Gene Cloning and Functional Analysis of MebZIP2 in Cassava
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摘要:
目的 bZIP转录因子在植物生长发育、淀粉合成和非生物胁迫等方面发挥重要作用。克隆木薯MebZIP2基因并进行亚细胞定位、表达分析和酵母单杂交分析，为进一步研究MebZIP2基因的功能提供参考。 方法 从木薯SC205中扩增MebZIP2基因编码区（CDS）序列，对其进行生物信息学分析，并构建pNC-Green-SubN融合表达载体，通过农杆菌介导转染烟草表皮细胞，观察荧光信号以确定MebZIP2蛋白的亚细胞定位情况。在木薯不同组织和块根不同发育阶段分析MebZIP2基因表达特征，利用酵母单杂交研究其与淀粉合成基因启动子的互作情况。 结果 MebZIP2基因CDS序列长度为465 bp，编码154个氨基酸，蛋白分子量为17 891.35 Da，理论等电点为5.23，属于不稳定亲水性蛋白。MebZIP2含有bZIP保守结构域，其氨基酸序列与橡胶树bZIP蛋白的相似性最高，为74.22%。MebZIP2基因启动子区域含有光响应元件，赤霉素、水杨酸和茉莉酸等激素响应元件，以及胚乳表达和胁迫响应元件。MebZIP2蛋白亚细胞定位于细胞核和细胞膜。MebZIP2基因在根尖分生组织、须根、茎和块根发育前期的表达量较高。MebZIP2与淀粉合成基因MeAPL5a、MeGBSS1和MeISA1共表达，而且MebZIP2蛋白可以与它们的启动子互作。 结论 MebZIP2基因属于bZIP基因家族成员，其表达具有组织特异性和块根发育阶段性，MebZIP2蛋白可以与淀粉合成基因MeAPL5a、MeGBSS1和MeISA1的启动子互作，可能参与了木薯块根发育和淀粉合成。 Abstract:Objective A functional analysis was conducted on the cloned bZIP transcription factor in cassava. Method The coding sequence (CDS) of ZIP2 in Cassava SC205 was amplified to perform a bioinformatic analysis in determining the subcellular localization and expression of MebZIP2. A pNC-green-subN fusion expression vector was constructed and transferred into tobacco epidermal cells using the Agrobacterium-mediated method. Fluorescence signals were observed for subcellular localization of the protein. Expressions of MebZIP2 in tissues at different stages of storage root development of the plant were determined. Interactions between MebZIP2 and the promoters of starch synthesis genes were analyzed by the yeast one-hybrid assay. Result The CDS of MebZIP2 was 465 bp long encoded 154 amino acids with a molecular weight of 17 891.35 Da and an isoelectric point of 5.23. The unstable hydrophilic protein contained a bZIP-conserved domain with the highest sequence similarity to that of one of Hevea brasiliensis at 74.22%. The promoter region of MebZIP2 included responsive elements to light, hormones (such as gibberellin, salicylic acid, and jasmonic acid), endosperm expression, and stresses. The protein localized in the cell membrane as well as the nucleus. The gene expressed highly in the root apical meristems, fibrous roots, stems, and at early development stages of storage roots and co-expressed with those involved starch synthesis, such as MeAPL5a, MeGBSS1, and MeISA1, whose promoters could interact with the protein. Conclusion Belonging to the bZIP family associated with plant growth, development, starch synthesis, and resistance to abiotic stress, MebZIP2 exhibited a tissue- and root development stage-specific expression. The protein interacted with the promoters of MeAPL5a, MeGBSS1, and MeISA1 related to starch synthesis in the plant. It might also be involved in cassava storage root development. -
Key words:
- Cassava /
- bZIP transcription factor /
- MebZIP2 /
- cloning /
- functional analysis
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图 1 MebZIP2基因扩增产物
M：DL2000 DNA标记；1：MebZIP2基因。
Figure 1. Electrophoresis of PCR amplified MebZIP2
M: DL2000 DNA marker; 1: MebZIP2.
图 2 MebZIP2与其他物种中同源氨基酸序列的比对结果
Figure 2. Alignment and homology of MebZIP2 amino acid sequences
图 3 MebZIP2与其他物种中同源的bZIP蛋白的系统发育进化树
Figure 3. Phylogenetic tree and homology of MebZIP2 amino acid sequences
图 6 MebZIP2基因表达分析
A. MebZIP2在木薯不同组织中的表达分析。B. MebZIP2在木薯块根不同发育阶段的表达分析。S1～S7表示木薯种植后第100、140、180、220、260、300、340天。不同字母表示均值之间显著差异（邓肯多重检验，P ＜ 0.05）。
Figure 6. MebZIP2 expressions
A. Expression analysis of MebZIP2 in different tissues of cassava. B. Expression analysis of MebZIP2 at different developmental stages of cassava storage root. S1–S7 indicate the 100th, 140th, 180th, 220th, 260th, 300th, 340th day after cassava planting. Different letters indicate significant differences between means (Duncan multiple test, P＜0.05).
图 7 MebZIP2与淀粉合成基因启动子的互作
A. MebZIP2与淀粉合成基因MeAPL5a、MeGBSS1、MeISA1在木薯块根不同发育阶段的表达趋势。S1～S7表示木薯种植后第100、140、180、220、260、300、340天。B. MebZIP2与淀粉合成基因MeAPL5a、MeGBSS1、MeISA1启动子的酵母单杂交结果。DDO：SD/-Trp-Leu培养基；TDO：不含3-AT的SD/-Trp/-Leu/-His培养基；TDO/5 mmol·L−1 3-AT：含5 mmol·L−1 3-AT的SD/-Trp/-Leu/-His培养基。
Figure 7. Interaction between MebZIP2 and promoters of starch biosynthesis genes
A. Expressions of MebZIP2 and MeAPL5a, MeGBSS1, and MeISA1 at cassava storage root development stages. S1-S7: 100th, 140th, 180th, 220th, 260th, 300th, and 340th day, respectively, after planting. B. Yeast one-hybrid assay results on interactions between MebZIP2 and promoters of MeAPL5a, MeGBSS1, and MeISA1. DDO: SD/-Trp-Leo medium; TDO: SD/-Trp/-Leu/-His medium without 3-AT; TDO/3-AT: SD/-Trp/-Leu/-His medium with 5 mmol·L−1 3-AT.
表 1 供试引物序列
Table 1. Primers applied
引物名称
Primer name引物序列
Primer sequence引物用途
Primer usageMebZIP2-F 5'-GCCATGGAGGCCAGTGAATTCATGGTCTCTTCTAGTGGGGCAT-3′ 基因克隆
Gene cloningMebZIP2-R 5′-CAGCTCGAGCTCGATGGATCCGAACAGGGCCATGTCCATG-3′ pNC-MebZIP2-F 5′-AGTGGTCTCTGTCCAGTCCTATGGTCTCTTCTAGTGGGGCAT-3′ 亚细胞定位
Subcellular locationpNC-MebZIP2-R 5′-GGTCTCAGCAGACCACAAGTGAACAGGGCCATGTCCATG-3′ qRTMebZIP2-F 5′-GGATCAGCTCGAGAAAGAAAAC-3′ 荧光定量PCR
Real-time PCRqRTMebZIP2-R 5′-TTAGGCCATTGCTGAAATTCAC-3′ Actin-F 5′-TGATGAGTCTGGTCCATCCA-3′ 内参基因
Reference geneActin-R 5′-CCTCCTACGACCCAATCTCA-3′ MeAPL5a-F 5′-GACTCACTATAGGGCGAATTCGGAAGTGAGCAATAAAAGTAGAAATG-3′ 启动子克隆
Promoter cloningMeAPL5a-R 5′-ATAATGCCAGGAATTACTAGTAGCAGCTGAATGTGGAGTGTTT-3′ MeGBSSI-F 5′-GACTCACTATAGGGCGAATTCGAAATGTAGGACCCTGGCGA-3′ 启动子克隆
Promoter cloningMeGBSSI-R 5′-ATAATGCCAGGAATTACTAGTCTCTATGCGGAAACAATTAAATCTG-3′ MeISA1-F 5′-GACTCACTATAGGGCGAATTCCTTGAAAATGAAAAAGATTAAAGAGAAA-3′ 启动子克隆
Promoter cloningMeISA1-R 5′-ATAATGCCAGGAATTACTAGTCGTTGGTATTACTTGGTGATAATCAC-3′ 
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