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    孙芝林, 刘冰, 李晓伟, 田玉珍, 张卿, 曹庆芹. 转录因子CmHAT1调控板栗体细胞胚胎发育的功能研究[J]. 北京林业大学学报. DOI: 10.12171/j.1000-1522.20230215
    引用本文: 孙芝林, 刘冰, 李晓伟, 田玉珍, 张卿, 曹庆芹. 转录因子CmHAT1调控板栗体细胞胚胎发育的功能研究[J]. 北京林业大学学报. DOI: 10.12171/j.1000-1522.20230215
    shu
    Sun Zhilin, Liu Bing, Li Xiaowei, Tian Yuzhen, Zhang Qing, Cao Qingqin. Functional research of transcription factor CmHAT1 regulates the development of somatic embryo in Castanea mollissima[J]. Journal of Beijing Forestry University. DOI: 10.12171/j.1000-1522.20230215
    Citation: Sun Zhilin, Liu Bing, Li Xiaowei, Tian Yuzhen, Zhang Qing, Cao Qingqin. Functional research of transcription factor CmHAT1 regulates the development of somatic embryo in Castanea mollissima[J]. Journal of Beijing Forestry University. DOI: 10.12171/j.1000-1522.20230215
    shu

    转录因子CmHAT1调控板栗体细胞胚胎发育的功能研究

    Functional research of transcription factor CmHAT1 regulates the development of somatic embryo in Castanea mollissima

      摘要
    • 摘要:
      目的 体细胞胚胎发生是板栗有效的离体再生方法。然而，目前板栗体细胞胚胎由于发育慢、再生效率低等问题，制约了板栗体细胞胚胎的再生进程，难以在后续基因功能的验证和新种质的创制过程中发挥作用。本文旨在通过研究HD-ZIPⅡ基因家族成员CmHAT1在板栗体细胞胚胎发育过程中的作用，进一步加速板栗体细胞的发育进程。
      方法 利用生物信息学的方法鉴定了板栗HD-ZIPⅡ基因家族成员，并分析了该家族成员在板栗体细胞胚胎不同发育时期的表达情况，筛选调控体细胞胚胎发育的关键候选基因CmHAT。进一步通过烟草亚细胞定位实验、荧光定量和遗传转化方法，分析关键基因CmHAT的亚细胞定位和基因功能。
      结果 （1）板栗HD-ZIPⅡ基因家族有7个成员，大部分CmHD-ZIP Ⅱ 成员在板栗体细胞胚胎发育时期均有表达，其中CmHAT1在愈伤组织时期表达较低，在球形胚时期表达上调，心形胚时期表达最高，而在子叶形胚时期表达下调，因此筛选出CmHAT1作为调控体细胞胚胎发育的关键候选基因。（2）CmHAT1定位在细胞核；与转化空载体相比，过表达CmHAT1使体细胞胚胎数量显著减少，体细胞胚胎体积显著增大，而沉默CmHAT1后体细胞胚胎的表型没有显著性差异。
      结论 在由同等质量的愈伤诱导体细胞胚胎的情况下，过表达CmHAT1后可以调控板栗体细胞胚胎的数量和体积，影响体细胞胚胎的发育。本研究为进一步优化板栗再生体系和遗传转化体系奠定了基础，为利用体细胞胚胎系统创制板栗新种质提供了科学依据。

       

      Abstract:
      Objective Somatic embryogenesisis an effective approach to in vitro regeneration of Chinese chestnut. However, the slow development of somatic embryos and the low reproduction efficiency of somatic embryos in Castanea mollissima Blume, which had limited the regeneration process of somatic embryo of Chinese chestnut, and it is difficult to provide technical support for the verification of gene function and the creation of new germplasm. The purpose of this study was to investigate the role of CmHAT1, a member of HD-ZIP Ⅱ gene family, in development of somatic embryo, and to further accelerate the development of somatic embryo in Chinese chestnut.
      Method The bioinformatics analysis was used to identify the Chinese chestnut HD-Zip gene family, the expression of the family members was analyzed in different developmental stages of Chinese chestnut somatic embryo, and the key candidate gene CmHAT was screened for regulating somatic embryo development. Further, the subcellular localization and gene function of CmHAT were analyzed by tobacco subcellular localization experiment, RT-qPCR and genetic transformation.
      Result (1) The results showed that there are 7 members of the Chinese chestnut HD-ZIP Ⅱ gene family, most CmHD-ZIP Ⅱ members were expressed in somatic embryo development of Chinese chestnut. CmHAT1 was lowly expressed in callus, up-regulated in Globular embryos, highly expressed in heart embryo, and down-regulated in cotyledonary embryo. Therefore, CmHAT1 was selected as a candidate gene for regulating somatic embryo development. (2) CmHAT1 was localized in the nucleus, Compared with empty vector transformation, overexpression of CmHAT1 significantly reduced the number of somatic embryos and increased the volume of somatic embryos, while there was no significant difference in phenotype of somatic embryos after CmHAT1 silencing.
      Conclusion In the case of somatic embryos induced by callus of the same quality, overexpression of CmHAT1 can regulates the number of somatic embryo and the volume of somatic embryo, and affecting the development of somatic embryo in Chinese chestnut. And lay a solid foundation for the establishment of regeneration system and genetic transformation system, and accelerate the creation of new and excellent germplasm of Chinese chestnut.

       

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