Cloning, subcellular localization and functional analysis of GmMADS4 in soybean
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摘要:目的
挖掘大豆Glycine max MADS转录因子家族成员GmMADS4基因信息，分析其结构及功能。
方法通过生物信息学分析，对GmMADS4基因进行基因结构、编码蛋白信息、保守结构域、系统进化树以及互作蛋白预测等分析。利用烟草叶片瞬时转化法分析亚细胞定位，通过RT-qPCR进行组织部位及响应缺素的表达模式分析，利用下胚轴复合植株转化法分析超量表达GmMADS4对转基因毛根生长的影响。
结果GmMADS4基因开放阅读框长732 bp，编码蛋白相对分子质量为28 000；保守结构域含有MADS-box和K-box，属于II型MADS家族成员，与拟南芥的AtAP3相似性较高；GmMADS4在大豆多个部位均有表达，且在花和种子中的表达量较高；缺氮和缺磷处理均显著增加GmMADS4在叶和根部的表达量；GmMADS4主要定位在细胞核，超量表达GmMADS4显著增加转基因毛根的可溶性磷含量。
结论GmMADS4属于大豆II型MADS家族成员，具有核定位功能，可能在大豆种子和花的发育过程中发挥作用，并参与大豆根部缺磷响应及磷稳态调节。
Abstract:ObjectiveTo explore the genetic information of GmMADS4, a member of soybean (Glycine max) MADS transcription factor family, and analyze its structure and function.
MethodThe structure, coding protein information, conserved domain, phylogenetic tree analysis and interaction protein prediction of GmMADS4 were analyzed by bioinformatics analysis. Transient transformation of tobacco leaves was used to analyze subcellular localization, RT-qPCR was used to analyze its expression patterns in different tissues and response to nutrient deficiency, and hypocotyl complex plant transformation method was used to analyze the effects of overexpression of GmMADS4 on transgenic hair root growth.
ResultThe length of open reading frame of GmMADS4 was 732 bp and the relative molecular mass of coding protein was 28 000. The conserved domain of GmMADS4 contained MADS-box and K-box, which was a member of type II MADS family, and had a high similarity with Arabidopsis AtAP3. GmMADS4 was expressed in many parts of soybean, especially in flowers and seeds. The expression of GmMADS4 in leaves and roots significantly increased by nitrogen and phosphorus deficiency induction. GmMADS4 was mainly located in the nucleus, and overexpression of GmMADS4 significantly increased soluble phosphate content in transgenic hair roots.
ConclusionGmMADS4 is a member of soybean type II MADS family, and has nuclear localization function, which may play a role in the development of soybean seeds and flowers, as well as participate in soybean root adaptive responses to phosphorus deficiency and the regulation of phosphate homeostasis.
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Keywords:
- Soybean /
- GmMADS4 /
- Subcellular localization /
- Expression pattern /
- Functional analysis
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图 1 GmMADS4基因结构及保守结构域预测
Figure 1. Gene structure and conserved domain prediction of GmMADS4
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图 2 GmMADS4蛋白二级结构预测
大写字母表示GmMADS4蛋白氨基酸序列；小写字母代表不同的二级结构，h代表α螺旋，c代表无规则卷曲，e代表延伸链，t代表β转角
Figure 2. Secondary structure prediction of GmMADS4
Capital letters indicate amino acid sequence of GmMADS4 protein; Lowercase letters represent different secondary structures, where h stands for α helix, c for random coil, e for extended strand, and t for β turn
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图 4 GmMADS4互作蛋白预测
不同颜色的圆球表示不同的蛋白，其中红色为GmMADS4；直线相连的2个蛋白预测存在相互作用
Figure 4. Interaction protein prediction of GmMADS4
Different colored spheres indicate different proteins, red is GmMADS4; Two proteins connected in a straight line are predicted to have an interaction
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图 5 GmMADS4基因克隆及酶切检测
A：GmMADS4基因PCR扩增，B：GmMADS4-pTF101s重组质粒酶切检测；M代表DNA marker，P代表PCR产物，泳道1为重组质粒酶切前，泳道2为重组质粒酶切后
Figure 5. Cloning and enzyme digestion of GmMADS4
A: PCR amplification of GmMADS4 gene, B: Digestion verification of GmMADS4-pTF101s recombinant plasmid; M represents DNA marker, P represents PCR product, lane 1 is the recombinant plasmid before digestion, lane 2 is the recombinant plasmid after digestion
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图 6 GmMADS4在大豆不同部位的表达分析
Figure 6. Expression pattern analysis of GmMADS4 in different soybean organs
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图 7 GmMADS4在大豆根和叶中响应缺素的表达模式分析
“*”表示缺素处理与对照相比差异显著(P < 0.05，t检验)
Figure 7. Expression pattern analysis of GmMADS4 in response to element deficiency in soybean roots and leaves
“*” indicates significant difference between element deficiency treatment and control (P < 0.05, t test)
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图 9 超量表达GmMADS4转基因复合植株的生长表型
Figure 9. Growth phenotypes of overexpressing GmMADS4 transgenic composite plants
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图 10 超量表达GmMADS4对大豆复合植株生物量和可溶性磷含量的影响
“*”表示对照和超量表达处理差异显著(P < 0.05，t检验)
Figure 10. Effects of overexpressing GmMADS4 on biomass and soluble phosphorus content of soybean composite plants
“*” indicates significant difference between control and overexpression treatment (P < 0.05, t test)
表 1 本研究所用到的引物
Table 1 Primers used in this study
引物名称 Primer name 引物序列(5′→3′)1)Primer sequence 注释 Annotation OE-GmMADS4-F C GAGCTCATGGGTCGTGGCAAGAT 超量表达 Overexpression OE-GmMADS4-R GC TCTAGATCAAGCAAGGCGAAGGTC GFP-GmMADS4-F GG GGTACCGATGGGTCGTGGCAAGAT 融合绿色荧光蛋白 Fused with GFP GFP-GmMADS4-R CG GGATCCCGAGCAAGGCGAAGGTCATG RT-GmMADS4-F GAGACAGATCAGGCATAGGA RT-qPCR RT-GmMADS4-R CCTACAGGTATCAGTCCGAG RT-GmEF-1α-F TGCAAAGGAGGCTGCTAACT RT-qPCR RT-GmEF-1α-R CAGCATCACCGTTCTTCAAA 1) 下划线为酶切位点 1) The restriction sites are underlined 
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