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作物学报 ›› 2024, Vol. 50 ›› Issue (6): 1628-1634.doi: 10.3724/SP.J.1006.2024.32041

• 研究简报 • 上一篇    

水稻OsCYP22互作蛋白的筛选及验证

张小芳1,2(), 朱琪1,2, 华芸堰1,2, 贾黎惠莹1,2, 邱士优1, 陈宇杰1, 马涛1,*(), 丁沃娜1,*()   

  1. 1宁波大学科学技术学院 / 宁波市农业种质资源挖掘与环境调控重点实验室, 浙江宁波 315300
    2宁波大学海洋学院, 浙江宁波 315211
  • 收稿日期:2023-09-27 接受日期:2024-01-31 出版日期:2024-06-12 网络出版日期:2024-02-21
  • 通讯作者: * 马涛, E-mail: matao08@163.com;丁沃娜, E-mail: dwn@zju.edu.cn
  • 作者简介:E-mail: zhangxiaofang916@163.com
  • 基金资助:
    国家自然科学基金项目(32071981);宁波市自然科学基金项目(202003N4016)

Screening and validation of OsCYP22 interacting proteins in rice

ZHANG Xiao-Fang1,2(), ZHU Qi1,2, HUA Yun-Yan1,2, JIA Li-Hui-Ying1,2, QIU Shi-You1, CHEN Yu-Jie1, MA Tao1,*(), DING Wo-Na1,*()   

  1. 1College of Science and Technology, Ningbo University / Ningbo Key Laboratory of Agricultural Germplasm Resources Mining and Environmental Regulation, Ningbo 315300, Zhejiang, China
    2School of Marine Science, Ningbo University, Ningbo 315211, Zhejiang, China
  • Received:2023-09-27 Accepted:2024-01-31 Published:2024-06-12 Published online:2024-02-21
  • Contact: * E-mail: matao08@163.com;E-mail: dwn@zju.edu.cn
  • Supported by:
    National Natural Science Foundation of China(32071981);Ningbo Natural Science Foundation(202003N4016)

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摘要：

为了探究水稻亲环素家族基因OsCYP22的功能, 构建OsCYP22酵母双杂交诱饵载体, 并利用水稻酵母双杂交cDNA文库筛选与OsCYP22相互作用蛋白。自激活试验结果表明, OsCYP22诱饵载体无自激活活性, 也对酵母细胞无毒性。通过对酵母双杂交文库的筛选, 共得到38个阳性菌落, 结合测序结果和生物信息学的方法筛选到20个可能与OsCYP22相互作用的蛋白。进一步对OsCYP22分别与调控植物根系生长相关基因OsCSN5和OsRUB1的全长互作验证, 结果显示OsCSN5与OsCYP22在酵母中相互作用。这为深入研究OsCYP22的生物学功能提供了理论依据。

关键词: 水稻, OsCYP22, 酵母双杂交, 互作蛋白

Abstract:

In order to understand the function of cyclophilin family OsCYP22 from Oryza sativa L., the yeast two hybrid bait vector of OsCYP22 was constructed and proteins interacting with OsCYP22 were screened by yeast two hybrid. The results showed that noself-activating activity and toxicity of OsCYP22 bait vector to yeast cells were detected., among which 38 positive colonies were obtained by yeast two hybrid screening. Combining sequencing analysis and bioinformatics methods, 20 candidates that may interact with OsCYP22 were screened. Further validation of the full-length interactions between OsCYP22 and OsCSN5 and OsRUB1 related to the regulation of plant root growth was conducted. The results showed that OsCSN5 interacted with OsCYP22 in yeast. This study provides a theoretical basis for further study of the biological function of OsCYP22.

Key words: rice, OsCYP22, Yeast two-hybird, interaction protein

表1

引物名称及序列"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 限制性内切酶
Restriction endonuclease
BD-OsCYP22-Forward GCATATGGCCATGGAGGCCGAATTCATGGCGACGGCGAGC EcoR I
BD-OsCYP22-Reverse GCGGCCGCTGCAGGTCGACGGATCCTGTCTTTGGAACTGTAACGTTC BamH I
AD-OsCSN5-Forward TATGGCCATGGAGGCCAGTGAATTCATGGAGCCCACCTCGT EcoR I
AD-OsCSN5-Reverse TCTGCAGCTCGAGCTCGATGGATCCTCATGCTTCAACCATAGGC BamH I
AD-OsRUB1-Forward TATGGCCATGGAGGCCAGTGAATTCATGCAGATCTTCGTGA EcoR I
AD-OsRUB1-Reverse TCTGCAGCTCGAGCTCGATGGATCCCTAATAACCACCCCTC BamH I

图1

OsCYP22基因扩增产物(A)及pGBKT7-OsCYP22诱饵质粒菌落PCR检测(B) M: 2000 bp DNA marker; 1~2: 阳性克隆, 3~12: 阴性克隆。"

图2

pGBKT7-OsCYP22诱饵载体自激活活性和毒性检测"

图3

酵母转化子生长图 A: pGBKT7-OsCYP22和pGADT7-cDNA文库质粒的Y2H gold在SD/-Ade/-His/-Leu/-Trp培养基生长的克隆; B: pGBKT7-OsCYP22和pGADT7-cDNA文库质粒的Y2H gold在SD/-Ade/-His/-Leu/-Trp (X-α-gal)培养基生长的克隆; +: 阳性对照(pGADT7-largeT+pGBKT7- p53); -: 阴性对照(pGADT7-largeT+pGBKT7-laminc)。"

表2

酵母双杂交筛选的候选OsCYP22互作蛋白"

编号
Number		 基因序列号
DNA sequence number		 编码蛋白
Coding protein		 包含结构域
Domain
1 XM_015770516.2 death-inducer obliterator 1 TFIIS central
2 XM_015771759.2 proliferating cell nuclear antigen Proliferating cell nuclear antigen PCNA
3 XM_015792790.2 dihydroneopterin aldolase 2 Dihydroneopterin aldolase
4 XM_015781704.1 COP9 signalosome complex subunit 5 CSN5 MPN
5 XM_015769167.2 probable splicing factor 3A subunit 1 Ubiquitin-like
6 XM_015756511.2 ubiquitin-NEDD8-like protein RUB1 Ubiquitin-like
7 XM_015789576.2 BTB/POZ domain-containing protein At4g08455 BTB
8 XM_015761445.2 suppressor of mec-8 and unc-52 protein homolog 2 protein RED C-terminal,
9 XM_015762019.2 probable serine/threonine-protein kinase SIS8 PAS
10 XM_015793158.1 peptidyl-prolyl cis-trans isomerase PPIase cyclophilin-type
11 XM_015767492.2 ADP-ribosylation factor GTPase-activating protein AGD12 Arf-GAP
12 XM_015765721.2 AMSH-like ubiquitin thioesterase 2 MPN
13 XM_026025881.1 pyruvate decarboxylase 1 Thiamine pyrophosphate enzyme central
14 XM_015784162.2 chaperone protein dnaJ A7A Saposin B-type
15 XM_015773025.2 probable ADP-ribosylation factor GTPase-activating protein Arf-GAP
16 XM_015784479.2 stromal 70 kD heat shock-related protein
17 XM_052311061.1 mitochondrial outer membrane protein porin 1
18 XM_015769697.2 DExH-box ATP-dependent RNA helicase DExH12
19 XM_015794278.1 tricin synthase 1
20 XM_015766015.2 raffinose synthase

图4

候选互作基因猎物载体阳性菌落PCR验证 A: pGADT7-OsCSN5菌落PCR; B: pGADT7-OsRUB1菌落PCR。"

图5

酵母双杂交验证OsCYP22与OsCSN5和OsRUB1"

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