Functional characterization of E3 ubiquity ligase Bra015092 in pollen development of Brassica campestris ssp. Chinensis.

Pollen development plays an important role in the sexual reproduction of seed-type plants. Ubiquitination of proteins is an essential link in the post-translational modification of proteins. E3 ubiquity ligase is a key protein that recognizes substrates in the protein ubiquitination pathway. The hybrid line "Bcajh97-01A/B" of Chinese cabbage (Brassica campestris L. ssp. Chinensis) was used as test material. The gene Bra015092, with a size of 642 bp, was amplified. Semi-quantitative (RT-PCR) and quantitative real-time PCR (qRT-PCR) techniques were utilized to analyze the expression of Bra015092 in the dual-purpose line of Chinese cabbage. It was found that Bra015092 had a higher expression level in inflorescence. Subcellular localization analysis showed that Bra015092 and GFP fusion expression protein widely exist in tobacco epidermal cells. Bra015092 was transformed into "Youqing49" cabbage to obtain Bra015092OE overexpressing transgenic lines. The morphological observation of Bra015092OE plants showed that the pollen of BcMF29OE plants became deformed and inactive, and the vegetative and reproductive nuclei were abnormally developed. The in vitro germination experiments showed that about 24.5% of the pollen in Bra015092OE plants could not germinate. The results of the semi-thin section showed that the pollen development of Bra015092OE plants was abnormal at the stage of binuclear pollen grains. Transmission electron microscopy revealed that the pollen grains of Bra015092OE plants gradually degraded from the binuclear to the trinucleate pollen grain stage, and the pollen inner wall was abnormally developed, indicating that Bra015092 plays a major role in the process of pollen development.

Full-length transcriptome analysis reveals the differences between floral buds of recessive genic male-sterile line (RMS3185A) and fertile line (RMS3185B) of cabbage.

MAIN CONCLUSION: Starch and sucrose metabolism and plant-pathogen interaction pathways play a dominate role in recessive genic male sterility (RGMS) of cabbage (Brassica oleracea L. var. capitata). RGMS is common in plants and has been widely applied as an effective and economic system for hybrid seed production in many crops. However, little is known regarding the molecular mechanisms of RGMS in cabbage. Hence, full-length transcriptomic and physiological analysis were performed in the spontaneous RGMS mutant RMS3185A and its near-isogenic fertile line (NIL) RMS3185B of small (< 1.6 mm in diameter), medium (~ 2.5 mm in diameter), and large floral buds (~ 3.4 mm in diameter) to identify the differentially expressed genes (DEGs) associated with RMGS. The pollen abnormalities between RMS3185B and RMS3185A appeared at the large floral bud stage. In contrast with RMS3185B, the mature anthers and stamens of RMS3185A were shorter than those of RMS3185B, and the anthers did not dehiscent. The concentrations of glucose, fructose, trehalose, starch, and cellulose in RMS3185A were all significantly lower than those in large floral buds of RMS3185B. PacBio sequencing results showed that DEGs were mainly concentrated in large floral bud stage. In combination with the KEGG enrichment analysis of DEGs in GO terms related to cell wall, pollen and anther, pentose and glucuronate interconversions (ko00040), starch and sucrose metabolism (ko00500), and plant-pathogen interaction (ko04626) were significantly enriched. Among which, cell-wall/pectin-related genes of eighteen PEI, twenty-two PEL, three PG, and fifteen PGL involved in ko00040, and one UGDH, one SPS, four CWINV, four TPP/TPS, and four EGL involved in ko00500, as well as plant-pathogen interaction genes, including sixteen calcium-dependent protein kinase (CDPK), one cyclic nucleotide-gated ion channel (CNGC), and twenty-three calcium-binding protein CML (CML), were significantly down-regulated in RMS3185A relative to that in RMS3185B. Besides, genes involved in ko04626, including two CML and one transcription factor WRKY33, were up-regulated in RMS3185A relative to that in RMS3185B. In conclusion, we hypothesized that the expression alterations of these genes were responsible for calcium signaling and sugar metabolism, thus affecting the occurrence of RGMS in cabbage.

Characterization of a male sterile related gene BcMF15 from Brassica campestris ssp. chinensis.

Data from cDNA-AFLP analysis based on the genome-wide transcriptional profiling on the flower buds of the male meiotic cytokinesis (mmc) mutant and its wild-type of Brassica campestris L. ssp. chinensis Makino, syn. B. rapa L. ssp. chinensis, indicated that mutation of the MMC gene resulted in changes in expression of a variety of genes. A transcript-derived fragment specifically accumulated in the wild-type flower buds was isolated, and the corresponding full-length cDNA and DNA was subsequently amplified. Bioinformatical analyses of this gene named BcMF15 (GenBank accession number EF600901) showed that it encoded a protein with 103 amino acids. The BcMF15 had a 88% nucleotide similarity to a lipid transfer protein-like gene. Moreover, sequence prediction indicated that BcMF15 might encode a membrane protein with a signal peptide at the N-terminus. Meanwhile, six domains were predicted in the deduced BcMF15 protein, such as the AAI domain existing in some crucial proteins of pollen development-preferential, signal peptide, transmembrane domain, vWF domain, ZnF_C4 domain, and Tryp_alpha_amyl domain. Spatial and temporal expression patterns analysis by RT-PCR indicated that BcMF15 was exclusively expressed in the fertile line, which indicated this gene is male sterile related. Phylogenetic analysis in Cruciferae revealed that the BcMF15 was relative conservative in evolution. We suppose BcMF15 may be a critical molecule in the transmembrane transportation and signal transduction during microspore development.