Functional characterization of phosphorus deficiency-responsive GmNTLs in soybean roots
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摘要:目的
低磷和铝毒胁迫是酸性土壤中限制作物生产的重要因素。植物NTL转录因子参与调控多种环境胁迫(包括铝毒胁迫)的适应性机制，本文探究GmNTLs调控大豆Glycine max 根系响应低磷胁迫的功能。
方法通过RT-qPCR分析大豆15个GmNTLs基因在根系响应低磷胁迫的表达模式，进一步构建了GmNTL1/4/7/8/10/12共6个GmNTLs基因的拟南芥超量表达材料，探究GmNTL成员在拟南芥根系中响应低磷胁迫的功能。
结果系统进化树及组织表达模式分析结果表明，GmNTLs家族分3个亚族，各亚族成员在大豆中组织表达模式不同。RT-qPCR结果表明，低磷处理12 d显著提高了GmNTL1/4/7/8/10/12在大豆根系中的表达。在拟南芥中超量表达不同GmNTL基因对低磷的响应不同。高磷处理下，超量表达GmNTL4/10/12拟南芥的鲜质量显著增加；低磷处理时，超量表达GmNTL4显著提高拟南芥鲜质量，而超量表达GmNTL1/12拟南芥的鲜质量显著降低。同时，仅超量表达GmNTL12拟南芥的主根长显著缩短，而超量表达其他基因对拟南芥植株的主根长无明显影响。
结论GmNTLs参与大豆根系对低磷胁迫的响应，该结果可为培育磷高效型大豆品种提供数据支持。
Abstract:ObjectiveLow phosphorus (P) availability and aluminum (Al) toxicity constrain crop production in acid soils. NTL transcription factors play an important role in the mechanisms of plant response to various abiotic stresses, including Al toxicity. This study focused on analyzing the function of GmNTLs in soybean roots responding to P deficiency.
MethodExpression pattern were performed on 15 members of the GmNTL family in the soybean by RT-qPCR assays. We further investigated the function of some GmNTLs members in adaptation to low P by overexpressing the genes of GmNTL1/4/7/8/10/12 in Arabidopsis thaliana.
ResultThe phylogenetic analysis and the tissue expression analysis of each subfamily members revealed that 15 GmNTLs were divided into three subgroups and different GmNTL family members had different tissue expression pattern in soybean. The RT-qPCR results showed that the expression levels of GmNTL1/4/7/8/10/12 in soybean roots significantly increased after 12 days of low P treatment. Overexpression of different GmNTLs in Arabidopsis showed different responses to low P. The fresh weight of transgenic Arabidopsis overexpressing GmNTL4/10/12 significantly increased compared to control lines under high P treatment. Overexpression of GmNTL4 significantly improved fresh weight of transgenic Arabidopsis plant under low P deficiency; Whereas, the plant fresh weight of transgene lines overexpressing GmNTL1/12 significantly decreased. Overexpression of GmNTL12 reduced the primary root length of transgenic plants; Whereas, overexpression of other GmNTLs had no significant effect on primary root length.
ConclusionGmNTLs involve in the response of soybean roots to low P stress, and these results can provide a theoretical basis for cultivating soybean varieties with high P efficiency.
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Keywords:
- Glycine max /
- Root /
- Low phosphorus stress /
- GmNTLs /
- NTL transcription factor
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图 1 大豆GmNTL蛋白系统进化树及组织表达模式分析
1：新叶；2：花；3：根；4：根瘤；5：花后14 d豆荚壳；6：花后14 d种子
Figure 1. Phylogenetic tree and tissue expression analysis of GmNTL proteins in soybean
1: Young leaf; 2: Flower; 3: Root; 4: Nodule; 5: Pod shell after flowering for 14 days; 6: Seed after flowering for 14 days
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图 2 不同磷处理时间对大豆生长的影响
Figure 2. Effect of different phosphorus treatment time on soybean growth
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图 3 不同磷处理时间对大豆生物量和根长的影响
“*”“**”“***”分别表示相同胁迫时间不同磷浓度处理在P < 0.05、0.01、0.001水平差异显著(t检验)
Figure 3. Effect of different phosphorus treatment time on soybean biomass and root length
“*” “**” “***” indicate significant differences between different phosphorus concentration treatments at the same stress time at P < 0.05, 0.01 and 0.001 levels, respectively (t test)
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图 4 不同磷处理时间对大豆根系GmNTLs表达模式的影响
“*”“**”“***”分别表示相同胁迫时间不同磷浓度处理在P < 0.05、0.01、0.001水平差异显著(t检验)
Figure 4. Effect of different phosphorus treatment time on relative expression of GmNTLs in soybean roots
“*” “**” “***” indicate significant differences between different phosphorus concentration treatments at the same stress time at P < 0.05, 0.01 and 0.001 levels, respectively (t test)
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图 5 不同磷处理浓度下大豆根系GmNTLs相对表达量
各小图中，柱子上方的不同小写字母表示不同磷处理浓度间差异显著(P < 0.05，Duncan’s法)
Figure 5. Relative expression of GmNTLs in soybean roots under different phosphorus concentrations
In each figure, different lowercase letters on bars indicate significant differences among different phosphorus concentrations (P < 0.05, Duncan’s method)
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图 6 不同磷浓度下转基因拟南芥生长情况
WT：拟南芥Col-0野生型；OX1和OX2：超量表达株系；标尺=1 cm
Figure 6. Growth of transgenic Arabidopsis under different phosphorus concentrations
WT: Arabidopsis Col-0 wild-type; OX1 and OX2: Overexpressed lines; Bar = 1 cm
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图 7 不同磷浓度下转基因拟南芥的生物量
WT：拟南芥Col-0野生型，OX1和OX2：超量表达株系；“*”“**”“***”分别表示相同磷浓度下超量表达株系和野生型在P < 0.05、0.01、0.001水平差异显著（t检验）
Figure 7. Biomass of transgenic Arabidopsis under different phosphorus concentrations
WT: Arabidopsis Col-0 wild-type, OX1 and OX2: Overexpressed lines; “*” “**” “***” indicate significant differences between overexpressed lines and wild-type under the same phosphorus concentration at P < 0.05, 0.01 and 0.001 levels, respectively (t test)
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图 8 不同磷浓度下转基因拟南芥的主根长
WT：拟南芥Col-0野生型，OX1和OX2：超量表达株系；“*”“**”“***”分别表示相同磷浓度下超量表达株系和野生型在P < 0.05、0.01、0.001水平差异显著(t检验)
Figure 8. Primary root length of transgenic Arabidopsis under different phosphorus concentrations
WT: Arabidopsis Col-0 wild-type, OX1 and OX2: Overexpressed lines; “*” “**” “***” indicate significant differences between overexpressed lines and wild-type under the same phosphorus treatment concentration at P < 0.05, 0.01 and 0.001 levels, respectively (t test)
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