耐冬山茶酵母双杂交文库构建及CjCBF1互作蛋白筛选与分析

doi:10.7668/hbnxb.20195482

摘要/Abstract

摘要：

低温是影响山茶在我国北方分布的主要因素,为进一步扩大山茶的分布范围,提升北方园林植物的多样性,以山茶科山茶属植物耐冬山茶岛城春早的叶片为材料,通过Gateway重组技术构建酵母文库,获得的初级文库总克隆数为1.44×10⁷ cfu,次级文库总克隆数为1.12×10⁷ cfu,重组阳性率为100%,酵母文库滴度为1.00×10⁸ cfu/mL,酵母克隆平均插入片段>1 000 bp,符合建库标准。利用双酶切同源重组方式构建诱饵载体pGBKT7-CjCBF1,其在酵母细胞中无毒性及自激活活性。使用质粒共转法进行筛库,获得46个与CjCBF1相互作用的候选蛋白,功能涉及植物生长发育、开花结实及响应胁迫等多个方面,选取CjRAV1作为候选蛋白。参考耐冬山茶转录组数据库和茶树基因组数据库设计引物,克隆得到CjRAV1基因,该基因CDS全长为1 023 bp。生物信息学分析结果显示,该基因编码341个氨基酸,相对分子质量为37.99 ku,蛋白质等电点9.10,不稳定指数34.74,脂肪指数76.60。选取与CjRAV1同源性较近的物种进行氨基酸序列比对并构建系统发育树,发现其与狭叶油茶、君迁子以及中华猕猴桃亲缘关系较近。为了降低筛库的假阳性概率,构建pGADT7-CjRAV1载体,与pGBKT7-CjCBF1进行点对点验证,证实二者之间存在互作关系,为后续深入研究耐冬山茶低温应答分子机制奠定基础。

关键词: 耐冬山茶, 酵母双杂交, CjCBF1, 低温胁迫, 蛋白互作

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

中图分类号:

S685.14

侯茂洋, 杨金霖, 崔灿, 陈博, 李伟, 殷恒福, 孙迎坤. 耐冬山茶酵母双杂交文库构建及CjCBF1互作蛋白筛选与分析[J]. 华北农学报, 2025, 40(3): 60-67. doi: 10.7668/hbnxb.20195482.

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.

http://www.hbnxb.net/CN/Y2025/V40/I3/60

图/表 8

表1 本研究所用引物

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

图1 耐冬山茶酵母文库的构建及鉴定 A.耐冬山茶初级文库库容量鉴定;B.耐冬山茶初级文库插入片段及重组率鉴定;C.耐冬山茶次级文库容量鉴定;D.耐冬山茶次级文库插入片段及重组率鉴定;E.耐冬山茶酵母文库滴度鉴定;F.耐冬山茶酵母克隆鉴定;M.DL2000 DNA Marker;1—24.PCR产物。

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.

图2 pGBKT7-CjCBF1的毒性及自激活鉴定 A.阴性对照;B.阳性对照;C.DDO/X培养基生长情况;D.TDO/X培养基生长情况;E.QDO/X/A培养基生长情况。

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.

图3 CjCBF1互作蛋白高严谨度筛选

Fig.3 Highly rigorous screening of CjCBF1 interacting proteins

表2 酵母双杂交文库筛选出的可能与CjCBF1互作的蛋白(部分)

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

图4 CjRAV1序列扩增 1—4.4个重复。

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

图5 CjRAV1生物信息学分析 A.CjRAV1保守结构域分析;B.CjRAV1二级结构预测;C.CjRAV1与其他物种RAV氨基酸序列对比;D.CjRAV1与其他物种RAV系统进化树。

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.

图6 CjCBF1与CjRAV1互作点对点验证

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

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