Identification of ChSEP genes in the whole genome of hazelnut, preparation of polyclonal antibody and expression in ovules

  Objective  Normal ovule (kernel) filling is the basis of hazel kernel formation. Insufficient ovule filling can frequently lead to the shriveled kernels or empty nuts in hazel. SEP belonging to the E class MADS-box gene plays an important role in ovule development of plants. To investigate the important role of ChSEP in hazelnut ovule development, the key ChSEP genes involved in the development of hazelnut ovules were identified. Then, polyclonal antibody against ChSEP was prepared, followed by immunohistochemical analysis of ChSEP using developing and aborted ovules as study materials. The present study is beneficial for better understanding of ovule development mechanism regulated by SEP in hazelnut.

  Method  All SEP gene family members in hazelnut genome were identified using genome and transcriptome data, and prokaryotic expression vector construction containing important SEP gene, gene expression, and polyclonal antibody preparation were carried out. Finally, important SEP involved in ovule development regulation was detected by immunohistochemistry.

  Result  (1) Three ChSEP sequences were identified in the genome of hazelnut, including Cor0054610.1, Cor0008190.1 and Cor0119400.1. All SEP proteins contained SRF-TF and K-box motifs. Polyclonal antisera of Cor0054610.1 was prepared, and the molecular mass of polyclonal antibody E18305 and E18306 was about 45 kDa, and 1−2 ng antigen could be detected after 1∶1000 dilution. (2) Immunohistochemical analysis showed that ChSEP was expressed in the integument of both developing and abortive ovules. The expression of ChSEP in integuments was higher than that in cotyledons. There was no significant difference in the distribution of ChSEP between developing and abortive ovules.

  Conclusion  This study provides evidence that ChSEP (Cor0054610.1) is involved in the regulation of hazelnut ovule development at both transcriptional and protein levels, and provides a scientific basis for better understanding of the ovule development mechanism in hazelnut.