龙眼体胚发生过程中DlAGO4基因的克隆及表达分析  

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

作　　者：陈荣珠 申序 林美珍[1] 陈晓慧[2] 徐小萍[2] 赖钟雄[2] CHEN Rongzhu;SHEN Xu;LIN Meizhen;CHEN Xiaohui;XU Xiaoping;LAI Zhongxiong(Department of Pharmacy, Zhangzhou Health Vocational College, Zhangzhou Fujian 363000,China;Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University, Fuzhou 350002, China)

机构地区：[1]漳州卫生职业学院药学系,福建漳州363000 [2]福建农林大学园艺植物生物工程研究所,福州350002

出　　处：《西北植物学报》2020年第5期747-755,共9页Acta Botanica Boreali-Occidentalia Sinica

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

摘　　要：该研究基于龙眼基因组数据库,采用RT-PCR技术,以‘红核子’品种龙眼松散型胚性愈伤组织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,并采用实时荧光定量技术分析其在龙眼体细胞胚胎发生不同阶段、不同组织部位、激素和非生物胁迫处理以及5-氮胞苷(5-azac)处理的表达模式。结果表明:(1)DlAGO4基因cDNA全长为3425 bp,包含开放阅读框长度为2781 bp,编码926个氨基酸。(2)生物信息学分析表明,DlAGO4蛋白为碱性亲水非分泌蛋白,含有AGO经典的保守结构域PAZ和Piwi,与克莱门柚CcAGO4的同源性最近,含有85个磷酸化位点和2个糖基化位点;亚细胞定位预测其最可能定位于细胞核;MicroRNA预测显示,DlAGO4基因受到4个miRNA靶向调控。(3)qRT-PCR结果表明,DlAGO4在龙眼球形胚阶段和种子中相对表达量最高;激动素、水杨酸、NaCl、甘露醇、PEG-4000和ABA处理均能促进DlAGO4基因的表达,而2,4-D和MeJA处理抑制其表达;不同浓度的5-azac处理1 d和3 d抑制DlAGO4的表达,但从处理第6天开始该基因呈上调表达,并于处理第12天相对表达量最高。研究认为,DlAGO4基因可能参与龙眼球形胚和种子的转录调控,且可能参与KT、SA的激素信号转导途径和NaCl、甘露醇、PEG-4000、ABA的逆境胁迫响应途径以及DNA甲基化调控机制。Based on the third generation genome of longan(Dimocarpus longan),we isolated the cDNA sequence of AGO4 gene from longan embryogenic callus(cultivar:‘Honghezi’)by reverse transcription polymerase chain reaction(RT-PCR)and analyzed the bioinformation.Furthermore,we analyzed the expression profiles in somatic embryogenesis,different tissues,hormones treatment,abiotic stress,and 5-azac(5-azacytidine)treatment.The results showed that:(1)The cDNA length of DlAGO4 was 3425 bp,which contained an open reading frame of 2781 bp and encoded 926 amino acids.(2)The bioinformatic analysis showed that it was basic,hydrophilic and non-secretory protein,containing PAZ and Piwi conserved domains,had 85 phosphorylation sites and 2 glycation sites,while shared high homology with CcAGO4 of Citrus clementina.The subcellular localization was predicted to be located in the nucleus,and microRNA prediction showed that DlAGO4 was regulated by four miRNAs.(3)qRT-PCR results indicated that the relative expression of DlAGO4 was up-expressed in the globular embryos(GE)and seeds in Longan,while KT,salicylic acid,NaCl,mannitol,PEG-4000 and ABA treatments could promote the expression,but 2,4-D and MeJA treatments inhibited the expression.After treated with 5-azac,the expression of DlAGO4 was down-regulated in cultured 1 d and 3 d,but up-regulated from cultured 6 d,and significantly increased in cultured 12 d.These results suggested that DlAGO4 might be involved in the transcriptional regulation of globular embryo stages and seeds development of longan.Furthermore,DlAGO4 might be involved in hormone,abiotic stress responsiveness and DNA methylation regulation.

关 键 词：龙眼体胚发生 AGO4 激素响应 非生物胁迫 5-氮胞苷 实时荧光定量PCR 

分 类 号：Q785[生物学—分子生物学] Q786