Molecular Cloning and Characterization of a Novel Polygalacturonase Gene, BcMF24, Involved in Pollen Development of Brassica campestris ssp. chinensis

In the present study, a polygalacturonase (PG) gene was isolated from Brassica campestris L. ssp. chinensis Makino (syn. Brassica rapa ssp. chinensis). Sequence analysis indicated that it contained three classical conserved domains (I, II, and IV) of PG proteins. Phylogenetic analysis demonstrated that it was clustered into the clade E of PG family with several other PGs from different plant species. The results obtained from quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ hybridization showed that the gene, B. campestris Male Fertility 24 (BcMF24), was specifically expressed in the microspores, in the partly degraded tapetum at binucleate stage and in the pollen grains at mature pollen stage. Bioinformatics analysis of a 1,489-bp sequence in the BcMF24 5′-upstream region illustrated that the said gene contained several typical cis-acting elements and pollen-specific elements. Transient expression analysis revealed that BcMF24 can drive the green fluorescent protein (GFP) expression in onion epidermal cells. β-glucuronidase (GUS) assay of the BcMF24 promoter–GUS constructs in transgenic Arabidopsis thaliana showed that it could specifically drive gene expression in the anther of flower buds at binucleate and mature pollen stages. To the best of our knowledge, the current study is the first demonstration of a PG gene, which belongs to clade E that is involved in pollen development.