The Expression Analysis And Functional Characterization Of E3 Ubiquitin Ligase Gene BcMF29 In Brassica Campestris

Pollen development is an important part in the process of sexual reproduction of seed plants.The mechanism of its development process is extremely complex,involving sophisticated genetic network regulation.In-depth analysis of the function and regulation of key genes during pollen development is of great significance for revealing the regulation mechanism of pollen development,thus providing a theory for the breeding of agricultural production and the solution of actual production problems.Protein ubiquitination is an important post-translational modification that is involved in various life processes of plants.E3 ubiquitin ligase is a key protein that recognizes substrates in the protein ubiquitination pathway.Studies have shown that some E3 ubiquitin ligases play an important role in plant pollen development.In this study,the'Aijiaohuang' genic male sterility line named'Bcajh97-01A/B' in Brassica campestris L.subsp.chinensis Makino,syn.B.rapa subsp.chinensis was used as a test material,and the gene BcMF29 was cloned.Bioinformatics software was used to analyze the sequence characteristics of BcMF29 to predict the structure and function of the encoded protein.The expression of BcMF29 in B.campestris was analyzed by qRT-PCR(quantitative real-time PCR)and RT-PCR(reverse transcription-PCR).The subcellular localization of tobacco in vivo was used to identify the distribution of the protein encoded by BcMF29 in cells.The overexpression vector and the gene knockout vector was transformed into 'B.campestris subsp.chinensis var.paracampestris cv.Youqing 49' and the overexpression and knockout transgenic lines were obtained,and then the biological function of BcMF29 in B.campestris pollen development was explored.The results obtained are as follows.(1)PCR amplification of DNA and CDS sequences yielded a gene of 624 bp intron-free,BcMF29.The gene encoded 207 amino acids,the encoded protein was insoluble,with a molecular weight of 41.65 kDa and an isoelectric point of 9.17.The amino acid sequence similarity with the Brassica oleracea homolog Bo1028200 and the Arabidopsis homolog of At3g25030 was 79.93%.NCBI noted that this gene encoded an E3 ubiquitin ligase,SignalP-4.1 predicted that the protein encoded by this gene had no signal peptide,and analysis of the transmembrane domain of BcMF29 by TMHMM Server v.2.0 revealed that BcMF29 did not have a transmembrane domain.The functional domain of the protein was predicted and found to contain the typical RING functional domain of E3 ubiquitin ligase.(2)The expression of BcMF29 in different tissues of B.campestris was detected by RT-PCR(reverse transcription-PCR)and qRT-PCR(quantitative real-time PCR).It was found that the transcript of BcMF29 was expressed in each tissue of B.campestris,but it was highly expressed in the inflorescence,and the expression of 'Bcajh97-01B' in the flower buds of 5 different developmental stages of B.campestris was higher than that of 'Bcajh97-01A'.Subcellular localization results showed that the fusion-expressed proteins formed by BcMF29 and GFP were widely distributed in tobacco epidermal cells.(3)A BcMF29 overexpression vector driven by CaMV35S(Cauliflower Mosaic Virus 35S)was constructed and overexpression transgenic lines BcMF29OE were obtained by using the mature genetic transformation system of our laboratory.Morphological and cytological observation was performed on BcMF29OE.BcMF29OE produced undeveloped,shrunken,deformed pollen.These malformed pollen were not viable,and showed abnormalities in the nutrient nucleus and reproductive nucleus.In vitro germination experiment,BcMF29OE showed that about 24.54%of the pollen could not germinate,compared with 10.21%in the control group.The results of semi-thin sections showed that pollen dysplasia of BcMF29OE began in the period of binuclear pollen grains.Transmission electron microscopy further observed that inclusion of the pollen grains in BcMF29OE was degraded in advance during the binuclear pollen grains,and the pollen grains were inwardly recessed from the germination groove.At the same time,the pollen inner wall was abnormally deposited.During the period of trinuclear pollen grains,the inclusion was almost completely degraded,and the inner wall of the pollen was still abnormal.These results indicate that BcMF29 plays an important role in pollen development.(4)The BcMF29 knockout vector KpBI35S::BcMF29 was constructed using CRISPR/Cas9 technology.By using Agrobacterium-mediated transformation of 'Youqing 49',a total of 17 positive bud lines were obtained,the genes in 4 positive transgenic bud lines were edited,and three editing types were produced.This indicates that the selected sgRNA plays a gene editing function in B.campestris,and the editing of the single gene BcMF29 can be realized in B.campestris.