华北农学报 ›› 2025, Vol. 40 ›› Issue (3): 60-67. doi: 10.7668/hbnxb.20195482

所属专题: 生物技术

• 作物遗传育种·种质资源·生物技术 • 上一篇    下一篇

耐冬山茶酵母双杂交文库构建及CjCBF1互作蛋白筛选与分析

侯茂洋1,2, 杨金霖1,2, 崔灿1,2, 陈博1,2, 李伟1,2, 殷恒福3, , 孙迎坤1,2,   

  1. 1 青岛农业大学 园林与林学院,山东 青岛 266109
    2 山东省耐盐碱草木种质创新重点实验室,山东 青岛 266109
    3 中国林业科学研究院 亚热带林业研究所,浙江 杭州 311400
  • 收稿日期:2024-11-20 出版日期:2025-06-28
  • 通讯作者:
    孙迎坤(1977—),女,山东潍坊人,教授,博士,主要从事山茶抗逆性分子机制研究。
    殷恒福(1981—),男,江苏扬州人,研究员,博士,主要从事山茶栽培性状的形成研究。
  • 作者简介:

    侯茂洋(2000—),男,山东临沂人,在读硕士,主要从事山茶抗寒性分子机制研究。

  • 基金资助:
    山东省自然科学基金面上项目(ZR2023MC156); 国家自然科学基金面上项目(32271839); 山东省青年创新团队项目(018-1622001)

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

HOU Maoyang1,2, YANG Jinlin1,2, CUI Can1,2, CHEN Bo1,2, LI Wei1,2, YIN Hengfu3, , SUN Yingkun1,2,   

  1. 1 College of Landscape Architecture and Forestry,Qingdao Agricultural University,Qingdao 266109,China
    2 Shandong Key Laboratory of Saline-alkali Tolerant Grass Germplasm Innovation,Qingdao 266109,China
    3 Research Institute of Subtropical Forestry,Chinese Academy of Forestry,Hangzhou 311400,China
  • Received:2024-11-20 Published:2025-06-28

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

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引用本文

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.