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

Abstract

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 Bra015092^OE overexpressing transgenic lines. The morphological observation of Bra015092^OE plants showed that the pollen of BcMF29^OE 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 Bra015092^OE plants could not germinate. The results of the semi-thin section showed that the pollen development of Bra015092^OE plants was abnormal at the stage of binuclear pollen grains. Transmission electron microscopy revealed that the pollen grains of Bra015092^OE 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.