Study on the tolerant function of soybean GmGST7 gene to acidic aluminum stress
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摘要:目的
耐酸铝基因GsMYB7过表达转化大豆品种‘华春6号’后，从转基因株系的表达谱中获得目标基因GmGST7，该基因受酸铝胁迫诱导上调，且位于GsMYB7基因下游，进一步分析其耐酸铝功能，以期提高大豆酸铝耐受能力。
方法采用生物信息学方法分析GmGST7基因的碱基序列、蛋白结构域和构建系统进化树。通过烟草叶片瞬时转化法完成亚细胞定位。通过RT-qPCR分析该基因组织表达特异性。设计0、25、50、75 和 100 µmol/L 5个AlCl3浓度梯度，研究GmGST7对酸铝胁迫的响应。在50 µmol/L AlCl3处理下，设计0、4、8、12、16、24、36、48和72 h共9个时间梯度，对GmGST7的表达模式进行分析。过表达GmGST7基因遗传转化拟南芥，鉴定阳性植株，并对转基因株系进行耐酸铝表型验证、氧化水平测定、耐酸铝标志基因及下游基因的表达分析。
结果GmGST7基因位于大豆第7号染色体，序列全长为1 128 bp。该基因含有2个外显子和1个内含子，2个外显子分别编码GST高度保守的N端和不保守的C端；GmGST7基因编码226个氨基酸，编码的蛋白为大豆GST蛋白的tau类家族成员，定位于细胞质和细胞核中；GmGST7基因在大豆根、茎、叶、花和幼荚中均有表达，且在根中的表达量最高；GmGST7基因在50 µmol/L AlCl3处理24 h时表达最高；AlCl3处理后，野生型拟南芥相对根伸长显著低于转基因株系的，野生型拟南芥氧化水平高于转基因株系的，耐酸铝标志基因和下游基因的表达量在转基因株系中较高。
结论GmGST7基因属于大豆GST tau类家族成员，在细胞核和细胞质中行使功能，呈组成型表达模式，且在大豆根中表达最高，对酸铝胁迫响应显著；GmGST7过表达通过激活酸铝胁迫标志基因及其下游基因的表达提高拟南芥的酸铝耐受能力。
Abstract:ObjectiveThe GmGST7 gene was obtained from the gene expression profile of the GsMYB7 overexpressed lines of soybean ‘Huachun 6’ which was tolerant to acidic aluminum stress. GmGST7 lied downstream of the GsMYB7 gene, and was up-regulated by acidic aluminum stress. Its function of acidic aluminum resistance was further investigated to enhance the tolerance to aluminum stress in soybean.
MethodThe bioinformatics of the GmGST7 gene was analyzed using the base sequence, protein domain and phylogenetic tree. Subcellular localization of GmGST7 protein was accomplished by transient transformation in tobacco leaves. The tissue expression specificity of the GmGST7 gene was analyzed by RT-qPCR. Five AlCl3 concentration gradients of 0, 25, 50, 75 and 100 µmol/L were designed to study the response of GmGST7 to aluminum stress. Under the treatment of 50 µmol/L AlCl3, nine time gradients of 0, 4, 8, 12, 16, 24, 36, 48 and 72 h were designed to investigate the expression patterns of GmGST7. Arabidopsis (Col-0) was transformed by overexpression of GmGST7, positive plants were identified by molecular technology. The phenotype identification of Arabidopsis tolerant to acidic aluminum stress were performed with the oxidation level determination, the expression analysis of the genes response to aluminum stress and downstream genes of GmGST7.
ResultThe full-length sequence of GmGST7 located on chromosome 7 of soybean was 1 128 bp. The GmGST7 gene contained two exons and one intron which encodes a highly conserved N domain and a unconserved C domain of GST, respectively. GmGST7 encoded 226 amino acids. The GmGST7 protein was a tau member of the GST family in soybean, and localized in the nuclear and cytoplasm. GmGST7 was expressed in soybean root, stem, leaf, flower and young pod, and rich in root. The GmGST7 gene was up-regulated by AlCl3 with the highest relative expression under 50 µmol/L AlCl3 for 24 h. The relative root elongation of wild type was significantly lower than that of the transgenic lines, the oxidation level was higher, and the expression levels of acidic aluminum stress response genes and downstream genes were higher.
ConclusionThe GmGST7 gene is a tau member of the GST family in soybean, locates in the nucleus and cytoplasm. The GmGST7 gene holds a constitutive expression pattern, and is rich in soybean root. GmGST7 is significantly up-regulated by acidic aluminum stress. Overexpression of GmGST7 enhances the tolerance to aluminum stress in Arabidopsis by activating the expression of the marker genes response to acidic aluminum stress and its downstream genes.
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Keywords:
- Soybean /
- GmGST7 /
- Acidic aluminum stress /
- Genetic transformation
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图 2 GmGST7蛋白序列对比(A)和进化树(B)分析
红色横线代表GST-N，蓝色横线代表GST-C，红色点标注为目标基因GmGST7
Figure 2. Sequence comparison (A) and evolutionary tree (B) analysis of GmGST7 protein
The red horizontal line represents GST-N, the blue horizontal line represents GST-C, and the red dot is labeled as the target gene GmGST7
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图 4 GmGST7基因的组织表达模式分析
“**”表示该组织与幼荚在P < 0.01水平差异显著(t检验)
Figure 4. Analysis of tissue expression pattern of the GmGST7 gene
“**” indicates that the tissue differed from the young pod at P < 0.01 level (t test)
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图 5 GmGST7基因酸铝胁迫响应
各图中，“**”表示处理与对照(CK)在P < 0.01水平差异显著(t检验)
Figure 5. Response of the GmGST7 gene to acidic aluminum stress
“**” indicates that the treatment differed from the control (CK) at P < 0.01 level in each figure (t test)
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图 6 GmGST7基因克隆及载体构建
A：GmGST7基因cDNA序列克隆，根据引物位置克隆大小为742 bp；B：连亚细胞定位载体后，根据载体引物位置PCR产物大小为1 416 bp；C：连接过表达载体后，根据载体引物位置PCR产物大小为870 bp
Figure 6. Cloning and vector construction of the GmGST7 gene
A: Cloning of GmGST7 gene cDNA sequence, according to the location of the primer, cloning size is 742 bp; B: After connecting the subcellular localization vector, according to the location of the carrier primer, the size of the PCR product is 1 416 bp; C: After connecting the overexpressed vector, according to the location of the vector primer, the size of the PCR product was 870 bp
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图 8 GmGST7转基因拟南芥阳性苗除草剂喷洒鉴定
阳性苗正常生长，假阳性苗则枯萎
Figure 8. Herbicide spraying identification of GmGST7 transgenic Arabidopsis positive seedlings
Positive seedlings grow normally, false positive seedlings wilt
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图 9 GmGST7转基因拟南芥阳性苗DNA鉴定
Figure 9. DNA identification of GmGST7 transgenic Arabidopsis seedlings
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图 10 转基因拟南芥T3代株系中GmGST7基因表达量
“**”表示株系与WT在0.01水平差异显著(t检验)
Figure 10. Expression of GmGST7 gene in T3 transgenic Arabidopsis thaliana
“**” indicates that the line differed from the WT at 0.01 level (t test)
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图 11 过表达GmGST7拟南芥耐酸铝表型
Figure 11. Overexpressed GmGST7 Arabidopsis aluminium-resistant phenotype
表 1 引物序列
Table 1 Primer sequence
引物名称
Primer name引物序列(5′→3′)
Primer sequencepTF101-GmGST7-F gagaacacgggggactctagaATGGCTGCTAATCAGGAAGATGTG pTF101-GmGST7-R cgatcggggaaattcgagctcTTTTGAAGCAGAAAGACTTTCATGG Super1300-GmGST7-F acgggggactcttgaccatggCTATGGCTGCTAATCAGGAAGATGTG Super1300-GmGST7-R aagttcttctcctttactagtTTTTGAAGCAGAAAGACTTTCATGG GmGST7-F GTCCTGATTCCCGGCTCAAT GmGST7-R AACTCACAAATGAGAGACCAGT RT-GmGST7-F TCAACCACCCTGTTGTCAAAC RT-GmGST7-R AAGACTTTCATGGCAGGCTTTGT 
下载: 导出CSV
表 2 荧光定量引物序列
Table 2 qPCR primer sequence
基因
Gene引物序列(5′→3′)
Primer sequenceAtALMT F: TCCCATGGGTAAAGACAAAG
R: ATAGTCTGCTTTCTGCCAAAAtMATE F: CATTCGAATCCATCGAGATT
R: CGAATGTTGCACTCTGTTTTAtALS3 F: AGCTTCGAGATGACATCAAA
R: ACGGTTTTGCAGCTATCTAAAtWAK1 F: TGGCCGCTGATATTACAAAT
R: CAGATTGGCTACTGGTTAGTAt1G78660 F: CAGGTTTGAGTGTATCGGTG
R: CATCTGATTCTTCTGCCCAAAt1G78670 F: TCCTCTGAGATGTGGAGATT
R: TAGTTGAGGTTTGGATCAGCAt1G78680 F: AAAATGGTGGATTTTGCAGG
R: ATAGGCTGACGTTCAAAGTTAt4G33090 F: TGGATCAGTTCAAAGGTGAG
R: GACTATGTCGAGATCGATGG
下载: 导出CSV
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