Diversity analysis of CdS-RNase genes and their encoding proteins in Camellia drupifera germplasm from Hainan island

The study aimed to investigate the diversities of CdS-RNase genes and their coding proteins in Camellia drupifera germplasm from Hainan island, explore the relationship between CdS-RNase protein types and the self-pollination setting rate of C. drupifera, and provide scientific reference for a rational combination of C. drupifera germplasm in the field conditions based on CdS-RNase protein types and to improve the fruit setting rate of C. drupifera.

Sixty local C. drupifera germplasm from Hainan island were selected, and their style cDNA was used as a template for RT-PCR-specific amplification of the CdS-RNase gene sequence. These CdS-RNase gene sequences were separated and re-purified with agarose gel electrophoresis, and further connected to the blunt sequencing vector. The constructed sequencing vector was transformed into E. coli competent cells and the positive clones were further screened with an LB solid plate with Kanamycin resistance. The resulting positive clones were sequenced to obtain CdS-RNase gene sequences. Subsequently, these CdS-RNase gene sequences were analyzed together with that from the “CV1”-“CV18” for gene haplotype classification, haplotype clustering analysis, obtain CdS-RNase protein amino acid sequences, categorize CdS-RNase protein types, and prediction of CdS-RNase protein secondary structure. Finally, based on the CdS-RNase protein types, these C. drupifera germplasm from Hainan island can be classified into three categories, including only detecting CdS-RNase protein (type I), co-detecting of CdS-RNase and CdS_m-RNase proteins (type II), and only detecting CdS_m-RNase protein (type III). Germplasms of each 5 of the three types were selected to do experiments of fruit setting percentage of self-pollination.

A total of 171 CdS-RNase gene haplotypes (Hap1-Hap171), which encoding 35 normal CdS-RNase proteins (CdS₁ RNase-CdS₃₅ RNase) and 22 mutant CdS_m-RNase proteins (CdS_m1 RNase-CdS_m22 RNase), were identified from these 78 C. drupifera germplasms from Hainan island. In the secondary structure CdS-RNase proteins and mutant CdS_m-RNase proteins, the ratios of α-helix were not significant difference, the ratios of β-turn and β-sheet were very significantly higher in the mutated CdS_m-RNase proteins, the ratio of the coil was significantly lower in the mutated CdS_m-RNase proteins. Additionally, the fruit setting percentage of self-pollination in type I germplasm was significantly lower than that of type II germplasm, which was further significantly lower than that of type III germplasm.

The diversity of CdS-RNase gene and its encoding protein is abundant in the local C. drupifera germplasm from the Hainan Island. The increase ratios of β-turn and β-sheet and decrease ratio of coil in the mutant CdS_m-RNase protein secondary structure probably affect CdS-RNase normal function, which lead to the improve of the self-pollination fruit setting rate of C. drupifera germplasm to some extent.