关键词:马铃薯; HD-Zip; ATHB12基因; 根; 丙二醛; 脯氨酸 Cloning and Functional Identification of the ATHB12Gene of HD-Zip IFamily in Potato ( Solanum tuberosum L.) WU Liang-Liang, YAO Lei, MA Rui, ZHU Xi, YANG Jiang-Wei, ZHANG Ning*, SI Huai-Jun Gansu Key Laboratory of Crop Genetic and Germplasm Enhancement, Gansu Provincial Key Laboratory of Aridland Crop Science / College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, China Fund:This study was supported by the National Natural Science Foundation of China (31460370), Specialized Research Fund for the Doctoral Program of Higher Education of China (20126202110007), International Science & Technology Cooperation Program of China (0102014DFG31570), and Gansu Key Laboratory of Aridland Crop Science of Gansu Agricultural University (GSCS-2012-02) AbstractHD-Zip I is a class of plant-specific transcription factors, which has an important role in response to adversity stress in plant. A ATHB12 gene of HD-Zip I transcription factors was cloned from potato cultivar Gannongshu 2, which contains a 759 bp open reading frame (ORF) encoding a protein of 252 amino acid residues. ATHB12 gene is located on potato chromosome 1, and its promoter region sequence contains cis-acting elements including ABRE, LTRECOREATCOR15, WBOXATNPR1 responsive to abiotic stresses (ABA, temperature, dehydration, and salt stress). ATHB12 gene expressed in root, stem and leaf of potato, with the highest expression in the root. qRT-PCR analysis confirmed that the gene was induced by PEG, NaCl, and ABA, but repressed by cold treatment. The overexpressed-vector of ATHB12 gene driven by the constitutive promoter CaMV 35S was constructed, and the transgenic plants were obtained using Agrobacterium-mediated transformation system. The malondialdehyde (MDA) content in the transgenic plant leaves was significantly lower ( P< 0.05), whereas the proline content was significantly higher ( P<0.05) than those of non-transgenic control under drought stress. The fresh and dry weight of the transgenic plant root was higher than that of non-transgenic plants. These results showed that ATHB12 gene may be involved in response to stress.
图2 马铃薯ATHB12氨基酸序列与其他物种同源氨基酸序列多重比对结果 下画线: HD结构域; 虚线: LZ基序。Fig. 2 Alignment of the potato ATHB12 amino acid sequence and its homologous amino acid sequences from other species Underline: HD domain; Dot line: LZ motif.
图3 马铃薯ATHB12基因在不同器官中的qRT-PCR表达检测内参基因: ef1a, n=3; 误差线表示3个重复间的标准偏差; 柱上不同字母表示差异显著(P< 0.05), 图4同。Fig. 3 qRT-PCR assay ofATHB12 gene in different organs of potatoReference gene: ef1a, n=3; Error bars indicate standard deviation for three replicates; Bars superscripted by different letters are significantly different at P< 0.05. The same as in Fig. 4.
图8 转基因植株的丙二醛和脯氨酸含量 A: 丙二醛含量, B: 脯氨酸含量; CK: 非转基因植株; L1, L7: 转基因植株。数据为5次生物学重复的平均值± 标准偏差, 柱上不同字母表示差异显著(P< 0.05)。Fig. 8 MDA and proline content in the transgenic potato plants A: MDA content, B: proline content; CK: non-transgenic plants; L1, L7: transgenic plants. Values are the mean ± SD of five biological replicates. Bars superscripted by different letters are significantly different at P< 0.05.
表2 Table 2 表2(Table 2)
表2 转基因植株的丙二醛和脯氨酸含量相对值 Table 2 Relative value of MDA and proline content in the transgenic potato plants
相对值(PEG处理后/未处理) Relative value (PEG treated/untreated)
CK
L1
L7
丙二醛MDA
2.43± 0.31 a
1.99± 0.34 a
1.95± 0.23 a
脯氨酸Proline
1.95± 0.26 a
2.76± 0.22 b
2.81± 0.40 b
Values followed by different letter within the same line are significant by different at P< 0.05; n = 5. 同行中具有不同字母的平均值间差异显著(P< 0.05); n = 5。
表2 转基因植株的丙二醛和脯氨酸含量相对值 Table 2 Relative value of MDA and proline content in the transgenic potato plants
表3 转基因植株试管苗株高和根重 Table 3 Plant height and root weight of the transgenic test-tube plant
株系Line
株高Plant height (cm)
根鲜重Root fresh weight (mg)
根干重Root dry weight (mg)
CK
6.0± 0.77 a
20.9± 1.5 a
1.3± 0.38 a
L1
6.2± 1.20 a
35.4± 2.3 b
2.7± 0.74 b
L7
6.5± 0.93 a
37.8± 4.2 c
3.2± 0.83 b
Values followed by different letter within the same line are significant different at P< 0.05. Statistics related indicators were calculated after three weeks of plantlets subculture. The data represent the mean ± standard deviation of five biological replicates. 同列中具有不同字母的平均值间差异显著(P< 0.05); 试管苗转接3周后统计相关指标。数据为5次生物学重复的平均值± 标准偏差。
表3 转基因植株试管苗株高和根重 Table 3 Plant height and root weight of the transgenic test-tube plant
图9 转基因植株试管苗表型差异 CK: 非转基因植株; L1, L7: 转基因植株。Fig. 9 Phenotype difference between the transgenic test-tube plants and the control CK: non-transgenic plants; L1, L7: transgenic plants
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