水稻腺苷5'-磷酰硫酸激酶编码基因的克隆及其酶活分析

doi:10.3969/j.issn.1001G7216.2015.06.002

摘要/Abstract

摘要：

硫是植物生长所必需的大量元素之一。植物通过根系吸收土壤中的硫酸盐,经ATP硫酸化酶活化为腺苷5'-磷酰硫酸(adenosine 5'-phosphosulfate,APS)。APS的代谢包括初级代谢和次级代谢,分别是APS还原酶催化APS生成亚硫酸盐;腺苷5'-磷酰硫酸激酶(adenosine 5'-phosphosulfate kinase,APSK)催化APS磷酸化生成3'-磷酸-腺苷5'-磷酰硫酸(3'-phosphoadenosine 5'-phosphosulfate,PAPS),PAPS进一步作为硫酸根供体参与胞内的硫酸化反应。近年来的研究表明,拟南芥APSK在调节硫的初级和次级代谢的过程中具有重要功能,且受氧化胁迫的APSK1在亚基C86和C119间形成二硫键降低其催化活性。水稻的同工酶是否也会受到氧化还原调控尚无相关报道。对APSK序列进行分析,发现水稻APSK1含有与拟南芥APSK1 C86和C69同源的半胱氨酸。本研究对水稻APSK1基因进行了克隆、双突变和酶活分析。OsAPSK1及双突变C36A/C69A经原核表达、纯化,获得了OsAPSK1及双突变蛋白。体外酶活分析表明,在氧化环境中OsAPSK1活性受到抑制,而C36A/C69A双突变蛋白不受氧化环境的影响。推测水稻胞内受到氧化胁迫时会降低OsAPSK1的活性,从而促进还原型谷胱甘肽的合成,提高水稻的抗氧化能力。通过分析PAPS的含量与氧化胁迫之间的相关性及不同OsAPSK亚型的活性调节机制等将有利于进一步阐明APSK在胞内的功能。

关键词: 水稻, 硫同化, 腺苷5', -磷酰硫酸激酶, 氧化还原环境

Abstract:

Sulfur is an essential element for plant growth. After absorbed from soil by plant roots, sulfate is firstly activated by ATP sulfurylase to form adenosine 5'-phosphosulfate (APS). There are primary and secondary pathways for further conversion of APS, namely reduction of APS by APS reductase to form sulfite, and the phosphorylation of APS by APS kinase (APSK) to form 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the sulfate donor for the sulfation reactions. Recent researches demonstrated that APSK from Arabidopsis thaliana has an important role in regulating the primary and secondary metabolism of sulfate, and that the formation of intersubunit disulfide bonds between C86 and C119 under oxidation state would decrease its catalytic efficiency dramatically. It had not been reported that whether the rice isozymes were affected by redox environments or not. Sequence analysis demonstrated that two rice APSK (OsAPSK) sequences contain corresponding cysteines homology to Arabidopsis thaliana involved in disulfide formation. In this study, OsAPSK1 and its C36A/C69A mutant were prokaryotically overexpressed, purified and assayed for activity of APS phosphorylation. Activity depression for OsAPSK1 under oxidative stress and no for its mutant C36A/C69A suggested that OsAPSK1 was regulated as proposed previously, and that depression of APSK activity in the secondary pathway would facilitate the primary pathway for sulfate reduction to enhance the cell redox buffering capacity. Further studies such as relations between PAPS contents and oxidative stress, and regulation of different APSK isoenzymes are being investigated.

Key words: rice, sulfate assimilation, adenosine 5'-phosphosulfate kinase, cysteine, redox environment

王德珍, 陆璐佳, 姜昭君, 饶玉春, 孙梅好. 水稻腺苷5'-磷酰硫酸激酶编码基因的克隆及其酶活分析[J]. 中国水稻科学, 2015, 29(6): 571-577.

De-zhen WANG, Lu-jia LU, Zhao-jun JIANG, Yu-chun RAO, Mei-hao SUN. Gene Cloning and Activity Analysis of Adenosine 5'-Phosphosulfate Kinase in Rice[J]. Chinese Journal OF Rice Science, 2015, 29(6): 571-577.

图/表 7

图1 硫酸盐在植物细胞内的同化过程

Fig. 1. Scheme of sulfate assimilation pathway in plant cells.

表1 OsAPSK1基因克隆及其定点突变所用引物序列

Table 1 Primer sequences for OsAPSK1 cloning and mutation.

引物 Marker	引物序列(5'-3') Sequence
OSAPSK1 -F	GAATTCATGGAGCAGCAGCAGC
OSAPSK1 -R	CTCGAGTTAAGCTTGCAAATAT
APSK1-C36A-F	CAATATACTGTGGCACAATGCGCCAATTGGACAATCTG
APSK1-C36A-R	CAGATTGTCCAATTGGCGCATTGTGCCACAGTATATTG
APSK1-C69A-F	GGGAAAAGCACTCTTGCAGCGGCACTGAATCGGGAG
APSK1-C69A-R	CTCCCGATTCAGTGCCGCTGCAAGAGTGCTTTTCCC

图2 不同物种APSK氨基酸序列比对

Fig. 2. Sequence alignment of APSKs from different species.

图3 OsAPSK1的APS磷酸化活性测定

Fig. 3. Phosphorylation of APS by OsAPSK1.

图4 OsAPSK1突变体C36A/C69A的APS磷酸化活性测定

Fig. 4. Activity assay for phosphorylation of APS by C36A/C69A.

表2 OsAPSK1与突变体C36A/C69A的动力学参数比较

Table 2 Comparison of kinetics parameters for OsAPSK1 and mutant C36A/C69A.

参数 Parameter	K_mAPS /(μmol·L^-1)	K_mATP /(mmol·L^-1)	K_iATP /(mmol·L^-1)	K_iAPS /(μmol·L^-1)	K_cat /(×10³min^-1)	K_cat/K_m /(×10³min^-1·μmol^-1·L)
WT	0.72±0.17	0.75±0.18	0.61±0.13	0.58±0.13	0.30±0.042	0.41
C36A/C69A	0.44±0.04	0.14±0.02	0.22±0.04	0.68±0.15	0.13±0.006	0.30

图5 OsAPSK1的结构

Fig. 5. Structural illustration of OsAPSK1.

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