脱落酸葡糖基转移酶参与果实成熟过程
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摘要
果实成熟是一个复杂的生理生化过程,近年来植物激素脱落酸(ABA)越来越成为研究果实成熟的一个热点。拟南芥中己证明葡糖基转移酶可以催化ABA的糖基化,从而调节ABA的内稳态。本实验主要以非跃变型果实草莓和跃变型果实番茄为材料来研究糖基转移酶在两种类型果实成熟过程中的作用。
在草莓果实中克隆得到ABA合成和代谢关键基因aNCED1、FaNCED2、FaCYP707A1。通过ABA含量测定和基因表达水平分析发现,ABA含量的变化和aNCED2基因的表达水平一致,均在白熟期草莓果实中出现峰值,并且在草莓果实成熟后期逐渐上升；FaCYP707A1的表达水平在白熟期草莓中出现峰值,随后的成熟过程中表达量逐渐下降,全红期达到最低水平。三个基因的表达水平在草莓果实和花器官中存在时空特异性,且被生长素处理下调。
葡糖基转移酶(GT)参与ABA的糖结合途径,本实验分别在草莓果实和番茄果实中研究其对果实成熟的作用。在草莓果实中,通过分析果实成熟过程中葡糖基转移酶的表达水平发现,许多GT基因的表达水平与成熟过程呈正相关。大部分葡糖基转移酶响应生长素和脱落酸的处理,且两种激素处理在草莓果实中对GT基因表达量的影响相反。在FaGTl和FaGT2沉默的草莓果实中,成熟过程被显著抑制,花青素代谢和细胞壁降解关键基因的表达量明显下调,表明FaGTl和FaGT2可能参与草莓果实的成熟。在番茄果实中,SIUGT75C1, SIUGT73C4, SIUGT76E1三个基因在果实成熟过程中高度表达,果肉中的表达量显著高于种子,生长素和脱落酸对其表达都有诱导作用。在SIUGT75C1基因沉默的番茄果实中,果实成熟进程受阻,乙烯生物合成关键基因、类胡萝卜素代谢关键基因的表达显著下调。三个基因编码的蛋白质都定位在叶绿体中。S1UGT75C1重组蛋白在体外能够催化ABA与尿苷二磷酸葡萄糖(UDPG)的结合,生成ABA葡萄糖酯(ABA-GE), Km值为3.24mM,最大反应速率为0.06uM/min。上述结果表明葡糖基转移酶可能通过影响果实中ABA的含量参与果实的成熟。
综上所述,本研究证明ABA葡糖基转移酶参与两种类型果实的成熟过程,为ABA调控果实成熟提供了新的证据。
Recently, plant hormone abscisic acid (ABA) is a new target for study the fruit ripening which is a complex physiological and biochemical progress. In Arabidopsis glucosyltransferases could glycosylate ABA and regulate ABA homeostasis. This paper focuses the role of glycosyltransferase in fruit ripening of non-climacteric fruit strawberry and climacteric furit tomato.
ABA biosynthetic and metabolic genes FaNCEDl, FaNCED2, and FaCYP707A1have been cloned from strawberry fruit. Analysis of ABA contents and genes expression during fruit ripening, results indicated that change of ABA content consistented with expression level of FaNCED2, both of which have peak on white stage then increase during ripening. Expression level of FaCYP707A1has peak on white stage, then decrease during ripening and has lowest level on red stage. Expression levels of three genes have temporal and spatial specific and down-regulated by NAA.
Glucosyltransferase (GT) takes part in ABA glucosylation. This paper focuses on the role of GTs during strawberry and tomato fruit ripening. In strawberry fruit, expression levels of mostly GTs have positive correlation with fruit ripening. Both auxin and ABA can regulate expression of GTs, which usually has opposite influence. Ripening process of FaGTl-or FaGT2-silencing strawberry fruit were interrupted, expression level of anthocyanin metabolic and cell wall degradation related genes were decreased, these suggested that FaGT1and FaGT2maybe involved in strawberry fruit ripening. In tomato fruit, SlUGT75C1, SlUGT73C4, and SlUGT76E1have high expression level during fruit ripening. Expression levels of three genes on pulp were higher than on seed. Auxin and ABA triggered expression of there genes. Ripening of SlUGT75C1-silencing tomato fruit was blocked, in which expression levels of ethylene biosynthesis and carotenoid metabolism related genes were down-regulated. Three glucosyltransferases located in chloroplast. Recombinase S1UGT75C1can catalyze ABA and UDPG to produce ABA-GE in vitro. Km is3.24mM and Vmax is0.06uM/min. The above results indicated that glucosyltransferase maybe involved in fruit ripening by affect ABA.
In conclusion, the study demostrated ABA glucosyltranferase involed in fruit ripening of two ripening type fruit, provided new evidence for ABA regulated fruit ripening.
在草莓果实中克隆得到ABA合成和代谢关键基因aNCED1、FaNCED2、FaCYP707A1。通过ABA含量测定和基因表达水平分析发现,ABA含量的变化和aNCED2基因的表达水平一致,均在白熟期草莓果实中出现峰值,并且在草莓果实成熟后期逐渐上升；FaCYP707A1的表达水平在白熟期草莓中出现峰值,随后的成熟过程中表达量逐渐下降,全红期达到最低水平。三个基因的表达水平在草莓果实和花器官中存在时空特异性,且被生长素处理下调。
葡糖基转移酶(GT)参与ABA的糖结合途径,本实验分别在草莓果实和番茄果实中研究其对果实成熟的作用。在草莓果实中,通过分析果实成熟过程中葡糖基转移酶的表达水平发现,许多GT基因的表达水平与成熟过程呈正相关。大部分葡糖基转移酶响应生长素和脱落酸的处理,且两种激素处理在草莓果实中对GT基因表达量的影响相反。在FaGTl和FaGT2沉默的草莓果实中,成熟过程被显著抑制,花青素代谢和细胞壁降解关键基因的表达量明显下调,表明FaGTl和FaGT2可能参与草莓果实的成熟。在番茄果实中,SIUGT75C1, SIUGT73C4, SIUGT76E1三个基因在果实成熟过程中高度表达,果肉中的表达量显著高于种子,生长素和脱落酸对其表达都有诱导作用。在SIUGT75C1基因沉默的番茄果实中,果实成熟进程受阻,乙烯生物合成关键基因、类胡萝卜素代谢关键基因的表达显著下调。三个基因编码的蛋白质都定位在叶绿体中。S1UGT75C1重组蛋白在体外能够催化ABA与尿苷二磷酸葡萄糖(UDPG)的结合,生成ABA葡萄糖酯(ABA-GE), Km值为3.24mM,最大反应速率为0.06uM/min。上述结果表明葡糖基转移酶可能通过影响果实中ABA的含量参与果实的成熟。
综上所述,本研究证明ABA葡糖基转移酶参与两种类型果实的成熟过程,为ABA调控果实成熟提供了新的证据。
Recently, plant hormone abscisic acid (ABA) is a new target for study the fruit ripening which is a complex physiological and biochemical progress. In Arabidopsis glucosyltransferases could glycosylate ABA and regulate ABA homeostasis. This paper focuses the role of glycosyltransferase in fruit ripening of non-climacteric fruit strawberry and climacteric furit tomato.
ABA biosynthetic and metabolic genes FaNCEDl, FaNCED2, and FaCYP707A1have been cloned from strawberry fruit. Analysis of ABA contents and genes expression during fruit ripening, results indicated that change of ABA content consistented with expression level of FaNCED2, both of which have peak on white stage then increase during ripening. Expression level of FaCYP707A1has peak on white stage, then decrease during ripening and has lowest level on red stage. Expression levels of three genes have temporal and spatial specific and down-regulated by NAA.
Glucosyltransferase (GT) takes part in ABA glucosylation. This paper focuses on the role of GTs during strawberry and tomato fruit ripening. In strawberry fruit, expression levels of mostly GTs have positive correlation with fruit ripening. Both auxin and ABA can regulate expression of GTs, which usually has opposite influence. Ripening process of FaGTl-or FaGT2-silencing strawberry fruit were interrupted, expression level of anthocyanin metabolic and cell wall degradation related genes were decreased, these suggested that FaGT1and FaGT2maybe involved in strawberry fruit ripening. In tomato fruit, SlUGT75C1, SlUGT73C4, and SlUGT76E1have high expression level during fruit ripening. Expression levels of three genes on pulp were higher than on seed. Auxin and ABA triggered expression of there genes. Ripening of SlUGT75C1-silencing tomato fruit was blocked, in which expression levels of ethylene biosynthesis and carotenoid metabolism related genes were down-regulated. Three glucosyltransferases located in chloroplast. Recombinase S1UGT75C1can catalyze ABA and UDPG to produce ABA-GE in vitro. Km is3.24mM and Vmax is0.06uM/min. The above results indicated that glucosyltransferase maybe involved in fruit ripening by affect ABA.
In conclusion, the study demostrated ABA glucosyltranferase involed in fruit ripening of two ripening type fruit, provided new evidence for ABA regulated fruit ripening.
引文
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