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林业科学 ›› 2022, Vol. 58 ›› Issue (3): 59-68.doi: 10.11707/j.1001-7488.20220307

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光皮桦BlBLH1基因的克隆、表达及互作蛋白筛选

庄和必,俞子承,林二培,黄华宏*   

  1. 浙江农林大学 亚热带森林培育国家重点实验室 杭州 311300
  • 收稿日期:2021-04-06 出版日期:2022-03-25 发布日期:2022-06-02
  • 通讯作者: 黄华宏
  • 基金资助:
    国家自然科学基金项目(31770641);浙江省重点研发计划(2021C02037)

Cloning, Expression and Interaction Protein Screening of BlBLH1 Gene in Betula luminifera

Hebi Zhuang,Zicheng Yu,Erpei Lin,Huahong Huang*   

  1. The State Key Laboratory of Subtropical Silviculture Zhejiang A&F University Hangzhou 311300
  • Received:2021-04-06 Online:2022-03-25 Published:2022-06-02
  • Contact: Huahong Huang

摘要:

目的: BLH基因家族是广泛存在于植物中的转录因子,其与KNOX等转录因子的互作被认为在植物的次生壁发育中起重要调控作用。本研究拟克隆光皮桦BlBLH1基因,分析其表达模式,并筛选能与其互作的蛋白。方法: 利用Blast等软件在光皮桦基因组序列中鉴定获得BlBLH1序列,克隆验证后对其进行多序列比对、系统进化等生物信息学分析;利用实时荧光定量PCR分析BlBLH1在光皮桦不同组织、器官和应拉木形成中的表达模式;通过酵母双杂交技术筛选BlBLH1互作的蛋白,并采用双分子荧光互补实验(BiFC)进一步验证其与部分蛋白在拟南芥细胞内的互作。结果: BlBLH1基因序列全长为2 128 bp,开放阅读框(ORF)为1 830 bp,编码609个氨基酸,具有SKY、BEL和HD 3个保守结构域。系统进化分析显示,BlBLH1与拟南芥BLH1有最近的同源关系。表达分析显示BlBLH1在雄花序中的表达量最高;在木质化茎段中的表达量次之;在应拉木诱导过程中,BlBLH1在应拉木中的表达量均显著高于对照。通过酵母双杂交筛选获得结构蛋白、酶、转录因子等47个与BlBLH1互作较强的蛋白;BiFC验证结果表明,BlBLH1与BlKNOX4、BlKNOX9在拟南芥原生质体内存在互作关系。而且,三者在茎段和应拉木的表达趋势基本一致。结论: 可从光皮桦中分离获得BlBLH1基因,生物信息学、表达和蛋白互作分析的结果表明BlBLH1参与光皮桦木材形成过程,且存在与KNOX蛋白的互作关系。

关键词: BlBLH1, 次生壁, 酵母双杂交, 双分子荧光互补

Abstract:

Objective: BLH (BEL1-like homeodomain) gene family belongs to a transcription factor widely existing in plant, and its interaction with KNOX and other transcription factors are considered to play an important regulatory role in the development of plant secondary cell wall (SCW). The aim of this study is to clone BlBLH1 gene of Betula luminifera, then analyze its expression pattern and identify proteins that can interact with BlBLH1. Method: The BlBLH1 sequence in the genome sequence of B. luminifera was first identified by blast and other software. After verification by cloning, further bioinformatic analyses, including multiple sequence alignment and phylogenetic analysis, were also carried out. Quantitative Real-time PCR (qRT-PCR) was used to analyze the expression pattern of BlBLH1 in different tissues, organs and tension wood (TW). Yeast two hybrid technology was used to screen the proteins interacting with BlBLH1, and bimolecular fluorescence complementary (BiFC) was used to verify its interactions with some identified proteins in Arabidopsis thaliana cells. Result: The full-length of BlBLH1 was 2 128 bp in length with an open reading frame (ORF) of 1 830 bp. It encoded 609 amino acids harboring three conserved domains of SKY, BEL and HD. Phylogenetic analysis showed that BlBLH1 had the closest homologous relationship with A. thaliana BLH1. Expression analysis showed that the expression level of BlBLH1 in male inflorescence was the highest, followed by that in lignified stem. During the induction of tension wood, the expression level of BlBLH1 in tension wood was significantly higher than that in control. Through yeast two hybrid, 47 proteins, including structural proteins, enzymes and transcription factors, with strong interaction with BlBLH1 were identified, and the interactions of BlBLH1 with BlKNOX4 and BlKNOX9 were also verified by BiFC in A. thaliana protoplasts. Moreover, the expression patterns of these three genes in stems and tension wood were similar. Conclusion: In this study, BlBLH1 gene is cloned from B. luminifera. Based on the results of bioinformatics, expression and protein interaction analysis, it is inferred that BlBLH1 may be involved in the process of wood formation in B. luminifera, and interacted with KNOX proteins.

Key words: BlBLH1, secondary cell wall, yeast two hybrid, bimolecular

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