Cloning And Functional Analysis Of AcAMS Gene Related To Cytoplasmic Male Sterility In Onion (Allium Cepa L.)

Onion(Allium cepa L.)is a biennial herb of the Liliaceae Allium,belonging to the cross-pollination crop,which is one of the major vegetables with a wide planting area in China.Onion has obvious heterosis,for its flower is so small,besides,scattered flowering period,low single flower seeding rate,costly and difficult artificial emasculation that the selection and utilization of onion male sterile line is extremely necessary.For the study of onion CMS,most focusing on the screening for the candidate genes related to CMS and molecular markers.However,the underlying molecular and regulation mechanism of sterility remain unclear.In our preliminary work of the laboratory,we defined that the reason for onion CMS was the early dissociation of tapetum cells,there was significant differential expression of the key candidate gene AMS(ABORTED MICROSPORES)related to the developing of tapetum between the male sterile line and the onion maintainer line.To further explore the function of the gene in the occurrence of CMS in onion,we cloned Ac AMS gene,analyzed the differential expression patterns during flower development,carried out the subcellular localization,constructed the expression vector of Ac AMS to transform the Arabidopsis thaliana,and detected the change of the gene expression level in upstream and downstream.The relationship was preliminarily discussed between Ac AMS and onion CMS.Simultaneously,a TRV-based p TRV2-Ac PDS vector was constructed to attempt to establish the onion VIGS system.The assay results are as follows:(1)Ac AMS(KY296301)was cloned from the anther c DNA at the tetrad stage of the onion maintainer line(SB2).Ac AMS had a full length of 1458 bp in CDS and encoded a putative protein with 485 amino acids.The Homologous between Ac AMS and other species(Asparagus officinalis L.,Musa acuminata subsp.malaccensis,Phoenix dactylifera L.,Elaeis guineensis)was more than 50% through BLASTp.Furthermore,the phylogenetic tree which showed that Ac AMS and Ao AMS of Asparagus officinalis L.were on the same branch with a closest relationship.The amino acid sequence alignment with other species demonstrated that the Ac AMS encoded a protein with a basic helix-loop-helix(b HLH)domain belonging to the MYC subfamily of b HLH transcription factors.(2)Using the Real-time fluorescent quantitative PCR to analyze the temporal and spatial expression of Ac AMS gene.It was discovered that Ac AMS had the highest expression in anthers,almost no expression in other floral organs with tissue specific,and compared its expression in anthers at different developmental stages of male sterile line SA2 and its maintainer line SB2.There was a significant difference in expression at the pollen mother cell stage and the tetrad stage.(3)We constructed the p GFP-Ac AMS vector and used PEG-Ga2+-mediated method to transform the Arabidopsis protoplast cells.The results verified that the fusion protein was transiently localized on the nucleus and had the same nuclear transcriptional characteristics as its transcription factor.(4)The over expression vector p1250-3301-Ac AMS was established,then we introduced the strain into the wild type Arabidopsis thaliana through inflorescence infection in order to obtain the T1 generation plants after PPT resistance screening and PCR identification.Compared with the wild type plants,the transgenic lines presented partial sterility or complete sterility,partial sterility plant had short pods,and the pods of complete sterility plant were extremely short and curved,the stamens and pistils of the flowers were more elongated,the anthers were dim and wizened,the viable pollen grains were significantly less than the wild type.The changes of At AMS and its upstream and downstream genes in transgenic lines were detected.The results showed that At AMS was significantly up-regulated,At TDF1 and At MS188 were significantly down-regulated,while At MGT5 expression level was not significantly different.The same method was used to genetically transform the ams mutant Arabidopsis thaliana,and the resistant seedlings were screened.(5)We constructed the CRISPR/Cas9 gene editing silencing vector p HUN411-Ac AMS.It will be utilized for genetic transformation of onions in the future.(6)The gene silencing vectors p TRV2-Ac PDS and p TRV2-Ac AMS were constructed by using the Ac PDS partial sequence(333 bp)and the Ac AMS partial sequence(384 bp)of onion to intend to establish the onion VIGS(Virus-induced gene silencing)transformation system.