[1]杨小林,杨卫华,蔡旋,等.水稻CYP81A6蛋白原核表达与活性检测[J].江苏农业学报,2025,(11):2081-2087.[doi:doi:10.3969/j.issn.1000-4440.2025.11.001]
　YANG Xiaolin,YANG Weihua,CAI Xuan,et al.Prokaryotic expression and activity detection of CYP81A6 protein in rice[J].,2025,(11):2081-2087.[doi:doi:10.3969/j.issn.1000-4440.2025.11.001]
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水稻CYP81A6蛋白原核表达与活性检测()

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2025年11期
页码:
2081-2087
栏目:
遗传育种·生理生化
出版日期:
2025-11-30

文章信息/Info

Title:
Prokaryotic expression and activity detection of CYP81A6 protein in rice
作者:
杨小林1; 2; 杨卫华3; 蔡旋1; 吕亮1; 刘新琼4; 张佑宏5
(1.湖北省农业科学院植保土肥研究所,湖北武汉430064；2.湖北洪山实验室,湖北武汉430070；3.湖北省浠水县农业农村局,湖北浠水438200；4.中南民族大学生命科学学院,湖北武汉430074；5.湖北省生物农药工程研究中心,湖北武汉430064)
Author(s):
YANG Xiaolin1; 2; YANG Weihua3; CAI Xuan1; LYU Liang1; LIU Xinqiong4; ZHANG Youhong5
(1.Institute of Plant Protection and Soil Science, Hubei Academy of Agricultural Sciences, Wuhan 430064, China;2.Hubei Hongshan Laboratory, Wuhan 430070, China;3.Bureau of Agriculture and Rural Affairs of Xishui County, Hubei Province, Xishui 438200, China;4.College of Life Sciences, South-Central Minzu University, Wuhan 430074, China;5.Hubei Biopesticide Engineering Research Center, Wuhan 430064, China)
关键词:
水稻; CYP81A6; 细胞色素P450; 原核表达; 蛋白纯化; 稻瘟病
Keywords:
rice; CYP81A6; cytochrome P450; prokaryotic expression; protein purification; rice blast
分类号:
S511.01
DOI:
doi:10.3969/j.issn.1000-4440.2025.11.001
文献标志码:
A
摘要:
在植物体中,植物细胞色素P450基因家族广泛参与了多种生理活动。笔者所在团队前期通过转录组测序发现,CYP81A6基因的相对表达量受到水杨酸、稻瘟病病菌的诱导而上调。尽管目前已知CYP81A6基因具备抗除草剂特性,但是至今还没有关于该基因参与植物产生病害反应的报道。因此,本研究以日本晴水稻叶片作为试验材料,用PCR技术对水稻CYP81A6基因片段进行扩增操作。随后,将成功扩增的基因片段克隆到pET32a原核表达载体上。在上述试验基础上,对该基因进行蛋白质表达与纯化处理。为了获得更多的可溶性重组蛋白,开展CYP81A6蛋白诱导条件的筛选试验。结果表明,当温度设定为16 ℃,并用07 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导20 h时,该蛋白质的表达量达到最佳水平。同时,用50 mmol/L咪唑洗脱,能够成功地将CYP81A6蛋白洗脱纯化出来。进一步利用纯化得到的CYP81A6蛋白与烟酰胺腺嘌呤二核苷酸磷酸(NADPH)进行酶促反应,并通过专业的检测方法测得CYP81A6的酶活性为1518 U/mL。综上,本研究通过对水稻CYP81A6基因的克隆、表达、纯化及酶活性的测定等,为后续深入探究CYP81A6基因的功能和作用机制奠定了基础。
Abstract:
In plants, the plant cytochrome P450 gene family is involved in a variety of physiological activities. Our previous transcriptome sequencing analysis revealed that the relative expression level of the CYP81A6 gene was up-regulated by the induction with salicylic acid and rice blast fungus. Although it is known that the CYP81A6 gene has herbicide resistance, there have been no reports so far on the involvement of this gene in plant disease resistance responses. Therefore, in this study, rice Nipponbare leaves were used as experimental materials. The CYP81A6 gene fragment of rice was amplified by PCR technology. Subsequently, the successfully amplified gene fragment was cloned into the prokaryotic expression vector pET32a. Based on the above experiments, the protein expression and purification were performed for this gene. In order to obtain more soluble recombinant protein, screening experiments for the induction conditions of CYP81A6 protein were carried out. The results showed that the optimal protein expression was achieved under induction conditions of 0.7 mmol/L isopropyl-β-D-thiogalactoside (IPTG) at 16 ℃ for 20 h. Meanwhile, the CYP81A6 protein could be successfully eluted and purified under the action of 50 mmol/L imidazole. Further, the purified CYP81A6 protein was used for enzymatic reaction with nicotinamide adenine dinucleotide phosphate (NADPH), and the enzyme activity of CYP81A6 was measured to be 1.518 U/mL by professional detection methods. In summary, this study has laid a foundation for in-depth exploration of the function and mechanism of the CYP81A6 gene in rice through cloning, expression, purification and enzyme activity determination.

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备注/Memo

备注/Memo:
收稿日期：2025-01-06基金项目：国家自然科学基金面上项目(31370306)作者简介：杨小林(1971-),女,湖北黄梅人,博士,研究员,主要从事水稻真菌病害研究。(Tel)027-88430557；(E-mail)xiaolinyangxly@163.com通讯作者：张佑宏,(Tel)027-88430557；(E-mail) 2430204775@qq.com
更新日期/Last Update: 2025-12-18