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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (6): 1628-1634.doi: 10.3724/SP.J.1006.2024.32041

• RESEARCH NOTES • Previous Articles    

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 Online:2024-06-12 Published: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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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

Table 1

Primer names and sequences"

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

Fig. 1

PCR product of OsCYP22 (A) and colony PCR of pGBKT7-OsCYP22 bait vector (B) M: 2000 bp DNA marker; 1-2: positive colonies; 3-12: negative colonies."

Fig. 2

Autoactivation and toxicity detection of pGBKT7-OsCYP22 bait protein"

Fig. 3

Growth diagram of yeast transformants A: colonies co-transformed Y2H gold with pGBKT7-OsCYP22 and pGADT7-cDNA library growing on SD/-Ade/-His/-Leu/-Trp; B: colonies co-transformed Y2H gold with pGBKT7-OsCYP22 and pGADT7-cDNA library growing on SD/-Ade/-His/-Leu/-Trp (X-α-gal); +: positive control; -: negative control."

Table 2

Candidate OsCYP22-interacting proteins screened by Y2H"

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

Fig. 4

PCR validation of positive colonies of candidate interacting gene prey vectors A: colony PCR of pGADT7-OsCSN5; B: colony PCR of pGADT7-OsRUB1, M: 2000 bp DNA marker."

Fig. 5

Yeast two-hybrid confirmed the interaction between OsCYP22 and OsCSN5 or OsRUB1"

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