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

Objective Somatic embryogenesis is 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, 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 the 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) There were 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 regulate the number of somatic embryo and the volume of somatic embryo, affecting the development of somatic embryo in Chinese chestnut. This study lays a solid foundation for the establishment of regeneration system and genetic transformation system, and accelerates the creation of new and excellent germplasm of Chinese chestnut.