Construction of a Yeast Two-Hybrid Library and Screening Analysis of CjCBF1 Interacting Proteins in Camellia japonica(Naidong)

doi:10.7668/hbnxb.20195482

Abstract

Abstract:

Low temperature is the main factor affecting the distribution of Camellia in Northern China.In order to further expand the distribution range of Camellia and enhance the diversity of garden plants in Northern China.The yeast library was constructed by Gateway recombination technology using the leaves of C.japonica(Naidong)Daochengchunzao as materials.The total number of clones in the primary library was 1.44×10⁷ cfu,the total number of clones in the secondary library was 1.12×10⁷ cfu,the positive rate of recombination was 100%,the titer of yeast library was 1.00×10⁸ cfu/mL,and the average insert fragment of yeast clone was more than 1 000 bp,which met the standard of library construction.The bait vector pGBKT7-CjCBF1 was constructed by double enzyme digestion and homologous recombination,which was non-toxicity and self-activation activity in yeast cells.The library screening was conducted by plasmid cotransfer method,and 46 candidate proteins interacting with CjCBF1 were obtained,whose functions involved plant growth and development,flowering and fruiting,and response to stress,etc.CjRAV1 was selected as a candidate protein.Primers were designed according to the transcriptome database of C.japonica(Naidong)and Camellia sinensis genome database.The CjRAV1 gene was cloned and the full length of the gene CDS was 1 023 bp.Bioinformatics results showed that the gene encoded 341 amino acids with a relative molecularity of 37.99 ku,a protein isoelectric point of 9.10,an instability index of 34.74 and a lipid index of 76.60.The amino acid sequence alignment and phylogenetic tree construction of the species with close homology to CjRAV1 were carried out.It was found that it was closely related to Camellia lanceoleosa,Diospyros lotus and Actinidia chinensis.In order to reduce the false positive probability of the library screening,the pGADT7-CjRAV1 vector was constructed and verified with pGBKT7-CjCBF1 by point-to-point.It was confirmed that there was an interaction between the two proteins,which laid a foundation for further study on the molecular mechanism of low temperature response of C.japonica(Naidong).

Key words: Camellia japonica(Naidong), Yeast two-hybrid, CjCBF1, Low temperature stress, Protein interaction

CLC Number:

S685.14

HOU Maoyang, YANG Jinlin, CUI Can, CHEN Bo, LI Wei, YIN Hengfu, SUN Yingkun. Construction of a Yeast Two-Hybrid Library and Screening Analysis of CjCBF1 Interacting Proteins in Camellia japonica(Naidong)[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 60-67. doi: 10.7668/hbnxb.20195482.

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Figures/Tables 8

Tab.1 Primers used in this study

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')
pGBKT7-CjCBF1-F	GACCTG catatgCATATGGATTCGAGCAAGAAAAAT
pGBKT7-CjCBF1-R	CTGCAG gtcgacGTCGACTTAAATTGAGAAGCTCCA
pGADT7-F	TAATACGACTCACTATAGGGC
pGADT7-R	GTGAACTTGCGGGGTTTTTC
CjRAV1-F	ATGGATGGTAGTTGCATAGATGAGAG
CjRAV1-R	TTACAAAGCTCCAATTACCCTTACTT
pGADT7-CjRAV1-F	GCCAGT gaattcATGGATGGTAGTTGCATAGATGAGAG
pGADT7-CjRAV1-R	CTCGAT ggatccTCCTCTTCCCCCCACAATTAC

Fig.1 Construction and identification of C.japonica(Naidong)yeast library A.Identification of C.japonica(Naidong)primary library capacity;B.Identification of insertion fragment size and recombination rate in C.japonica(Naidong)primary library;C.Identification of C.japonica(Naidong)secondary library capacity;D.Identification of insertion fragment size and recombination rate of C.japonica(Naidong)secondary library;E.C.japonica(Naidong)yeast library titer identification;F.C.japonica(Naidong)yeast clone identification; M. DL2000 DNA Marker; 1—24.PCR products.

Fig.2 Toxicity and self-activation identification of pGBKT7-CjCBF1 A.Negative control;B.Positive control;C.Growth in DDO/X medium;D.Growth in TDO/X medium;E.Growth in QDO/X/A medium.

Fig.3 Highly rigorous screening of CjCBF1 interacting proteins

Tab.2 Yeast two-hybrid library screening for possible interacting proteins of CjCBF1(Part)

编号 Number	NCBI登录号 Accession	功能描述 Description
1	XM_028202723	进化氧促进蛋白
2	XM_028246211	转录因子VRN1
3	XM_028267934	光系统Ⅱ稳定性因子HCF136
4	XM_028234794	含U-box结构域蛋白13
5	XM_028236090	2-氧戊二酸依赖双加氧酶AOP1
6	XM_028267884	BTB/POZ结构域蛋白NPY2
7	XM_028198090	SRC2蛋白
8	XM_028232116	转录因子RAV1
9	XM_028262014	转录因子WRKY35
10	XM_028262837	茎特异性蛋白TSJT1
11	XM_028201251	果胶甲酯酶CGR3
12	XM_028205965	异丁香酚合酶1
13	XM_028237531	转录因子ILR3
14	XM_028252767	泛素蛋白连接酶UPL1
15	XM_028271234	转录因子WRKY20
16	XM_028196337	锌指蛋白COL4
17	XM_057610170	丝氨酸/苏氨酸蛋白激酶TOR
18	XM_028266474	糖脂转运蛋白1
19	XM_028266516	BURP结构域蛋白RD22
20	XM_028244846	低温诱导半胱氨酸蛋白酶

Fig.4 Sequence amplification of CjRAV1 fragment 1—4.Four replicates.

Fig.5 Bioinformatics analysis of CjRAV1 A.Conserved domain analysis of CjRAV1;B.Secondary structure prediction of CjRAV1;C.Comparison of amino acid sequence of CjRAV1 with other species;D.Phylogenetic tree of CjRAV1 and other species.

Fig.6 Point-to-point verification of the interaction between CjCBF1 and CjRAV1

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