【作者】 李睿；

【导师】 李晶；

【作者基本信息】 东北农业大学 ， 植物学， 2021， 博士

【摘要】 长期以来,植物体内特殊（次生）代谢途径一直被认为是以初生代谢产物为前体来产生具有生物活性的终产物的单向途径。然而当遭遇营养胁迫时,在植物组织中可以观察到这类“终”产物的内源性降解。因此,次生代谢产物尤其是富含氮和（或）硫的化合物在受到环境胁迫时能否重新回到初生代谢中以重新获得利用,这一问题受到广泛关注。在本研究中,主要对在十字花目植物中广泛存在的次生代谢产物—芥子油苷（glucosinolates）展开研究。芥子油苷通常由β-D-硫葡萄糖基、硫化肟基团和来源于不同氨基酸的侧链R基组成。由于在芥子油苷中至少包含2个硫原子,因此是一类富硫的植物代谢物。当植物生长在硫缺乏条件下,体内芥子油苷含量会大幅减少。在之前的研究中认为,芥子油苷的减少是由于黑芥子酶水解其结构中的硫代葡萄糖苷键,使芥子油苷发生降解,然后将结构中的硫原子释放出来重新形成含硫的初生代谢产物,以弥补植物生长过程中对硫的需求。然而在这一过程中哪种黑芥子酶发挥作用及芥子油苷中硫原子重新回到初生代谢产物的途径都尚不明确。本研究以十字花科植物拟南芥（Arabidopsis thaliana）为研究对象,对其在硫缺乏条件生长时,芥子油苷降解后硫的再分配过程进行探究,主要实验结果如下:（1）通过对已发表的转录组数据进行分析,发掘在硫缺乏条件下,β-葡萄糖苷酶（β-glucosidase）BGLU28和BGLU30可能行使黑芥子酶的功能。为了验证前人的转录组数据是否可信,利用实时荧光定量PCR方法对拟南芥BGLU家族全部基因进行转录水平分析,实验结果表明,在BGLU1-BGLU47基因的表达变化中,只有BGLU28和BGLU30的表达水平显著升高。（2）在通过对在不同硫浓度条件下生长的bglu28、bglu30和bglu28 bglu30双缺失突变体中芥子油苷含量变化进行分析发现:与野生型（Col-0）、bglu28和bglu30单突变体相比,在bglu28 bglu30双缺失突变体中芥子油苷含量明显高于Col-0、bglu28和bglu30单缺失突变体中的含量,表明在bglu28 bglu30双缺失突变体中,芥子油苷的降解受到明显抑制,此结果说明在缺硫条件下BGLU28和BGLU30参与芥子油苷的降解过程。（3）通过体外酶活实验,以多种芥子油苷为底物,进一步验证BGLU28和BGLU30的黑芥子酶活性。（4）通过在培养液中添加带有氘标记的芥子油苷确定芥子油苷降解后的代谢途径。芥子油苷经BGLU28和BGLU30黑芥子酶降解后形成异硫氰酸盐（isothiocyanates,ITCs）,ITCs与谷胱甘肽（glutathine,GSH）结合形成共轭物,在这一过程PAD2参与GSH的合成。随后逐步从ITC-GSH中裂解甘氨酸（glycine,Gly）和γ-谷氨酸（γ-glutamine,γ-Glu）残基,形成ITC-半胱氨酸（Cysteine,Cys）共轭物,最后环化形成ITC-NH₂和萝卜氨酸（raphanusamic acid,RA）,而后RA经OXP1被降解进一步生成Cys。（5）通过在培养液中添加带有34S标记的芥子油苷对代谢过程中的中间体进行进一步的验证,并进一步明确硫原子的去向。（6）通过对微阵列数据分析,获得可能参与代谢途径的候选基因,并通过对候选基因突变体幼苗的培养液中添加芥子油苷,并对中间产物的含量变化进行分析以确定代谢途径中的功能基因。根据本实验中的发现,证实了初生代谢产物和次生代谢产物之间的相互作用,加深了我们对植物适应环境的代谢机制的理解。更多还原

【Abstract】 The specialized（secondary）metabolic pathway in plants has long been considered as one-way routes to produce bioactive end products with primary metabo lites as precursors.However,endogenous degradation of these"end products"can be observed in plant tissues under nutritional stress.Therefore,there is a matter of general interest whether the secondary metabolites,especially the compounds rich in nitrogen-and/or sulfur-atoms,can be reintegrated into the primary metabolism for reuse under environmental stress.In the study,glucosinolates（GLS）,the secondary metabolites widely existing in the order Brassicales,are usually composed ofβ-D-thioglucosyl,sulfoxime groups and side chain R groups which are derived from different amino acids.GLSs are sulfur rich plant metabolites containing at least two sulfur atoms.When plants suffer sulfur deficiency conditions,the content of GLSs in vivo will be greatly reduced.Previous studies suggested that the decrease of GLSs was due to the hydrolysis by myrosinase（s）,and then released sulfur atoms from the structure to form sulfur-containing primary metabolites to compensate the sulfur demand in plant growth.However,it is not clear which myrosinase plays the important role in this process and how sulfur atoms in GLSs reintegrate to primary metabolites.The model plant,Arabidopsis thaliana（hereafter Arabidopsis）was used in our study.The main results are as follows:（1）Based on the analysis of published transcriptome data,BGLU28 and BGLU30 genes may perform the function of myrosinases under sulfur deficiency condition.In order to veri fy the hypothesis,real-time quantitative PCR was used to analyze the transcriptional level of all BGLU family genes in Arabidopsis.The results showed that only the expression of BGLU28 and BGLU30was significantly increased in all BGLU 47 genes.（2）The GLS contents of bglu28,bglu30 and bglu28 bglu30 double knockout mutant grown under different sulfur contents were analyzed.The content of GLSs in bglu28 bglu30 mutant was significantly higher than that in Col-0,bglu28 and bglu30 single mutants,which indicated that the degradation of GLSs in bglu28 bglu30 mutant was significantly inhibited,our experiments showed that BGLU28 and BGLU30 were involved in the degradation of GLSs under sulfur deficiency.（3）Enzyme activity in vitro using various glucosinolates as substrates were determined to verify that at least BGLU28 has the myrosinase activity.（4）The metabolic pathway of glucosinolates was determined by adding deuterium labeled（d ₅）glucosinolates to the culture medium.Glucosinolates were degraded b y myrosinases BGLU28 and BGLU30 to form isothiocyanates（ITCs）and ITCs combine with glutathione（GSH）to form conjugates.In this process,PAD2 were involved in the biosynthesis of GSH.Then,glycine（Gly）andγ-glutamine（γ-Glu）residues were cleaved from ITC-GSH to form ITC-cysteine（Cys）conjugate,and finally cyclized to form ITC-NH₂ and Raphanus amic acid（RA）,which were further degraded by OXP1 to form Cys.（5）By adding ³⁴S labeled glucosinolates in the culture medium,the intermediates in the meta bolic process were further verified,and the fate of sulfur atoms was further determined.（6）Based on the analysis of microarray data,candidate genes that may be involved in the metabolic pathway were obtained,and the functional genes in the metabolic p athway were identified by analyzing the content changes of intermediate products through adding glucosinolates to the culture medium growing candidate mutants.Overall,our findings demonstrate the bidirectional interaction between primary and specialized metabolism,which enhances our understanding of the underlying metabolic mechanisms via which plants adapt to their environments.更多还原