龙眼体胚发生过程中DlAGO4基因的克隆及表达分析
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1.漳州卫生职业学院;2.福建农林大学园艺植物生物工程研究所

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漳州卫生职业学院科技创新团队培育项目(Kjcx-3)、福建农林大学科技创新专项基金(CXZX2017189;CXZX2016118、CXZX2017314、CXZX2018076、KF2015108)*国家自然科学(31672127,31572088)、福建省高原学科建设经费(102/71201801101)和福建农林大学科技创新专项基金(CXZX2017189;CXZX2016118、CXZX2017314、CXZX2018076、KF2015108)


Cloning and Expression Analysis of DlAGO4 Gene from Embryogenic Callus in Dimocarpus longan Lour.
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    摘要:

    本研究基于龙眼基因组数据,采用RT-PCR技术,以‘红核子’品种龙眼胚性愈伤组织cDNA为模板,进行龙眼胚性愈伤组织DlAGO4基因的克隆和生物信息学分析,生物信息学分析包括蛋白结构域及特性、启动子顺式作用元件、分子进化树分析,并对可能互作的miRNA进行预测分析,结果表明DlAGO4蛋白为碱性亲水非分泌蛋白,含有经典的保守结构域PAZ和Piwi,与柑橘的CcAGO4同源性较高,亚细胞定位预测位于细胞核,含有85个磷酸化位点和2个糖基化位点;5’端调控序列预测分析表明DlAGO4含有1个CpG岛,启动子序列含有大量光响应元件、多种激素响应元件和非生物胁迫应答元件,还含有胚乳特异表达元件及损伤修复元件;MicroRNA预测显示,DlAGO4受到3个miRNA靶向调控。利用实时荧光定量PCR技术检测DlAGO4在龙眼不同体胚阶段、不同组织部位、不同激素处理、非生物胁迫处理以及不同浓度5-氮胞苷处理下的相对表达量。从定量结果可知,在龙眼体胚发生不同阶段,DlAGO4在早期体胚阶段相对表达量较高,尤其在球形胚阶段,推测其在龙眼体胚发生早期可能发挥重要作用;在‘红核子’品种龙眼不同组织部位中DlAGO4在种子中的相对表达量最高,其次是果实中,这说明该基因可能参与龙眼种子和果实的发育;不同种类激素和非生物胁迫处理的结果表明,激动素、水杨酸、NaCl、甘露醇、PEG-4000和ABA处理均能促进该基因的表达,而2,4-D和MeJA处理确能抑制该基因的表达,推测DlAGO4可能参与不同激素和不同非生物胁迫的调控机制;不同浓度5-azac不同天数处理下,在培养1d和3d时均对DlAGO4的表达起到抑制的作用,从培养第6d开始开始促进该基因的表达,且在第12d时有最大的相对表达量,推测一定浓度的5-azac能降低龙眼EC的甲基化水平,促进体胚的发生。

    Abstract:

    Based on the genome of Dimocarpus longan, the cDNA sequences of argonaute4 gene was isolated from longan embryogenic callus (cultivar:‘Honghezi’) by reverse transcription polymerase chain reaction (RT-PCR). The protein characteristics, protein domain, cis-acting element of promoter, phylogenetic trees, and possibly interacted with miRNAs in the longan genome were predicted by bioinformatic analysis. The results showed that it was basic, hydrophilic and non-secretory protein, containing classical conserved domains PAZ and Piwi. DlAGO4 shared high homology with AGO4 gene of Citrus clementina. The subcellular localization was predicted to be located in the nucleus and which contained 85 phosphorylation sites and 2 glycation sites. The prediction analysis of the promoter sequences showed that it contained one CpG island, multiple light hormone and abiotic stress response elements, as well as endosperm specific expression element and damage repair elements. MicroRNA prediction showed that DlAGO4 was regulated by three miRNAs. QPCR analysis indicated that during somatic embryogenesis(SE) in longan, the relative expression of DlAGO4 is relatively high in the early somatic embryo stage, especially in the globular embryos, and suggested that the gene may be involved in the early stage of somatic embryo development. It was detected that DlAGO4 abundantly accumulated in seeds, followed by fruits, indicating that it may be involved in the development of longan seeds and fruits. The expression of DlAGO4 disposed hormone and abiotic stress treatment showed that KT, salicylic acid, NaCl, mannitol, PEG-4000 and ABA treatment could promote the expression, while 2, 4-D and MeJA treatment could inhibit the expression, so it may be involved in the regulation mechanism of different hormones and abiotic stress. The expression of DlAGO4 was down-regulated in cultured 1d and 3d, but up-regulated from cultured 6d, and significantly increased in cultured 12d, so it showed that certain concentration of 5-azac could reduce the methylation level of longan EC and promote the occurrence of somatic embryos.

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  • 收稿日期:2020-01-03
  • 最后修改日期:2020-03-16
  • 录用日期:2020-04-03
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