桑树脱落酸生物合成相关基因的鉴定及转录表达分析

doi:10.3864/j.issn.0578-1752.2015.05.19

摘要/Abstract

摘要： 【目的】9-顺式环氧类胡萝卜素双加氧酶（NCED）、醛氧化酶（AAO）和玉米黄质环氧化酶（ZEP）在脱落酸（abscisic acid，ABA）间接途径合成中发挥主要调节作用，它们是ABA合成相关基因。以桑树栽培品种嘉陵40号（Morus atropurpurea Roxb.）果实为材料，测定其发育过程中ABA的含量，分析桑椹成熟过程中ABA合成相关基因的转录表达、ABA及其合成抑制剂对果实发育的影响，为研究ABA在桑椹成熟和衰老过程中的作用机制提供基础数据。【方法】根据从单倍体川桑（Morus notabilis Schneid.）基因组数据库（http://morus.swu. edu.cn/morusdb）下载的ABA合成相关基因序列，对其DNA及推导的氨基酸序列进行分析。使用RNAiso Plus（TaKaRa）提取总RNA。以cDNA为模板，利用real-time PCR方法检测不同时期和不同处理桑椹中ABA合成相关基因的转录表达差异。利用高效液相色谱法，测定桑椹发育过程中的ABA含量。【结果】在川桑基因组数据库筛选鉴定到6个ABA合成相关基因：1个MnAAO、2个MnZEP和3个MnNCED。MnNCED1-3氨基酸序列高度保守，聚类分析表明它们与双子叶果树NCEDs序列同源性较高，与单子叶植物同源性较低；转录分析表明它们在叶中转录水平相对高于其他组织，在根中最低。其中MnNCED2和MnNCED3在根、皮、冬芽、雄花、叶中的转录水平较高。随着果实的发育，ABA含量在转色期开始逐渐上升。外源ABA处理后，桑椹脱落率升高，而氟啶酮（fluridone）可以抑制桑椹的脱落。MnNCED1-3在果实发育中后期的转录水平较高，与ABA含量的升高相一致，而MnAAO和MnZEP1-2在中后期下调。离体桑椹经ABA处理后，MnNCED1、MnAAO和MnZEP1的转录水平直到第4天才出现上调，MnNCED3在第3天和第4天被上调，MnZEP2一直上调；氟啶酮处理后MnNCED1和MnZEP2的转录表达量只在第1天和第3天被下调，MnAAO和MnZEP1只在第4天升高，MnZEP1只在处理后第1天被下调，MnNCED2在ABA和氟啶酮处理后均被下调。【结论】从桑树中获得了6个ABA合成相关基因MnAAO、MnZEP1—MnZEP2和NCED1—NcED3，MnNCEDs在果实发育中后期的转录表达相对较高，并与ABA的含量变化相一致，可能对ABA的合成起主要调控作用，外源ABA处理能够促进桑椹的成熟和脱落，氟啶酮处理能够抑制桑椹的成熟和脱落。

关键词: 桑树, 脱落酸, NCED , AAO , ZEP , 转录表达, 氟啶酮

Abstract: 【Objective】 9-cisepoxycarotenoid dioxygenase (NCED), ABA-aldehyde oxidase(AAO), Zeaxanthin epoxidase (ZEP) are the key enzymes involved in biosynthesis of abscisic acid (ABA) via indirect pathway. The aim of this study was to analyze the transcriptional expression of ABA biosynthesis related genes and detect the content of ABA during Jialing 40 (Morus atropurpurea Roxb.) fruit development. The effects of ABA and its synthesis inhibitors on fruit development were also explored. This work will lay a foundation for further studying the role of ABA on mulberry (Morus alba L.) fruit maturity and senescence. 【Method】In this study, the sequences of six putative ABA biosynthesis related genes were obtained from Morus notabilis genome database and its deduced amino acid sequences were analyzed by using bioinformatics tools. Total RNA was extracted from mulberry fruit using RNAiso Plus (TaKaRa) and reverse transcribed to synthesize cDNA. The relative transcriptional expression of ABA biosynthesis related genes in different developmental stages and treated mulberry fruits was investigated by using qRT-PCR. The content of ABA was determined by using HPLC.【Result】Six ABA biosynthesis related genes were isolated from mulberry, including one AAO gene, two ZEP genes and three NCED genes. Multiple-sequence alignment results showed that MnNCEDs amino acids are relatively conserved in higher plants. Cluster analysis revealed that MnNCED1-3 are closely related with dicotyledons plants and farther with monocotyledons plants. Transcription analysis indicated that the expression of ABA biosynthesis related genes was higher in leaves and the lowest expression was detected in roots. Particularly, the expression level of MnNCED2 and MnNCED3 was higher in different plant tissues compared to other genes. The concentration of ABA was strongly increased after the conversion stage of fruit. Exogenous ABA significantly promoted fruit maturity, whereas fluridone remarkably inhibited fruit ripening compared with the negative control treated with distilled water. The results of real-time PCR showed that the expression abundance of MnNCEDs was higher in the middle and latter periods than the early stage. Again, ABA biosynthesis related genes have shown different expression patterns under the same treatment. The expression level of ABA biosynthesis related genes was up-regulated in the fourth day after ABA treatment while down-regulated in the first day after fluridone treatment, unlikely the expression level of MnNCED2 was down-regulated after both ABA and fluridone treatments. 【Conclusion】 In this study six putative ABA biosynthetic genes in M. notabilis genome were obtained. The expression levels of MnNCEDs were higher in middle and later stages than early fruit developmental stage, which is in coincidence with the trend of ABA content during fruit development. Thus, MnNCEDs may play important roles in ABA biosynthesis. Exogenous ABA significantly promoted fruit maturity, while fluridone markedly inhibited fruit ripening.

Key words: mulberry, ABA, NCED, AAO, ZEP, transcriptional expression analysis, fluridone

朱攀攀，刘长英，赵爱春，裴汭超，李军，王晓红，李镇刚，王茜龄，鲁成，余茂德. 桑树脱落酸生物合成相关基因的鉴定及转录表达分析[J]. 中国农业科学, 2015, 48(5): 1011-1022.

ZHU Pan-pan, LIU Chang-ying, ZHAO Ai-chun, PEI Rui-chao, LI Jun, WANG Xiao-hong, LI Zhen-gang, WANG Xi-ling, LU Cheng, YU Mao-de. Characterization and Transcriptional Expression Analysis of ABA Biosynthesis Related Genes from Mulberry (Morus alba L.)[J]. Scientia Agricultura Sinica, 2015, 48(5): 1011-1022.

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