10.1007/BF02772687
[20] 张玉芳, 赵丽娟, 曾幼玲. 基因表达研究中内参基因的选择与应用[J]. 植物生理学报, 2014, 50(8): 1119−1125. Zhang Y F, Zhao L J, Zeng Y L. Selection and application of reference genes for gene expression studies[J]. Plant Physiology Journal, 2014, 50(8): 1119−1125.
[21] 王彦杰, 董丽, 张超, 等. 牡丹实时定量PCR分析中内参基因的选择[J]. 农业生物技术学报, 2012, 20(5): 521−528. Wang Y J, Dong L, Zhang C, et al. Reference gene selection for real-time quantitative PCR normalization in tree peony (Paeonia suffruticosa Andr.)[J]. Journal of Agricultural Biotechnology, 2012, 20(5): 521−528.
[22] Untergasser A, Ruijter J M, Benes V, et al. Web-based LinRegPCR: application for the visualization and analysis of (RT)-qPCR amplification and melting data[J]. BMC Bioinformatics, 2021, 22(1): 398.
[23] Ramakers C, Ruijter J M, Deprez R H L, et al. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data[J]. Neuroscience Letters, 2003, 339(1): 62−66. doi: 10.1016/S0304-3940(02)01423-4
[24] 王雨, 裴行杰, 傅钇钧, 等. 基于全基因组鉴定华石斛优良内参基因[J]. 热带作物学报, 2023, 44(7): 1365−1372. Wang Y, Pei X J, Fu Y J, et al. Genome-wide identification of superior reference genes in Dendrobium sinense[J]. Chinese Journal of Tropical Crops, 2023, 44(7): 1365−1372.
[25] Vandesompele J, de Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biology, 2002, 3(7): 341.
[26] Andersen C L, Ledet-Jensen J, Ørntoft T F. Normalization of real-time quantitative RT-PCR data: a model based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets[J]. Cancer Research, 2004, 64(15): 5245−5250. doi: 10.1158/0008-5472.CAN-04-0496
[27] Pfaffl M W, Tichopád A, Prgomet C, et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations[J]. Biotechnology Letters, 2004, 26(6): 509−515. doi: 10.1023/B:BILE.0000019559.84305.47
[28] Xie F, Wang J, Zhang B. RefFinder: a web-based tool for comprehensively analyzing and identifying reference genes[J]. Functional & Integrative Genomics, 2023, 239(2): 125.
[29] Shabanian S, Esfahani N M, Karamian R, et al. Physiological and biochemical modifications by postharvest treatment with sodium nitroprusside extend vase life of cut flowers of two gerbera cultivars[J]. Postharvest Biology and Technology, 2018, 137: 1−8. doi: 10.1016/j.postharvbio.2017.11.009
[30] 李文阳, 马梦迪, 郭红卫. 植物激素乙烯作用机制的最新进展[J]. 中国科学: 生命科学, 2013, 43(10): 854−863. doi: 10.1360/052013-284 Li W Y, Ma M D, Guo H W. Advances in the action of plant hormone ethylene[J]. Scientia Sinica Vitae, 2013, 43(10): 854−863. doi: 10.1360/052013-284
[31] Stéphanie G, Mélanie M, Jérôme P, et al. Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references[J]. Journal of Experimental Botany, 2009, 60(2): 487−493. doi: 10.1093/jxb/ern305
[32] Zhang L, He L L, Fu Q T, et al. Selection of reliable reference genes for gene expression studies in the biofuel plant Jatropha curcas using real-time quantitative PCR[J]. International Journal of Molecular Sciences, 2013, 14(12): 24338−24354. doi: 10.3390/ijms141224338
[33] Luciana D, Barry C, Julia W, et al. FORMOSA controls cell division and expansion during floral development in Antirrhinum majus[J]. Planta, 2009, 229(6): 1219−1229. doi: 10.1007/s00425-009-0910-x
[34] Li C Q, Hu L Z, Wang X Q, et al. Selection of reliable reference genes for gene expression analysis in seeds at different developmental stages and across various tissues in Paeonia ostii.[J]. Molecular Biology Reports, 2019, 46(6): 6003−6011. doi: 10.1007/s11033-019-05036-7
[35] Li M, Xing Q, Li H G, et al. Selection of reference genes for quantitative real-time PCR analysis in cucumber (Cucumis sativus L.), pumpkin (Cucurbita moschata Duch.) and cucumber-pumpkin grafted plants[J]. Peer Journal, 2019, 7: e6536. doi: 10.7717/peerj.6536
[36] 尚申申, 樊琳婷, 周爽, 等. 伊藤牡丹‘巴茨拉’内参基因筛选及花瓣衰老相关基因的表达分析[J]. 植物生理学报, 2023, 59(1): 153−164. Shang S S, Fan L T, Zhou S, et al. Screening of reference genes and expression analysis of petal senescence associated genes in Itoh peony ‘Bartzella’ cut flowers[J]. Plant Physiology Journal, 2023, 59(1): 153−164.
[37] Depaepe T, van der Straeten D. Tools of the ethylene trade: a chemical kit to influence ethylene responses in plants and its use in agriculture[J]. Small Methods, 2020, 4: 1900267. doi: 10.1002/smtd.201900267
[38] Yan X Y, Zhao J H, Deng M H, et al. Two key enzyme genes associated with ethylene bio-synthesis, LbACS and LbACO, are involved in the regulation of lily flower senescence[J]. Scientia Horticulturae, 2024, 330: 113045. doi: 10.1016/j.scienta.2024.113045
[39] Pattyn J, John V H, van de Poel B. The regulation of ethylene biosynthesis: a complex multilevel control circuitry[J]. New Phytologist, 2021, 229: 770−782. doi: 10.1111/nph.16873
[40] Ryo N, Niki T, Ichimura K. Differential regulation of two 1-aminocyclopropane-1-carboxylate oxidase (ACO) genes, including the additionally cloned DcACO2, during senescence in carnation flowers[J]. Postharvest Biology and Technology, 2022, 183: 111752. doi: 10.1016/j.postharvbio.2021.111752
[41] Yokotani N, Nakano R, Imanishi S, et al. Ripening-associated ethylene biosynthesis in tomato fruit is autocatalytically and developmentally regulated[J]. Journal of Experimental Botany, 2009, 60(12): 3433−3442. doi: 10.1093/jxb/erp185
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