程玉豆, 张亚光, 关军锋^,, 冯云霄, 何近刚河北省农林科学院遗传生理研究所/河北省植物转基因中心,石家庄 050051

Effects of 1-MCP and Delayed Cold-Storage on Quality and Expression of Softening Related Genes in ‘Doyenne du Comice’ Pear During Shelf-Life

CHENG YuDou, ZHANG YaGuang, GUAN JunFeng^,, FENG YunXiao, HE JinGangInstitute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences/Plant Genetic Engineering Center of Hebei Province, Shijiazhuang 050051

通讯作者: 关军锋,E-mail： junfeng-guan@263.net

责任编辑: 赵伶俐
收稿日期:2020-03-7接受日期:2020-05-19网络出版日期:2020-11-16

基金资助:

河北省重点研发计划.19227111D 河北省现代农业产业技术体系.HBCT2018100207 河北省创新工程项目.2019-2-1 河北省农林科学院创新团队建设项目.F20E06001-1

Received:2020-03-7Accepted:2020-05-19Online:2020-11-16
作者简介 About authors
程玉豆,E-mail： chengyudouyn@163.com。







摘要
【目的】针对‘早红考密斯’梨因果肉软化和果实腐烂导致损耗严重等问题,通过分析1-MCP和延迟冷藏处理对‘早红考密斯’梨果实腐烂和货架期软化的影响,解析‘早红考密斯’梨中果实软化相关基因表达模式,旨在为延长‘早红考密斯’梨货架期提供新的理论依据。【方法】‘早红考密斯’梨经1.0 μL?L ^-1 1-甲基环丙烯（1-MCP）处理后,分别在（20±2）℃下放置0、3和6 d后再进行（0±0.5）℃冷藏。冷藏95 d后转入货架期（（20±2）℃）分析果实品质变化,利用实时荧光定量PCR（qRT-PCR）分析多聚半乳糖醛酸酶基因（PG1、PG2）、α-阿拉伯呋喃糖苷酶基因（ARF1、ARF2）和β-半乳糖苷酶基因（GAL4）的表达模式。 【结果】延迟冷藏易导致‘早红考密斯’梨果实软化和腐烂,并且随着处理时间延长,果实腐烂率加剧;与空气密封处理后直接冷藏（CK）相比,1-MCP结合延迟冷藏处理能有效维持货架期果实硬度,抑制果实呼吸速率和乙烯生成速率,减少果实腐烂,保持果实品质,但随着延迟冷藏处理时间延长,果实货架期呼吸速率和乙烯生成速率增加,果实软化进程加快;1-MCP结合延迟冷藏处理可显著抑制果实软化相关基因PG1、PG2、ARF1、ARF2和GAL4等的表达,但随着延迟冷藏处理时间延长,这种抑制效果减弱。【结论】延迟冷藏易导致‘早红考密斯’梨腐烂,因此采后需尽快冷藏;1-MCP抑制软化和腐烂效果明显,结合延迟冷藏处理,在一定程度上有利于‘早红考密斯’梨冷藏后货架期果实软化;PG1、PG2、ARF1和GAL4参与了‘早红考密斯’梨冷藏后货架期果实的软化过程。
关键词： ‘早红考密斯’梨;;1-甲基环丙烯;延迟冷藏;品质;软化

Abstract
【Objective】Focusing on the serious loss of ‘Doyenne du Comice’ (Pyrus communis L.) pear fruit that was resulted from rapid softening and decay, the effects of 1-methylcyclopropene (1-MCP) and delayed cold-storage on fruit decay and softening at shelf-life was studied, and the expression pattern of the softening-related genes of ‘Doyenne du Comice’ pear was also detected, so as to provide a theoretical basis for extending the shelf-life of ‘Doyenne du Comice’ pear.【Method】The ‘Doyenne du Comice’ pear fruit were firstly treated with 1.0 μL?L ^-1 1-MCP, and then were respectively placed at 20 ± 2℃ for 0, 3 and 6 d before storing at (0 ± 0.5)℃, which was considered as the delayed cold-storage. After 95 d of cold storage, the fruit quality was detected at shelf-life (20 ± 2℃), and the expression patterns of softening associated genes, such as polygalacturonase genes (PG1, PG2), α-arabinofuranosidase genes (ARF1, ARF2) and β-galactosidase gene (GAL4), were studied by the real-time quantitative PCR (qRT-PCR).【Result】The delayed cold-storage treatment could easily lead to fruit softening and decay in ‘Doyenne du Comice’ pear, and the fruit decay rate increased with the extending of delayed cold-storage. Compared with the treatment that the fruit were directly stored at 0 °C after air sealing (CK), the treatments of 1-MCP and 1-MCP combined with delayed cold-storage could maintain a higher fruit firmness, and reduce the rates of fruit decay, respiration and ethylene production, but it would increase in the rates of respiration and ethylene production, and accelerate the fruit softening with the extending of delayed cold-storage. The expression of softening related genes, such as PG1, PG2, ARF1, ARF2 and GAL4, were significantly inhibited by 1-MCP and 1-MCP combined with delayed cold-storage, while it would be weakened with the delayed cold-storage extended.【Conclusion】The decay of ‘Doyenne du Comice’ pear fruit was easily induced by delayed cold-storage treatment, so it was proposed that the postharvest fruit should be rapidly stored under the cold condition. The inhibition of the fruit softening and decay by 1-MCP treatment was more obvious, and the treatment that 1-MCP combined with delayed cold-storage was beneficial to fruit softening. The genes of PG1, PG2, ARF1 and GAL4 were involved in the softening of ‘Doyenne du Comice’ pear at shelf-life after cold storage.
Keywords：‘Doyenne du Comice’ pear;;1-methylcyclopropene;delayed cold-storage;quality;softening


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本文引用格式
程玉豆, 张亚光, 关军锋, 冯云霄, 何近刚. 1-MCP和延迟冷藏对‘早红考密斯’梨货架期间品质 和软化相关基因表达的影响[J]. 中国农业科学, 2020, 53(22): 4658-4666 doi:10.3864/j.issn.0578-1752.2020.22.012
CHENG YuDou, ZHANG YaGuang, GUAN JunFeng, FENG YunXiao, HE JinGang. Effects of 1-MCP and Delayed Cold-Storage on Quality and Expression of Softening Related Genes in ‘Doyenne du Comice’ Pear During Shelf-Life[J]. Scientia Agricultura Sinica, 2020, 53(22): 4658-4666 doi:10.3864/j.issn.0578-1752.2020.22.012


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0 引言

【研究意义】近年来,西洋梨在我国市场上日益受到欢迎,其中,‘早红考密斯’梨为代表性品种。它原产于英国,属于早熟品种,果实呈葫芦状,果皮呈紫红色,果面平滑有光泽,果肉白色,具有肉质细腻、柔软多汁、风味香甜的特点,果实品质上乘^[1]。但因其果实采后易软化和腐烂,货架期较短,无法满足广大消费者的需求,因此,常需要冷藏来延长供应时间。果实受采后处理、周转与运输等因素的影响,常常会导致果实品质劣变,降低商品价值,损耗严重^[2]。因此,探究‘早红考密斯’梨采后冷藏和货架期品质变化规律及其相关调控机理,对果实采后贮藏保鲜新技术的研发具有重要指导意义。【前人研究进展】研究表明,在一些苹果和西洋梨品种上,适当推迟冷藏能减少果实冷害,改善品质^[3,4,5],但在部分白梨、猕猴桃和李子等水果中,延迟冷藏则会加重组织褐变^[6,7,8]。1-甲基环丙烯（1-methylcyclopropene,1-MCP）,可以与乙烯竞争结合受体,从而抑制乙烯介导的果实后熟相关生理生化反应。因此,1-MCP作为一种新型保鲜剂,在果蔬贮藏保鲜领域得到了广泛应用^[9,10],如1-MCP处理可有效抑制常温货架期间果肉软化、腐烂,维持果实品质^[11,12,13];1-MCP可减少果实冷藏及后续货架期间虎皮病、黑心病的发生^{[14,15,16,17]}。综上所述,1-MCP在延长果实贮藏和货架期中的作用及其机制值得重视。软化是引起果实衰老的重要因素,已有研究表明,果实软化与多聚半乳糖醛酸酶（PG）、α-阿拉伯呋喃糖苷酶（α-ARF）和β-半乳糖苷酶（β-GAL）等细胞壁水解酶活性及其编码基因表达量上调密切相关^{[18,19,20,21,22,23]}。进一步研究发现,1-MCP处理可以有效降低果实PG、α-ARF和β-GAL等酶活性及其编码基因表达,进而延缓果实软化^[22,23]。【本研究切入点】尽管延迟冷藏和1-MCP处理在多种果实贮藏中得到研究,但不同品种间的效果存在差异^{[3,4,5,6,7,8]},并且,在‘早红考密斯’梨上,延迟冷藏的效果如何,目前还不清楚,有关1-MCP保鲜效果及其具体调控机制也缺乏系统研究。【拟解决的关键问题】通过研究1-MCP和延迟冷藏处理对‘早红考密斯’梨货架期间品质和软化的影响,解析‘早红考密斯’梨中果实软化相关基因表达模式,为延长‘早红考密斯’梨贮藏及货架期提供新的理论依据。

1 材料与方法

1.1 材料与处理

供试‘早红考密斯’梨（Pyrus communis L. cv. ‘Doyenne du Comice’）于2018年7月22日采收自河北省深州市唐奉镇东蒲疃村果园（北纬N 38°08′15.03″,东经E 115°34′31.34″）,采收当天运回实验室。选取大小适中、成熟度相对一致、无机械损伤和病虫害的果实并平均分成2组。室温（20±2）℃下,一组进行1-MCP（有效成分0.14%,美国陶氏公司生产）密封熏蒸处理24 h（最终浓度为1.0 μL?L^-1）;另一组以空气密封24 h作为对照,每组处理360个果实。结束后取出2组果实,再分别进行如下处理：（1）直接进行（0±0.5）℃冷藏;（2）（20±2）℃下放置3 d后再进行（0±0.5）℃冷藏（记作20℃ 3 d）;（3）（20±2）℃下放置6 d后再进行（0±0.5）℃冷藏（记作20℃ 6 d）。空气密封和1-MCP熏蒸处理后直接进行（0±0.5）℃冷藏果实分别记做：CK和1-MCP;处理后20℃下放置3 d后再进行0℃冷藏果实分别记做：20℃ 3 d和1-MCP+20℃ 3 d;（20±2）℃下放置6 d后再进行（0±0.5）℃冷藏果实分别记做：20℃ 6 d和1-MCP+20℃ 6 d,每个处理设置3个重复,每个重复40个果实。以直接进行（0±0.5）℃冷藏计,果实贮藏95 d后取出,置于（20±2）℃下货架贮存,分别测定其0、3和6 d品质指标（硬度、可溶性固形物、可滴定酸含量）。同时,取果肉样品经液氮冷冻后于-80℃冻存,备用。

1.2 品质与腐烂率测定

硬度：将果实去皮后,采用GY-4型果实硬度计（浙江托普云农科技股份有限公司）测定。可溶性固形物含量（SSC）：采用PAL-1型手持数字糖度计（日本ATGAO公司）测定。可滴定酸（TA）含量：采用酸碱滴定法测定。每个处理重复3次,每次重复5个果实。腐烂率：统计腐烂的果实数,腐烂率计算公式如下：腐烂率（%）=（腐烂果数/总果数）×100。

1.3 呼吸速率和乙烯释放速率测定

将果实放在气密性良好的真空干燥器中密封3 h后,采用HWF-1 CO₂红外分析仪（江苏金坛市科析仪器有限公司）测定CO₂含量,计算呼吸速率。采用GC9790ⅡB型气相色谱仪（浙江福立分析仪器股份有限公司）测定乙烯含量,计算乙烯释放速率。色谱条件：载气为氮气,进样量1 mL。测定条件为：柱箱温度90℃,汽化室温度140℃,FID温度200℃;氮气流量30 mL?min^-1,氢气流量30 mL?min^-1,空气流量300 mL?min^-1。每个处理重复3次,每次重复10个果实。

1.4 RNA提取与定量分析

RNA提取采用改良CTAB法^[24];反转录试剂盒（PrimeScript^TM RT reagent Kit with gDNA Eraser）和荧光定量试剂盒（TB Green^TM Premix Ex Taq^TM II）均购自宝生物工程（大连）有限公司。定量分析采用ABI 7500型荧光定量PCR仪（美国应用生物系统公司）,反应体系：10.0 μL SYBR Green PCR Premix Ex Taq?,0.5 μL ROX Reference DyeⅡ,正、反向引物（10 μmol?L）各0.5 μL,5 ng cDNA模板,6.0 μL ddH₂O。反应条件：95℃预变性30 s;95℃ 5 s,60℃ 34 s,40个循环。反应结束后做熔解曲线确定目的基因片段的特异性。以ACT2为内参基因,采用2^-ΔCT法计算目标基因表达量,PG1、PG2、ARF1、ARF2和GAL4定量分析引物参考WEI等^[22]文献报道,具体如表1所示。

Table 1
表1
表1果实软化相关基因qRT-PCR引物
Table 1Primers of the fruit softening-related gene for qRT-PCR

基因 Gene	登录号 Access No.	正向引物 Forward primer	反向引物 Reverse primer
PG1	KC855751	5′-GCTTATCCTTCATACATGGACACC-3′	5′-CTGATGATGTTGCTATCCCACC-3′
PG2	KC855752	5′-AAATCAGAAAATCCTAAAACCAGC-3′	5′-GCTTATAGCCATCATCAACAACG-3′
ARF1	KC855732	5′-CTTTTTTCCACAATCCTCATTCC-3′	5′-GCTCTCGACGGTATAGAATTTGC-3′
ARF2	KC855733	5′-TAGCAACATTCCCATCCACC-3′	5′-AACATTACACTGCCTATGATCTCG-3′
GAL4	KC855745	5′-GATCAATTCACTATCCCAGAAGC-3′	5′-GTATAACATCCAAGCCTCCAGC-3′
ACT2	GU830958	5′-GGACATTCAACCCCTCGTCT-3′	5’-ATCCTTCTGACCCATACCAACC-3’

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1.5 数据分析和处理

数据采用Excel 2007和SPSS 18.0软件进行作图和统计分析,方差分析采用Duncan法,结果用3次重复的平均值表示。

2 结果

2.1 1-MCP和延迟冷藏对‘早红考密斯’梨冷藏后腐烂的影响

由表2可知,出库当天（货架0 d）时,空气密封处理后再延迟3 d（20℃ 3 d）和6 d（20℃ 6 d）冷藏的果实明显加剧腐烂,分别是空气密闭处理后（0±0.5）℃直接冷藏果实（CK）的3.8和10.4倍,说明在不进行1-MCP处理时,延迟冷藏明显不利于果实贮藏保鲜;相反,1-MCP处理可明显降低‘早红考密斯’梨出库时腐烂率,尽管1-MCP处理果实在延迟冷藏后也加剧果实腐烂,但与20℃ 3 d和20℃ 6 d处理相比,果实腐烂明显减少,在生产上具有实际意义。总体来说,保鲜效果最好的是1-MCP处理后再（0±0.5）℃直接冷藏处理（1-MCP）果实。20℃ 3 d和20℃ 6 d处理果实由于腐烂严重,无法再进行后续研究。其他处理在货架6 d内均未出现腐烂,可用于后续研究。

Table 2
表2
表21-MCP和延迟冷藏对‘早红考密斯’梨果实腐烂的影响
Table 2Effect of 1-MCP and delayed cold-storage on the decay of 'Doyenne du Comice' pear fruit

处理 Treatment	腐烂率 Decay rate (%)
CK	8.8±2.4c
1-MCP	1.3±2.2d
20℃ 3 d	33.7±0.6b
1-MCP+20℃ 3 d	8.7±2.1c
20℃ 6 d	91.3±5.6a
1-MCP+20℃ 6 d	6.3±2.2cd

Different small letters in the table indicated significant differences (P<0.05)
不同小写字母表示差异显著（P<0.05）

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2.2 1-MCP和延迟冷藏对‘早红考密斯’梨货架期品质的影响

随着货架期延长,各处理间果实硬度均呈下降趋势,并且延迟冷藏会加速果实软化;与CK相比,‘早红考密斯’梨经1-MCP处理后,果实具有较高的硬度,直接冷藏处理的果实硬度最高,1-MCP处理后延迟6 d冷藏（1-MCP+20℃ 6 d）果实硬度下降程度最大,1-MCP处理后延迟3 d冷藏（1-MCP+20℃ 3 d）果实硬度介于二者之间（图1-A）。SSC在处理与对照果实间差异较小,除货架3 d时,1-MCP+20℃ 3 d和1-MCP+20℃ 6 d处理果实SSC显著高于CK外,其他货架时间各处理则无显著差异（图1-B）;TA含量在各处理果实中均呈缓慢下降趋势,其中,1-MCP处理果实在货架3 d时显著高于CK,1-MCP+20℃ 3 d和1-MCP+20℃ 6 d处理果实在货架6 d时显著高于CK（图1-C）。说明1-MCP能明显延缓‘早红考密斯’梨果肉的软化速度,1-MCP结合延迟冷藏后有一定的促进软化作用,并能保持较好的风味。

图1

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图11-MCP和延迟冷藏对‘早红考密斯’梨货架期硬度（A）、可溶性固形物（B）和可滴定酸含量（C）的影响

图中不同小写字母表示差异显著（P<0.05）。下同
Fig. 1Effects of 1-MCP and delayed cold-storage treatments on the firmness (A), SSC (B) and TA content (C) of 'Doyenne du Comice' pear during shelf-life

Different small letters in the figure indicated significant differences (P<0.05). The same as below

2.3 1-MCP和延迟冷藏对‘早红考密斯’梨冷藏后货架期呼吸速率和乙烯生成速率的影响

‘早红考密斯’梨在冷藏后货架0 d时,1-MCP结合延迟冷藏处理果实呼吸速率显著低于CK;随着货架期延长,CK果实呼吸速率迅速上升,1-MCP结合延迟冷藏处理果实呼吸速率上升则较缓慢。在货架6 d时,1-MCP+20℃ 3 d和1-MCP+20℃ 6 d处理果实呼吸速率有所下降（图2-A）。货架0 d时,CK果实的乙烯释放速率最高,随着货架时间延长,乙烯释放速率大幅降低;与CK相比,1-MCP结合延迟冷藏处理果实乙烯释放速率变化不明显,在货架0 d和3 d时低于对照,但在6 d时与对照接近（图2-B）。

图2

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图21-MCP和延迟冷藏对‘早红考密斯’梨货架期呼吸速率（A）和乙烯释放速率（B）的影响

Fig. 2Effects of 1-MCP and delayed cold-storage treatments on the rates of respiration (A) and ethylene production (B) of 'Doyenne du Comice' pear during shelf-life

2.4 1-MCP和延迟冷藏对‘早红考密斯’梨冷藏后货架期PGs表达的影响

实时荧光定量PCR分析结果显示,CK果实中,PG1表达呈先上升后下降趋势,在货架期3 d时表达量最高;而1-MCP结合延迟冷藏处理果实中PG1表达量则呈持续增加趋势,但在货架期0 d和3 d时,PG1表达量显著低于CK;货架6 d时,1-MCP+20℃ 3 d和1-MCP+20℃ 6 d处理果实中PG1表达量高于CK,并以1-MCP+20℃ 6 d处理的PG1表达量最高（图3-A）。与PG1相似,CK果实中,PG2表达模式也呈先上升后下降趋势。1-MCP结合延迟冷藏处理果实在货架期0 d时,PG2表达量显著低于CK,然后逐渐升高;货架3 d时,1-MCP+20℃ 6 d处理果实中PG2表达量开始显著高于CK;货架6 d时,1-MCP结合延迟冷藏处理果实中PG2表达量均显著高于CK,并表现为1-MCP+20℃ 3 d处理的最高,1-MCP+ 20℃ 6 d处理的次之,1-MCP处理最低（图3-B）。

图3

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图31-MCP和延迟冷藏对‘早红考密斯’梨货架期PGs表达的影响

Fig. 3Effects of 1-MCP and delayed cold-storage treatments on the expression of PGs in the 'Doyenne du Comice' pear during shelf-life

2.5 1-MCP和延迟冷藏对‘早红考密斯’梨冷藏后货架期ARFs表达的影响

ARF1在CK果实中同样呈现先缓慢上升再下降的趋势,并且,1-MCP结合延迟冷藏处理果实中ARF1表达量在整个货架期均显著低于CK;在货架3 d时,1-MCP+20℃ 6 d处理果实ARF1表达量显著高于1-MCP和1-MCP+20℃3 d处理果实,而在货架6 d时,1-MCP+20℃ 3 d处理果实ARF1表达量则显著高于1-MCP和1-MCP+20℃ 6 d处理果实（图4-A）。与ARF1表达模式不同,ARF2在所有处理中均表现出随着货架时间延长而表达量逐渐下降的趋势,与CK相比,1-MCP结合延迟冷藏处理显著抑制了货架期间ARF2表达;并且,在货架0 d和3 d时,1-MCP处理果实ARF2表达量均为最低（图4-B）。

图4

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图41-MCP和延迟冷藏对‘早红考密斯’梨货架期ARFs表达的影响

Fig. 4Effects of 1-MCP and delayed cold-storage treatments on the expression of ARFs (A and B) in the 'Doyenne du Comice' pear during shelf-life

2.6 1-MCP和延迟冷藏对‘早红考密斯’梨冷藏后货架期GAL4表达的影响

在1-MCP结合延迟冷藏处理的果实中,GAL4表达量在货架期间均显著低于CK果实,并且货架0 d和3 d时,GAL4表达量表现为1-MCP+20℃ 6 d处理的最高,1-MCP+20℃ 3 d处理的次之,1-MCP处理最低;而在货架6 d时,1-MCP+20℃ 6 d处理果实的GAL4表达量则显著高于1-MCP和1-MCP+20℃ 3 d处理的果实（图5）。

图5

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图51-MCP和延迟冷藏对‘早红考密斯’梨货架期GAL4基因表达的影响

Fig. 5Effects of 1-MCP and delayed cold-storage treatments on the expression of GAL4 in the 'Doyenne du Comice' pear during shelf-life

3 讨论

乙烯在调控呼吸跃变型果实成熟衰老过程中发挥着重要的作用^[25]。本研究中,1-MCP处理可显著抑制‘早红考密斯’梨果实冷藏后货架期呼吸速率和乙烯释放速率,这与其抑制梨果实乙烯合成酶活性及相关基因表达有直接关系^[26,27],表明1-MCP可通过降低‘早红考密斯’梨内源乙烯合成延缓果实成熟衰老。西洋梨果肉软化是其成熟衰老的重要指标之一。研究表明,一些西洋梨品种如‘巴梨’‘安久’‘阿巴特’和‘康复伦斯’梨等使用1-MCP处理后,贮藏和货架期果肉软化减缓,果实腐烂和虎皮病减少^{[27,28,29,30,31]}。

与上述研究结果相似,本研究中,1-MCP处理也可有效延迟果实冷藏后货架期硬度下降,减少果实腐烂,因此,1-MCP处理有利于‘早红考密斯’梨采后贮藏保鲜。本研究还发现,随着延迟冷藏时间延长,‘早红考密斯’梨果实腐烂率明显加剧,但经1-MCP处理可明显减少腐烂,与此同时,1-MCP结合延迟冷藏处理的果实在货架期硬度下降速度相对较快,因此,从西洋梨自身品种特性——需后熟软化的意义上来讲,对于1-MCP处理过的果实,可适当延长常温下运输或周转时间。如果单纯为了减少腐烂,在‘早红考密斯’梨采后贮藏中,无论是否使用1-MCP处理,果实采收后应尽快入冷库贮藏。

果实软化过程主要由细胞壁结构破坏及果胶物质降解引起,其中以PG研究最为深入。研究表明,PG参与了梨果实采后软化过程^[22-23,32]。在‘南果’梨中,PuPG1、PuPG4和PuPG7在采收后的果实中开始表达,且随着贮藏时间的延长,表达量逐渐升高,1-MCP处理明显抑制3个基因的表达^[33]。‘京白’梨中,PG1和PG2随着硬度下降表达量迅速升高,1-MCP处理在显著抑制果实乙烯生成和硬度下降的同时,极显著抑制了PG1和PG2表达量升高^[18]。本研究结果与‘南果’和‘京白’梨中的报道相似,即货架期果实软化越快,PG1和PG2表达量在软化前期就越高,反之亦然,这说明PG1和PG2因参与了‘早红考密斯’冷藏后货架期果实软化过程。相比于PG1,各处理果实中PG2在货架期0 d时稍低,但在后续货架期变化幅度较PG1大,推测PG1和PG2在‘早红考密斯’梨冷藏后货架期果实软化过程的作用可能略有不同,PG1可能在果实软化前期起主要作用,而PG2则可能在果实软化中期起主要作用。

α-ARF和β-GAL与果实成熟时细胞壁的修饰过程密切相关,α-ARF可以通过水解果胶上的阿拉伯糖苷键而引起细胞壁完整性遭到破坏;而β-GAL则通过降解果胶多聚醛酸链上的半乳糖苷键使细胞壁变得膨胀而不稳定,这两种酶活性及其基因表达量升高均可导致果实软化^{[21, 34-35]}。本研究中,ARF1表达量高,果实硬度下降迅速,并且1-MCP处理抑制了ARF1的表达,硬度下降缓慢,这与‘京白’梨常温贮藏中的研究结果相似^[22],表明ARF1参与了‘早红考密斯’梨货架期果实软化过程,1-MCP通过抑制其表达延迟果实软化进程,达到果实保鲜效果。ARF2由于在各处理中随着货架期延长表现出表达量逐渐下降,与果实硬度下降的趋势不同,因此,推测其可能不参与‘早红考密斯’梨货架期软化过程。已有研究证实,西洋梨中GAL1/2在果实软化前期表达量迅速升高,并受1-MCP处理抑制^[21];在日本梨中,JP-GAL只在成熟果实中表达,并且与果实软化密切相关^[35];‘京白’梨采后货架期间GAL4表达量伴随着果实硬度下降而逐渐升高,1-MCP处理可以显著抑制其表达和硬度下降^[22,23]。本研究发现,GAL4表达模式也与果实软化密切相关,即在果实硬度下较降快的处理中,GAL4表达量高;果实硬度下降缓慢的处理中,GAL4表达量相对较低,结合日本梨和‘京白’梨的研究结果,说明GAL4参与了‘早红考密斯’梨果实货架期软化过程,1-MCP可以通过抑制GAL4表达延迟果实货架期软化进程。

4 结论

‘早红考密斯’梨延迟冷藏易导致果实贮藏期软化速度加快,果实腐烂严重,建议采收后尽快入库冷藏。1-MCP处理能够有效延迟‘早红考密斯’梨货架期果实硬度下降,抑制果实呼吸和内源乙烯生成,减少果实腐烂,保持果实品质,起到了很好的保鲜作用。与1-MCP处理后直接冷藏相比,1-MCP结合延迟冷藏6 d处理可一定程度促进货架期果实软化。1-MCP通过抑制软化相关基因PG1、PG2、ARF1和GAL4等的表达,维持‘早红考密斯’梨货架期果实硬度,达到保鲜效果。

参考文献 View Option 原文顺序
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[1] 祝美云, 李小月, 梁丽松, 马庆华, 王贵禧 . 1-MCP处理和打蜡处理对‘早红考密斯’西洋梨贮藏期和货架期保护性酶活性的影响
食品科学 2015,36(24):307-312.
DOI:10.7506/spkx1002-6630-201524057URL [本文引用: 1]
Purpose: To study the effect of different postharvest treatments with 1-methylcyclopropene (1-MCP) or wax on
the activities of protective enzymes in Pyrus communis L. cv. ‘Doyenne du Comice’. Methods: The pear fruits were treated
respectively with 1-MCP at 0.5 and 1 μL/L and wax after harvest and then stored in a cold room at (0 ± 0.5) ℃. The activities
of protective enzymes in flesh and peel during 90 days of storage at low temperature and during shelf life storage at 25 ℃
after 60 and 90 days of cold storage were assayed. Results: During cold storage, both 1-MCP and wax treatments improved
superoxide dismutase (SOD) activity in flesh and reduced the activities of ascorbate peroxidase (APX) and peroxidase (POD)
in flesh and catalase (CAT) activity in peel. During shelf life storage, wax treatment could improve SOD activity in flesh
and POD activity in peel. Conclusions: 1-MCP treatment has a positive effect on the physiology of pear fruits during cold
storage, which is more effective at 0.5 μL/L than at 1 μL/L for flesh, while the contrary result was observed for peel. Wax
treatment shows a better effect on peel during shelf life. In addition, the activities of protective enzymes SOD, CAT, APX
and POD in peel are much higher than in flesh during 90 days of cold storage and also during 5 days of subsequent storage at
room temperature.
ZHU M Y, LI X Y, LIANG L S, MA Q H, WANG G X . Effect of 1-Methylcyclopropene (1-MCP) and wax treatments on the activities of protective enzymes in Pyrus communis L. cv. ‘Doyenne du Comice’ during cold storage and shelf life
Food Science, 2015,36(24):307-312. (in Chinese)
DOI:10.7506/spkx1002-6630-201524057URL [本文引用: 1]
Purpose: To study the effect of different postharvest treatments with 1-methylcyclopropene (1-MCP) or wax on
the activities of protective enzymes in Pyrus communis L. cv. ‘Doyenne du Comice’. Methods: The pear fruits were treated
respectively with 1-MCP at 0.5 and 1 μL/L and wax after harvest and then stored in a cold room at (0 ± 0.5) ℃. The activities
of protective enzymes in flesh and peel during 90 days of storage at low temperature and during shelf life storage at 25 ℃
after 60 and 90 days of cold storage were assayed. Results: During cold storage, both 1-MCP and wax treatments improved
superoxide dismutase (SOD) activity in flesh and reduced the activities of ascorbate peroxidase (APX) and peroxidase (POD)
in flesh and catalase (CAT) activity in peel. During shelf life storage, wax treatment could improve SOD activity in flesh
and POD activity in peel. Conclusions: 1-MCP treatment has a positive effect on the physiology of pear fruits during cold
storage, which is more effective at 0.5 μL/L than at 1 μL/L for flesh, while the contrary result was observed for peel. Wax
treatment shows a better effect on peel during shelf life. In addition, the activities of protective enzymes SOD, CAT, APX
and POD in peel are much higher than in flesh during 90 days of cold storage and also during 5 days of subsequent storage at
room temperature.

[2] JOHNSTON J W, HEWETT E W, HERTOG M L . Apple (Malus domestica) softening in the postharvest coolchain: Effects of delayed cooling and shelf-life temperatures
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[4] DEELL J R, EHSANI-MOGHADDAM B . Timing of postharvest 1-methylcyclopropene treatment affects Bartlett pear quality after storage
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Abstract

The recent availability of the inhibitor of ethylene perception, 1-methylcyclopropene (1-MCP), has resulted in an explosion of research on its effects on fruits and vegetables, both as a tool to further investigate the role of ethylene in ripening and senescence, and as a commercial technology to improve maintenance of product quality. The commercialization of 1-MCP was followed by rapid adoption by many apple industries around the world, and strengths and weaknesses of the new technology have been identified. However, use of 1-MCP remains limited for other products, and therefore it is still necessary to speculate on its commercial potential for most fruits and vegetables. In this review, the effects of 1-MCP on fruits and vegetables are considered from two aspects. First, a selected number of fruit (apple, avocado, banana, pear, peaches and nectarines, plums and tomato) are used to illustrate the range of responses to 1-MCP, and indicate possible benefits and limitations for commercialization of 1-MCP-based technology. Second, an outline of general physiological and biochemical responses of fruits and vegetables to the chemical is provided to illustrate the potential for use of 1-MCP to better understand the role of ethylene in ripening and senescence processes.

[11] 冯云霄, 何近刚, 程玉豆, 李丽梅, 李学营, 冯建忠, 关军锋 . 1-MCP处理对早熟苹果常温贮藏生理及品质的影响
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[本文引用: 1]

FENG Y X, HE J G, CHENG Y D, LI L M, LI X Y, FENG J Z, GUAN J F . Effects of 1-MCP treatment on the physiology and quality of early-maturing apples stored at ambient temperature
Modern Food Science and Technology, 2019,35(12):130-136. (in Chinese)
[本文引用: 1]

[12] 姜云斌, 佟伟, 王志华, 王文辉, 都兴日, 夏玉静 . 1-MCP处理对明珠、贵妃梨货架期间品质的影响
中国农学通报 2010,26(21):67-70.
URL [本文引用: 1]
Mingzhu pear and Guifei pear was used to investigate the effect of 1-MCP on shelf-life quality . The result showed that 1.0&mu;l/L 1-MCP treatment could significantly delay ripening of the fruit, obviously keep the fruit quality and flavor,reduce the rates of fruit rot.
JIANG Y B, TONG W, WANG Z H, WANG W H, DU X R, XIA Y J . Effects of 1-MCP treatment on shelf-life quality of Mingzhu pear and Guifei pear
Chinese Agricultural Science Bulletin, 2010,26(21):67-70. (in Chinese)
URL [本文引用: 1]
Mingzhu pear and Guifei pear was used to investigate the effect of 1-MCP on shelf-life quality . The result showed that 1.0&mu;l/L 1-MCP treatment could significantly delay ripening of the fruit, obviously keep the fruit quality and flavor,reduce the rates of fruit rot.

[13] 王晓飞, 杨艳青, 任小林, 孙海亭, 向春燕, 孙炜尚 . 1-MCP对‘粉红女士’苹果果实采后生理及其品质的影响
食品科学, 2014,35(18):219-223.
DOI:10.7506/spkx1002-6630-201418042URL [本文引用: 1]
This study was aimed to investigate the effects of 1-methylcyclopropene (1-MCP) on the postharvest physiology
and storage quality of ‘Pink Lady’ apple fruits. The apples were stored at ambient temperature (20 ℃) after being fumigated
with 1.0 μL/L 1-MCP. The firmness, titratable acidity content, the greasiness of the epicuticular wax, respiration rate,
ethylene production rate, malondialdehyde content, and the activities of peroxidase (POD), superoxide dismutase (SOD)
and catalase (CAT) were measured regularly during storage. The results indicated that 1-MCP delayed the decrease of
firmness and titratable acidity content of apples, and inhibited the greasiness of epicuticular wax. Fruits treated with 1-MCP
experienced a significant reduction of respiration rate and ethylene production compared with control fruits. Treatment
with 1-MCP not only retarded the changes in the activities of CAT and POD, but also increased the activities of SOD and
CAT. Malondialdehyde content of apples was delayed obviously by 1-MCP. In conclusion, 1-MCP treatment could inhibit
physiological metabolism, maintain fruit quality, and delay the senescence of ‘Pink Lady’ apple fruits.
WANG X F, YANG Y Q, REN X L, SUN H T, XIANG C Y, SUN W S . Effects of 1-MCP on postharvest physiology and quality of ‘Pink Lady’ apple fruits
Food Science, 2014,35(18):219-223. (in Chinese)
DOI:10.7506/spkx1002-6630-201418042URL [本文引用: 1]
This study was aimed to investigate the effects of 1-methylcyclopropene (1-MCP) on the postharvest physiology
and storage quality of ‘Pink Lady’ apple fruits. The apples were stored at ambient temperature (20 ℃) after being fumigated
with 1.0 μL/L 1-MCP. The firmness, titratable acidity content, the greasiness of the epicuticular wax, respiration rate,
ethylene production rate, malondialdehyde content, and the activities of peroxidase (POD), superoxide dismutase (SOD)
and catalase (CAT) were measured regularly during storage. The results indicated that 1-MCP delayed the decrease of
firmness and titratable acidity content of apples, and inhibited the greasiness of epicuticular wax. Fruits treated with 1-MCP
experienced a significant reduction of respiration rate and ethylene production compared with control fruits. Treatment
with 1-MCP not only retarded the changes in the activities of CAT and POD, but also increased the activities of SOD and
CAT. Malondialdehyde content of apples was delayed obviously by 1-MCP. In conclusion, 1-MCP treatment could inhibit
physiological metabolism, maintain fruit quality, and delay the senescence of ‘Pink Lady’ apple fruits.

[14] ARGENTA L C, MATTHEIS J P, FAN X, AMARANTE C V T. Managing ‘Bartlett’ pear fruit ripening with 1-methylcyclopropene reapplication during cold storage
Postharvest Biology and Technology, 2016,113:125-130.
DOI:10.1016/j.postharvbio.2015.11.009URL [本文引用: 1]

[15] SAQUET A A, ALMEIDA D P F. Ripening physiology and biochemistry of ‘Rocha’ pear as affected by ethylene inhibition
Postharvest Biology and Technology, 2017,125:161-167.
DOI:10.1016/j.postharvbio.2016.11.008URL [本文引用: 1]

[16] CHENG Y D, LIU L Q, FENG Y X, DONGY , GUAN J F,. Effects of 1-MCP on fruit quality and core browning in ‘Yali’ pear during cold storage
Scientia Horticulturae, 2019,243:350-356.
DOI:10.1016/j.scienta.2018.08.041URL [本文引用: 1]

[17] TAO D B, WANG J W, ZHANG L, JIANG Y G, LV M . 1-Methylcyclopropene alleviates peel browning of ‘Nanguo’ pears by regulating energy, antioxidant and lipid metabolisms after long term refrigeration
Scientia Horticulturae, 2018,247:254-263.
DOI:10.1016/j.scienta.2018.12.025URL [本文引用: 1]

[18] MWANIKI M W, MATHOOKO F M, MATSUZAKI M, HIWASA K, TATEISHI A, USHIJIMA K, NAKANO R, INAB A A, KUBO Y . Expression characteristics of seven members of the β-galactosidase gene family in ‘La France’ pear(Pyrus communis L.) fruit during growth and their regulation by 1-methylcyclopropene during postharvest ripening
Postharvest Biology and Technology, 2005,36(3):253-263.
DOI:10.1016/j.postharvbio.2005.02.002URL [本文引用: 2]

[19] SEKINE D, MUNEMURA I, GAO M, MITSUHASHI W, TOYOMASU T, HIDEKI M . Cloning of cDNAs encoding cell-wall hydrolases from pear ( Pyrus communis) fruit and their involvement in fruit softening and development of melting texture
Physiologia Plantarum, 2006,126(2):163-174.
DOI:10.1111/ppl.2006.126.issue-2URL [本文引用: 1]

[20] FONSECA S, MONTEIRO L, BARREIRO M G, PAIS M S . Expression of genes encoding cell wall modifying enzymes is induced by cold storage and reflects changes in pear fruit texture
Journal of Experimental Botany, 2005,56(418):2029-2036.
DOI:10.1093/jxb/eri201URLPMID:15955791 [本文引用: 1]
Preclimacteric 'Rocha' pears stored under chilling conditions, had a larger increase of ACO (1-aminocyclopropane-1-carboxylate oxidase) activity and softened faster than those treated with ethylene. Non-treated fruit did not ripen or soften, acquired a rubbery texture, and showed barely detectable levels of ACO activity. The transcript accumulation of seven genes encoding cell wall modifying enzymes was followed during fruit growth, ripening, and senescence, and in fruit that failed to ripen, by quantitative real-time PCR. Transcripts from 'Rocha' pear polygalacturonase1 and 2 (PcPG1, PcPG2), beta-galactosidase (PcbetaGAL) and beta-xylosidase (PcXYL) genes accumulated up to 1000-fold at the climacteric onset, while low transcript levels were detected in growing fruit. In fruit that did not ripen, this transcript accumulation was lower compared with fruits that ripened normally. Transcripts for expansin1 and 2 (PcEXPA1, PcEXPA2) accumulated in growing fruit, but about 10-fold more in fruit after rewarming. Xyloglucan endotransglucosylase/hydrolase (PcXTH) had the highest basal expression levels in all samples, showing only a small increase during fruit growth and ripening. PcEXPA2 and PcXTH transcripts accumulated in untreated fruit, 21 d after harvest, to levels similar to those of fruit that ripened normally. Since in untreated fruit ACO activity was barely detectable, it is likely that the activation of these genes might occur at very low ethylene levels. Results suggest that PcXTH and PcEXPA2 gene induction might be associated with cell wall maintenance during 'Rocha' pear development and ripening, while PcEXPA1, PcPG1, PcPG2, PcbetaGAL, and PcXYL expression is likely to be related to cell wall disassembly and loosening.

[21] SONG L Y, WANG Z G, WANG Z M, MENG G, ZHAI R, CAI M, MA F W, XU L F . Screening of cell wall-related genes that are expressed differentially during ripening of pears with different softening characteristics
Postharvest Biology and Technology, 2016,115:1-8.
DOI:10.1016/j.postharvbio.2015.12.012URL [本文引用: 3]

[22] WEI J M, QI X D, CHENG Y D, GUAN J F . Difference in activity and gene expression of pectin-degrading enzymes during softening process in two cultivars of Chinese pear fruit
Scientia Horticulturae, 2015,197:434-440.
DOI:10.1016/j.scienta.2015.10.002URL [本文引用: 6]

[23] WEI J M, CHENG Y D, FENG Y X, QI X D, HE J G, GUAN J . Different response to 1-methylcyclopropene in two cultivars of Chinese pear fruit with contrasting softening characteristics
Journal of Applied Botany and Food Quality, 2019,92:138-142.
[本文引用: 4]

[24] GASIC K, HERNANDEZ A, KORBAN S S . RNA extraction from different apple tissues rich in polyphenols and polysaccharides for cDNA library construction
Plant Molecular Biology Reporter, 2004,22(4):437-438.
DOI:10.1007/BF02772687URL [本文引用: 1]
Recovering RNA of high quality and quantity is a prerequisite for ensuring representation of all expressed genes in a cDNA library. An efficient procedure for isolating RNA from bud, internodal shoot, flower, and fruit tissues of apple has been developed. This protocol does not involve the use of phenol, lyophilization, or ultracentrifugation. In addition, this protocol overcomes problems of both RNA degradation and low yield attributed to oxidation by polyphenolic compounds and coprecipitation with polysaccharides, both abundant components in apple fruit and flower tissues. Isolated RNA is of high quality and is undegraded as assessed by spectrophotometric readings and electrophoresis in denaturing agarose gels. RNA quality is further assessed following its use in reverse transcription and cDNA library construction, and it can be used for a number of downstream analyses, including Northern blot hybridization and reverse transcription-polymerase chain reaction (RT-PCR). With this modified protocol, 25–900 μg of total RNA is routinely obtained from 1 g of fresh material. This method is of low cost and easy to perform.

[25] PECH J, PURGATTO E, BOUZAYEN M, LATCHé A . Ethylene and fruit ripening
Annual Plant Reviews, 2012,44:275-304.
[本文引用: 1]

[26] XIE X B, ZHAO J, WANG Y . Initiation of ripening capacity in 1-MCP treated green and red ‘Anjou’ pears and associated expression of genes related to ethylene biosynthesis and perception following cold storage and post-storage ethylene conditioning
Postharvest Biology and Technology, 2016,111:140-149.
DOI:10.1016/j.postharvbio.2015.08.010URL [本文引用: 1]

[27] TRINCHERO G D, SOZZI G O, COVATTA F, FRASCHINA A A . Inhibition of ethylene action by 1-methylcyclopropene extends postharvest life of ‘Bartlett’ pears
Postharvest Biology and Technology, 2004,32(2):193-204.
DOI:10.1016/j.postharvbio.2003.11.009URL [本文引用: 2]

[28] 贾晓辉, 李振茹, 王文辉, 佟伟, 夏玉静, 张志云, 王志华 . 1-MCP处理对巴梨果实采后生理效应及黑皮病的影响
保鲜与加工, 2010,10(4):13-16.
[本文引用: 1]

JIA X H, LI Z R, WANG W H, TONG W, XIA Y J, ZHANG Z Y, WANG Z H . Influence of 1-Methylcyclopropene on postharvest physiology and skin brown of ‘Bartlett’ Pears
Storage and Process, 2010,10(4):13-16. (in Chinese)
[本文引用: 1]

[29] ARGENTA L C, FAN X, MATTHEIS J P . Influence of 1- methylcyclopropene on ripening, storage life, and volatile production by d’Anjou cv. pear fruit
Journal of Agricultural and Food Chemistry, 2003,51(13):3858-3864.
DOI:10.1021/jf034028gURLPMID:12797756 [本文引用: 1]
d'Anjou cv. pear fruit (Pyrus communis L.) exposed at harvest to 0, 0.42, 4.2, or 42 micromol m(-)(3) 1-methylcyclopropene (1-MCP) for 12 h at 20 degrees C were stored at 1 degrees C for up to 8 months. After storage, half of the fruit was continuously exposed to ethylene (0.45 or 4-18 mmol m(-)(3)) for 7 days at 20 degrees C. All fruit treated with 1-MCP had lower respiration and ethylene production compared to untreated controls. Fruit quality changes were delayed following 1-MCP treatment, as was development of superficial scald and peel yellowing. The duration of 1-MCP-induced responses was dependent on 1-MCP treatment concentration. When 1-MCP-treated fruit began to ripen, softening and production of volatile compounds proceeded similar to that of untreated fruit. Post-storage ethylene exposure did not consistently stimulate ripening of fruit previously treated with 1-MCP. Efficacy of ethylene treatment depended on 1-MCP concentration and storage duration.

[30] 祝美云, 李小月, 梁丽松, 马庆华, 王贵禧 . 不同采后处理对‘阿巴特’西洋梨果实冷藏后货架期品质的影响
中国农学通报, 2015,31(33):273-278.
[本文引用: 1]

ZHU M Y, LI X Y, LIANG L S, MA Q H, WANG G X . Effect of different postharvest treatments on quality of ‘Abate’ pear during shelf life after cold storage
Chinese Agricultural Science Bulletin, 2015,31(33):273-278. (in Chinese)
[本文引用: 1]

[31] 侯玉茹, 李文生, 王宝刚, 苗飞, 石磊 . 1-MCP处理对贮藏后西洋梨货架期品质的影响
食品工业科技, 2015,36(20):335-338.
[本文引用: 1]

HOU Y R, LI W S, WANG B G, MIAO F, SHI L . Effect of 1-MCP treatment on quality of Pyrus communis L. during shelf-life of storage
Science and Technology of Food Industry, 2015,36(20):335-338. (in Chinese)
[本文引用: 1]

[32] HIWASATANASE K, KINUGASA Y, AMANO S, HASHIMOTO A, NAKANO R, INABA A, KUBO Y . Ethylene is required for both the initiation and progression of softening in pear (Pyrus communis L.) fruit
Journal of Experimental Botany, 2003,54(383):771-779.
DOI:10.1093/jxb/erg073URLPMID:12554720 [本文引用: 1]
In order to investigate the physiological role of ethylene in the initiation and subsequent progression of softening, pear fruit were treated with propylene, an analogue of ethylene or 1-methylcyclopropene (1-MCP), a gaseous inhibitor of ethylene action at the preclimacteric or ripening stages. The propylene treatment at the pre-ripe stage stimulated ethylene production and flesh softening while the 1-MCP treatment at the same stage markedly retarded the initiation of the ripening-related events. Moreover, 1-MCP treatment after the initiation of ripening markedly suppressed the subsequent flesh softening and ethylene production. These results clearly indicate that ethylene is not merely a by-product, but plays a crucial role in both the initiation and maintenance of regulating the softening process during ripening. The observations also suggest that ethylene in ripening is regulated entirely in an autocatalytic manner. The mRNA accumulation of pear polygalacturonases (PG) genes, PC-PG1 and PC-PG2, was in parallel with the pattern of fruit softening in both propylene and 1-MCP treatments. However, the expression pattern of pear endo-1,4-beta-D-glucanases (EGase) genes, PC-EG1 and PC-EG2, was not affected in both treatments. The results suggest that ethylene is required for PGs expression even in the late ripening stage, but not for EGases.

[33] 袁晖, 张利超, 卜海东, 王爱德 . 南果梨成熟相关PG基因筛选及表达分析
沈阳农业大学学报, 2018,49(5):522-528.
[本文引用: 1]

YUAN H, ZHANG L C, BU H D, WANG A D . The screening and expression analysis of ripening-related PG genes in ‘Nanguo’ pear
Journal of Shenyang Agricultural University, 2018,49(5):522-528. (in Chinese)
[本文引用: 1]

[34] TATEISHI A, MORI H, WATARI J, NAGASHIMA K, YAMAKI S, INOUE H . Isolation, characterization, and cloning of α-L- Arabinofuranosidase expressed during fruit ripening of Japanese pear
Plant Physiology, 2005,138(3):1653-1664.
DOI:10.1104/pp.104.056655URLPMID:15965025 [本文引用: 1]
alpha-L-Arabinofuranosidase (alpha-L-arafase) was purified from fruit of Japanese pear (Pyrus pyrifolia). The enzyme solubilized from the cell wall by NaCl and Triton X-100 had the homogeneity of a single 62-kD polypeptide on SDS-PAGE after purification through the steps of hydroxyapatite, anion-exchange chromatography, and size-exclusion chromatography. A related cDNA clone was isolated (PpARF2). The transcript and related protein were detected solely in the ripening fruit corresponding to the increase of alpha-L-arafase activity. Transcripts of PpARF2 were not detected in buds, leaves, roots, or shoots of the Japanese pear. The deduced amino acid sequences of PpARF2 had low identity with those of other plants or bacteria. This alpha-L-arafase belonged to glycoside hydrolase family 3, which includes some beta-xylosidases. The purified enzyme hydrolyzed mainly p-nitrophenyl alpha-L-arabinofuranoside and also reacted bifunctionally with p-nitrophenyl beta-d-xylopyranoside. However, it released only arabinose from native cell wall polysaccharides prepared from Japanese pear and from sugar beet arabinan. The enzyme did not release xylose from arabinoxylan and xylan. The only activity of the alpha-L-arafase presented here was hydrolyzing the arabinosyl residue from native polysaccharides, whereas it showed bifunctional activity against artificial substrates. According to the expression pattern and properties of the enzyme, it is a new member of the glycoside hydrolase family 3 isolated from fruit, and it may be responsible for modification of the cell wall architecture during fruit softening.

[35] TATEISHI A, INOUE H, SHIBA H, YAMAKI S . Molecular cloning of β-galactosidase from Japanese pear(Pyrus pyrifolia) and its gene expression with fruit ripening
Plant Cell Physiology, 2001,42(5):492-498.
DOI:10.1093/pcp/pce059URLPMID:11382815 [本文引用: 2]
We have cloned a cDNA fragment encoding a beta-galactosidase from Japanese pear (Pyrus pyrifolia) fruit (JP-GAL). It contained an untranslated sequence of 182 nucleotides at the 5' end, a presumptive coding sequence of 2,193 nucleotides and an untranslated sequence of 268 nucleotides including a polyadenylation signal and a poly (A) tail at the 3' end. It encoded a protein with a calculated molecular weight of 80.9 kDa which consists of 731 amino acids. Both the nucleotide and the deduced amino acid sequences showed a 98% sequence identity with that obtained from the apple beta-galactosidase cDNA. The peptide sequence obtained from the purified Japanese pear beta-galactosidase III matched the deduced amino acid sequence of SVSYDHKAIIINGQKRILISG (amino acid 25-45). Northern blot analysis showed that the probe derived from JP-GAL hybridized to a single 2.6 kb RNA. The mRNA was detected solely in the fruit; none was detected in the buds, leaves, roots or shoots of the Japanese pear. The steady-state level of the beta-galactosidase mRNA was measured during fruit ripening in three cultivars, Housui, Kousui (early ripening) and Niitaka (late ripening). The results showed that regardless of the cultivar, no JP-GAL mRNA was detected in the immature fruit. Increment of the mRNA level with fruit ripening coincided with the increase in the beta-galactosidase III activity. Our results showed that the expression of JP-GAL correlated with fruit softening and JP-GAL may be beta-galactosidase III.