GRAS转录因子AtSCL4负调控拟南芥应答渗透胁迫

doi:10.13560/j.cnki.biotech.bull.1985.2021-0963

摘要/Abstract

摘要：

探讨拟南芥GRAS转录因子AtSCL4（Arabidopsis thaliana SCL4）在渗透胁迫中发挥的生物学功能,为GRAS蛋白在非生物胁迫中的功能研究奠定基础。以野生型和AtSCL4突变体拟南芥为试验材料,通过生理指标测定和qRT-PCR方法研究渗透胁迫下AtSCL4调控植物抗逆的生物学机制。研究发现AtSCL4受渗透胁迫诱导后显著上调表达,且AtSCL4突变体的抗渗能力强于野生型。在渗透胁迫下,AtSCL4可以负调节ATMYB6的表达,减小气孔开放度,降低叶片水分流失;AtSCL4通过负调控P5SC1和BADH的转录来提高植物体内脯氨酸和甜菜碱的含量;AtSCL4通过负调节AtSOD1和PER4的表达来增加抗氧化酶活性而降低活性氧含量。AtSCL4可负向调控抗逆基因表达和生理变化应答渗透胁迫。

关键词: 拟南芥, 渗透胁迫, GRAS转录因子, 气孔, 活性氧

Abstract:

Exploring the biological function of Arabidopsis thaliana GRAS transcription factor AtSCL4 in response to osmotic stress would lay foundation for studying the function of GRAS protein in abiotic stresses. Wild-type and AtSCL4 mutant were used as experimental materials,and physiological index determination and qRT-PCR methods were applied to study the biological mechanism of AtSCL4 regulating plant osmosis resistance. The study revealed that AtSCL4 was significantly up-regulated by osmotic stress,and mutants of AtSCL4 showed improved osmotic resistance than WT. In response to osmotic stress,AtSCL4 negatively regulated AtMYB61 expression to increase stomatal closure,resulting in reducing water loss. Furthermore,AtSCL4 improved proline and betaine levels by regulating the transcripts of P5SC1 and BADH. In addition,AtSCL4 increased antioxidant enzyme activity by negatively regulating AtSOD1 and PER4 expressions,leading to the decrease of reactive oxygen. AtSCL4 negatively modulated osmotic stress tolerance via the induction of stress-responsive genes and physiological changes.

Key words: Arabidopsis thaliana, osmotic stress, GRAS transcription factor, stomatal aperture, ROS

徐红云, 张明意. GRAS转录因子AtSCL4负调控拟南芥应答渗透胁迫[J]. 生物技术通报, 2022, 38(6): 129-135.

XU Hong-yun, ZHANG Ming-yi. AtSCL4,an Arabidopsis thaliana GRAS Transcription Factor,Negatively Modulates Plants in Response to Osmotic Stress[J]. Biotechnology Bulletin, 2022, 38(6): 129-135.

图/表 7

表1 qRT-PCR所用引物序列

Table 1 Primer sequences used in qRT-PCR

引物名称 Primer name	引物序列Primer sequence（5'-3'）
AtSCL4-F	ATGGCTTACATGTGCACTGATAG
AtSCL4-R	TTATCGCCAGGAAGAAAGAGTG
ATMYB61-F	GATTGCGTCAAGGCTTCCG
ATMYB61-R	CGCAGAAGAGGAACTAGGAG
P5CS1-F	GTCAAGTCTATGCTTGATTTG
P5CS1-R	GATTTGTCGCCGAATGTAATC
BADH-F	GCTGACCTAGCTGAAGGCTTG
BADH-R	CACCTCGCGGCAAATATCAGC
SOD1-F	GTCCACATTTCAACCCCGATG
SOD1-R	GAGACCAATGATGCCGCAAGC
PER4-F	GAAGGTTGGTCGAAGAGATTC
PER4-R	CGTATCTCCACCGTTGACCGG
Act7-F	ATGTTCACCACTACCGCAG
Act7-R	ACCTCAGGACAACGGAATCTC
Tub2-F	GTCTCCAAGGGTTCCAGGTT
Tub2-R	GACAGAGTAGCGTTGTAAGGC

图1 甘露醇处理下AtSCL4基因在不同时间段的表达分析 *：在P<0.05水平差异显著。下同

Fig. 1 Expression profiles of AtSCL4 at different times in response to mannitol stress *：Indicates difference significant at P< 0.05. The same below

图2 AtSCL4在突变体中的表达量分析 KO1-KO8为不同突变体株系

Fig. 2 Expression analysis of AtSCL4 in mutants KO1-KO8 are different mutant lines

图3 渗透胁迫下AtSCL4调控苗的萌发和生长分析 A：平板培养基观察萌发率情况;B：发芽率统计;C：根长观察;D：根长和鲜重统计

Fig. 3 Analysis of seedling germination and growth regulated by AtSCL4 under osmotic stress A：Observation of the germination phenotypes on plate medium. B：Statistics of germination. C：Observation of root length. D：Statistics of root length and fresh weight

图4 渗透胁迫下AtSCL4调控苗的气孔开放度分析 A：叶片失水率测定;B：气孔观察;C：气孔开放度统计;D：气孔调控相关基因AtMYB61表达量分析

Fig. 4 Stomatal aperture assay of seedlings regulated by AtSCL4 under osmotic stress A：Determination of water loss rate. B：Observation of stomatal aperture.C：Determination of stomatal aperture. D：Expression of AtMYB61 related to stomatal aperture regulation

图5 渗透胁迫下AtSCL4调控苗渗透物质合成分析 A：脯氨酸含量;B：甜菜碱含量;C：脯氨酸合成相关基因P5CS1表达量分析;D：甜菜碱合成相关基因BADH表达量分析

Fig. 5 Synthesis analysis of osmolyte in seedlings mediated by AtSCL4 under osmotic stress A：Proline content. B：Betaine content. C：Expressions of of P5CS1 related to proline synthesis. D：Expressions of BADH related to betaine synthesis

图6 渗透胁迫下AtSCL4调控苗活性氧清除能力分析 A：DAB染色观察H2O2含量;B：NBT染色观察O2-·含量;C：SOD酶活;D：POD酶活;E：SOD酶合成相关基因AtSOD1表达量分析;F：POD酶合成相关基因PER4表达量分析

Fig. 6 ROS scavenging capacity assay of seedlings regul-ated by AtSCL4 under osmotic stress A：Observation of H2O2 content by DAB staining. B：Observation of O2-· content by NBT staining. C：SOD activities. D：POD activities. E：Expression of AtSOD1.F：Expression of PER4

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