草莓生长素合成关键酶FveTAA1保守氨基酸位点T111的生物学功能研究

doi:10.16420/j.issn.0513-353x.2020-0688

摘要/Abstract

摘要：

结合生物信息学、生物化学和遗传学等方法对森林草莓（Fragaria vesca）生长素合成通路色氨酸转氨酶TAA1（Tryptophan aminotransferase of Arabidopsis 1）进化过程中保守的氨基酸位点T111的生物学功能进行了研究。通过基因表达分析发现,草莓FveTAA1在果实膨大及果实成熟阶段均上调表达,暗示其在果实发育过程中可能具有重要的生物学功能。通过生物信息分析发现,FveTAA1蛋白的第111位苏氨酸（T111）在进化过程中高度保守,推测该位点可能具有重要的生物学功能。通过生物化学方法检测发现,该位点突变成模拟非磷酸化状态的丙氨酸T111A及模拟磷酸化状态的天冬氨酸T111D,其蛋白均丧失色氨酸转氨酶活性,因此推断该位点对其酶活性至关重要。通过遗传学方法发现,FveTAA1^WT能够回补拟南芥TAA1/TAR突变体的遗传表型,而FveTAA1^T111A与 FveTAA1^T111D均无法回补,进一步证明了FveTAA1 T111氨基酸位点在体内重要的生物学功能。本研究的结果不仅证明了生长素合成途径在进化上存在保守的调控机制,也为研究生长素合成在草莓果实发育和成熟过程中的调控机制提供了理论基础。

关键词: 草莓, 生长素, TAA1, 转氨酶活性, 果实生长发育

Abstract:

In this study,combined with bioinformatics,biochemistry and genetics methods,the biological function of the evolutionary conserved site T111 of tryptophan transaminase TAA1（Tryptophan aminotransferase of Arabidopsis 1）in strawberry（Fragaria vesca）were studied. Though gene expression analysis,it was found that strawberry FveTAA1 was up-regulated during fruit expansion and fruit ripening stages,suggesting that it may have important biological function during fruit development. Based on bioinformatics analysis,it was found that the 111th threonine（T111）of FveTAA1 protein,which is highly evolutional conserved,may have an important biological function. Through biochemical detection,it was found when T111 was mutated to alanine T111A,a simulated non-phosphorylation version,or aspartic acid T111D,a simulated phosphorylation version,FveTAA1 will lose its tryptophan aminotransferase activity,which suggests that this site maybe significant for its function. Using genetic methods,it was found that FveTAA1 ^WT could complement the genetic phenotype of TAA1/TAR mutant in Arabidopsis whereas FveTAA1 ^{T111A / T111D} could not,which further proved that FveTAA1 T111 site plays a key role for its biological function. Overall,these results not only support the evolutionary conservative regulation mechanism of auxin synthesis,but also provide a theoretical basis for the study of the regulation mechanism of auxin synthesis in fruit development and ripening process in strawberry.

Key words: strawberry, auxin, TAA1, transaminase activity, fruit growth and development

中图分类号:

S668.4

杨丽媛, 王倩, 王许会, 徐通达, 马军. 草莓生长素合成关键酶FveTAA1保守氨基酸位点T111的生物学功能研究[J]. 园艺学报, 2021, 48(9): 1695-1705.

YANG Liyuan, WANG Qian, WANG Xuhui, XU Tongda, MA Jun. The Important Biological Function of T111,an Evolutional Conserved Amino Acid in FveTAA1,a Key Enzyme in Strawberry Auxin Biosynthesis[J]. Acta Horticulturae Sinica, 2021, 48(9): 1695-1705.

图/表 9

表1 本试验中所用的引物

Table 1 Primers used in the study

引物用途 Usage	引物名称 Primer name	引物序列（5′-3′） Sequence
纯化蛋白 Purified protein	pET-FveTAA1-F	AAGAAGGAGATATACCATGGGATCCATGTGCGGCACTGATC
纯化蛋白 Purified protein	pET-FveTAA1-R	CAGTGATGATGATGATGATGAGGCCTGTGCCCATTACTGATGGC
T111点突成T111A Mutating T111 to T111A	pET-FveTAA1T111A-F	ACAGGCTCCGCGCAGCTCTA
T111点突成T111A Mutating T111 to T111A	pET-FveTAA1T111A-R	TAGAGCTGCGCGGAGCCTGT
T111点突成T111D Mutating T111 to T111D	pET-FveTAA1T111D-F	ACAGGCTCCGAGCAGCTCTA
T111点突成T111D Mutating T111 to T111D	pET-FveTAA1T111D-R	TAGAGCTGCTCGGAGCCTGT
基因表达量检测 Detecting gene expression	qPCR-ACTIN-F	GCCAGAAAGATGCTTATGTCGGTG
	qPCR-ACTIN-R	TGGGGCAACACGAAGCTCAT
	qPCR-FveTAA1-F	GTTCCCGTATTGGATGGGCT
	qPCR-FveTAA1-R	CTCGGTGCTGAGAGTCCTTG
蛋白功能验证 Protein function verification	pDONR-FveTAA1-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTATGTGCGGCACTGATCATAACC
蛋白功能验证 Protein function verification	pDONR-FveTAA1-R	GGGGACCACTTTGTACAAGAAAGCTGGGTGTGCCCATTACTGATGGCTTT

图1 草莓不同发育时期果实中FveTAA1的相对表达量 S1：开花 Flowering;S2：2 ~ 4 d;S3：5 ~ 7 d;S4：8 ~ 10 d;S5：11 ~ 13 d;S6：14 ~ 16 d;S7：17 ~ 19 d;RS1：20 ~ 22 d;RS2：23 ~ 25 d;RS3：26 ~ 28 d;RS4：29 ~ 31 d;RS5：32 ~ 34 d。

Fig. 1 Relative expression of FveTAA1 in different fruit development stages

图2 FveTAA1（紫色）蛋白与AtTAA1（黄色）蛋白的三级结构分析绿色为PLP辅酶;红色为T111位点。

Fig. 2 The third-order structure analysis of FveTAA1 protein（purple）and AtTAA1 protein（yellow） PLP coenzyme was green;T111 site was red.

图3 通过邻接法对FveTAA1蛋白及其他物种同源蛋白的进化分析

Fig. 3 Analysis of the evolution of FveTAA1 and other homologous proteins by adjacency method

图4 17个物种TAA1同源蛋白的序列比对分析橙色显示保守的修饰位点,对应草莓FveTAA1的T111位点。

Fig. 4 The sequence alignment of TAA1 homologous proteins of 17 species Orange shows the conservative modification site,corresponding to T111 site of FveTAA1 in strawberry.

图5 凝胶内酶反应染色法,检测FveTAA1各突变蛋白的色氨酸转移酶（TRP）活性

Fig. 5 In-gel tryptophan aminotransferase（Trp-AT）activity assay was used to detect the tryptophan transferase （TRP）activity of FveTAA1 and mutant proteins

图6 Salkowski试剂测量IPA产量方法检测FveTAA1各突变蛋白色氨酸转氨酶催化活性

Fig. 6 Using salkowski reagent to measure IPA production method to detect the transaminase activity of FveTAA1 mutant protein

图7 草莓FveTAA1的T111位点调控拟南芥植株高度

Fig. 7 T111 locus of FveTAA1 of strawberry regulates plant height in Arabidopsis

图8 草莓FveTAA1的T111位点调控拟南芥花器官发育

Fig. 8 T111 locus of FveTAA1 in strawberry regulates flower organ development in Arabidopsis

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