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2025年4月6日 星期日
农业生物技术学报  2020, Vol. 28 Issue (7): 1211-1220    DOI: 10.3969/j.issn.1674-7968.2020.07.007
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
血橙果肉MYB转录因子的克隆与表达分析
夏荣娜1, 李凛1, 杨鑫1, 王建辉2,*
1 成都理工大学 生态环境学院,成都 610059;
2 四川省农业科学院园艺研究所 农业农村部西南地区园艺作物生物学与种质创制重点实验室,成都 610066
Cloning and Expression Analysis of MYB Transcription Factor in Blood Orange (Citrus sinensis)
XIA Rong-Na1, LI Lin1, YANG Xin1, WANG Jian-Hui2,*
1 College of Ecology and Environment, Chengdu University of Technology, Chengdu 610059, China;
2 Key Laboratory of Horticultural Crops Biology and Germplasm Enhancement in Southwest, Ministry of Agriculture and Rural Affairs, Horticulture Research Institute of Sichuan Academy of Agricultural Sciences, Chengdu 610066, China
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摘要 花青苷是一种高活性的黄酮多酚类化合物,广泛存在于各种植物中。MYB转录因子调控植物体内花青苷生物合成。本研究从血橙(Citrus sinensis)果肉中克隆了MYB转录因子,并进行生物信息学分析及基因表达研究;同时,构建原核表达载体PET28-CsMYB,所获得重组蛋白经新西兰大白兔(Oryctolagus cuniculus)免疫制备多克隆抗体,进而使用Western blot技术分别检测了不同样本之间MYB的差异表达。研究结果表明,血橙MYB型转录因子的核酸全长表达序列为789 bp,编码262个氨基酸,编码蛋白的1~56位与57~111位氨基酸分别具有MYB保守结构域R2和R3,将其命名为CsMYB (GenBank No. KT757348)。将不同植物来源的MYB氨基酸全长序列进行多重比对分析并构建系统进化树,结果表明,CsMYB编码蛋白与已报道的荔枝(Litchi chinensis)花青苷调控蛋白(LcMYB1)聚在同一个进化支上,两个蛋白的氨基酸序列同一性为56.1%。对血橙不同组织进行基因差异表达分析,结果显示,CsMYB在果肉组织中高表达;同时参与花青苷生物合成与运输途径的查尔酮合成酶基因(chalcone synthase, CHS)与谷胱甘肽巯基转移酶基因(glutathione S-transferase, GST)在果肉中也呈现高表达;CsMYB基因表达随着果实发育与成熟而逐渐升高;与低花青苷含量的血橙芽变突变体比较,正常花青苷积累的果肉组织中CsMYB基因表达更高,并且CHS表达变化趋势与CsMYB一致。Western blot结果表明,只在血橙果肉样本中有36 kD的MYB蛋白杂交条带。综上所述,CsMYB可能参与调控血橙果肉花青苷的生物合成,本研究可为血橙果实着色机制研究与遗传育种提供参考依据。
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夏荣娜
李凛
杨鑫
王建辉
关键词 血橙花青苷CsMYB原核表达蛋白免疫印迹    
Abstract:Anthocyanins belong to a kind of flavonoid and are widely found in different plants. The biosynthesis of anthocyanin is transcriptionally regulated by MYB transcription factor. In this study, the full length of MYB type transcription factor was cloned from flesh of blood orange (Citrus sinensis), then bioinformatic analysis and differential expression of the gene were performed. Furthermore, the prokaryotic expression vector PET28-CsMYB was developed and used to induce expression of MYB recombinant protein, which was used to immune rabbit (Oryctolagus cuniculus) to prepare polyclonal antibody. Then Western blot was used to detect MYB expression between different samples. The results showed that the full-length of MYB nucleotide sequence was 789 bp, which encoded a protein with 262 amino acids. The protein had R2 and R3 MYB conserved domains at the position of 1~56 and 57~111 of amino acids residues, so that it was designated by CsMYB (GenBank No. KT757348). Multiple alignments were performed using different homologous MYB-type proteins. The phylogenetic tree showed that CsMYB protein was clustered with Litchi chinensis, an anthocyanin biosynthesis regulator (LcMYB1), and the amino acid identity between the two proteins was 56.1%. Comparison of differential gene expression in different tissues of blood orange showed that higher transcription level was found in the flesh only. In addition, chalcone synthase (CHS) and glutathione S-transferase (GST) also exhibited higher expression levels in the flesh. The relative expression of CsMYB significantly increased during fruit development. Compared with a mutant fruit with less anthocyanin contents, the relative expression of CsMYB in the red-fleshed fruit with normal anthocyanin contents was significantly higher, and the expression change of CHS was consistent with CsMYB. Western blot results showed that a weak hybridization band at 36 kD was occurred only in the red-fleshed fruit. Taken together, CsMYB might be involved in the anthocyanin regulation of blood orange. The present study could provide a reference for further studies on fruit coloring mechanism and genetic breeding of blood orange.
Key wordsBlood orange    Anthocyanin    CsMYB    Prokaryotic expression    Western blot
收稿日期: 2019-12-13     
ZTFLH:  S666.4  
基金资助:四川省财政创新能力提升工程项目高新技术专项(2016GXTZ-004); 四川省农业科学院青年基金(2018QNJJ-010); 四川省农业科学院优秀论文基金专项(2017LWJJ-003); 2017年成都理工大学中青年骨干教师培养计划项目(2017JXGG09)
通讯作者: * kevin_wangjh@126.com   
引用本文:   
夏荣娜, 李凛, 杨鑫, 王建辉. 血橙果肉MYB转录因子的克隆与表达分析[J]. 农业生物技术学报, 2020, 28(7): 1211-1220.
XIA Rong-Na, LI Lin, YANG Xin, WANG Jian-Hui. Cloning and Expression Analysis of MYB Transcription Factor in Blood Orange (Citrus sinensis). 农业生物技术学报, 2020, 28(7): 1211-1220.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2020.07.007     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2020/V28/I7/1211
 
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