Cloning and expressions analysis of CmWOX2 from Citrus maxima (Burm.) Merr. during embryo development

Abstract:【Objective】The development of plant embryo is an extremely important part in the process of individual development. The morphogenesis of plant embryo is jointly determined by the MAPK (MAP KINASE)/GRD (GROUNDED) pathway and the WOX (WUSCHEL RELATED- HOMEBOX) transcription factor family members. The WOX transcription factor family members are differentially expressed in apical cells and basal cells, leading to the differentiation of different cells. WOX2 plays an important role in the early development of embryonic cells and the morphogenesis of stem apical meristems. This study aimed to explore the regulatory role of the CmWOX2 in seed development of Citrus maxima (Burm.) Merr.【Methods】The materials of this experiment were Majiayou trees from Shangrao, Jiangxi Province. The unirradiated and 60Co-γ irradiated sour pomelo anthers were used for artificial pollination. The ovarys and seeds from the fruits were collected before and after pollination. The seeds derived from the pollination with the unirradiated pollens were sown in test tubes containing MS basic medium, and the roots, stems and leaves of the young plants were collected after 30 d culture (20 d dark culture, 10 d light culture, culture temperature 26±1 ℃). The WOX2 gene sequence of the pomelo was obtained through search and comparison, and the full-length sequence of the CmWOX2 gene was obtained through amplification by designing specific primers. The PCR products were recovered and pu-rified, and then were connected to the PMD18-T vector, and the positive clones were obtained through the blue- white screening for further sequencing analysis. DNAMAN, ExPASy ProtParam tool, TMHMM 2.0, SignalP 4.1 software were used for bioinformatics analysis. The expression of the CmWOX2 gene in degraded seeds obtained by pollination after pollen radiation treatment and full seeds in normal fruits were compared and analyzed by the qRT- PCR technology at different developmental stages. WPS2020 was used to sort and graph the data, SPSS 26.0 was used for significance analysis, the Duncan’s method (p＜0.05) was used for multiple comparisons of means, and the Student’s t-test (p＜ 0.01) was used for comparison.【Results】The CmWOX2 gene were identified in the Citrus maxima (L.) Osbeck genome. The length of the CmWOX2 cDNA was 1177 bp, the length of open reading frame (ORF) was 750 bp, encoding 249 amino acids. The molecular formula of the protein was C1202H1859N345O383S14, and the molecular mass was 27.719 9 ku, the highest content was glutamic acid (8.8%). The predicted isoelectric point was 6.30, the instability index was 58.73, the fat solubility index was 55.26, and the total average hydrophilicity (GRAVY) was -0.770, which is speculated to be an unstable hydrophilic protein. The prediction of secondary structure showed that the CmWOX2 protein contains 50.6% helical structure, 60.2% folding, 16.1% circular structure, and the prediction of tertiary structure also had similar conclusions, showing that most of the CmWOX2 proteins had helical structure and folded structure, with less loop structure. Further analysis of the evolutionary relationship of the CmWOX2 protein showed that the CmWOX2 gene and the WOX2 genes of other species were highly similar in the 5’homeobox region, such as CsWOX2, VvWOX2, JrWOX2, HbWOX2, MeWOX2, StWOX2, AtWOX2, while there were some differences in the 3’WUS- box region compared with the VvWOX2, JrWOX2 and StWOX2; the transmembrane domain and protein signal peptide of the amino acid sequence of CmWOX2 were predicted, and the results showed that the CmWOX2 is not involved in the process of cell signal transduction. The phylogenetic tree analysis showed that the CsWOX2 from Citrus sinensis had the highest similarity with the CmWOX2, suggesting that the CsWOX2 was homologous with the CmWOX2; while the ThWOX2 and CmWOX2 had the farthest relationship. qRT-PCR results showed that the low- level expression of the CmWOX2 in roots, stems and leaves of seedings, while there was high level expression in the seeds. The CmWOX2 gene was also detected in the unfertilized ovaries, and the expression level of the CmWOX2 began to increase in the development process of the seeds after fertilization, the highest expression level was seen at the 10th week, while the expression level of the CmWOX2 gradually decreases with the development of the seeds; the expression level of the CmWOX2 in degraded seeds of the seedless fruits was significantly lower than that of plump seeds in the normal fruits.【Conclusion】The CmWOX2 plays a vital role in the formation and development of the Majiayou embryos, and the decrease expression of the CmWOX2 in the seedless fruits obtained by pollen radiation is closely related to the abnormal embryo development.