| Citrus is distributed widely and has become the world’s largest fruit inducstry.The crucial breeding goal of citrus is seedlessness,and male sterility is a main cause to produce seedless fruit.The seedless cytoplasmal cybrid of pummelo(Citrus.Grandis)‘Huayou No.2’(G1+HBP)was obtained by somatic fusion,using the seedless variety ‘Guoqing No.1’satsuma mandarin(G1)of cytoplasmic male sterility(CMS)as callus parent and the seeded variety HB pomelo(Hirado Buntan pummelo)as mesophyll parent.It shows male sterility and is a vital material for uncovering CMS mechanisms in citrus.We previously identified the stamen primordium initiation defect in G1+HBP.AP3,a class B gene that regulates the development of stamen,is down-regulated compared with the fertile parent HB pummelo(HBP)in the flower of G1+HBP at stamen primordium development stage.However,the function of AP3 gene in citrus and the interaction of AP3 with ABCE model proteins remain to be uncovered.In this study,the function of AP3 gene was validated by genetic transformation in the precocious wild kumquat(Fortunella hindsii)and protein interaction assay,and the male sterile kumquat was produced.The mechanism of AP3 mediated regulation on stamen development was analyzed,which would provide a theoretical basic and materials for citrus seedless breeding.The results are as follows:1.ABCE model genes identification and expression pattern analysis.The ABCE model genes of citrus genome were identified by aligning protein sequences.By constructing the phylogenetic tree of AP3 genes,it was confirmed that citrus AP3 differentiated into three homologous genes,namely AP3.1,AP3.2 and AP3.3,in which AP3.2 and AP3.3 were highly homologous to Arabidopsis AP3 with a reliability of 86%,while AP3.1 was highly homologous to TM6 in strawberry and tomato with a reliability of82%.The flower buds and anthers of citrus cytoplasmic hybrid G1+HBP and its parent HBP at different stages of stamen development were collected to detecte the expression of ABC genes by RT-q PCR.There was significant difference in the expression of Cg AP2.1and Cg AP3.2 between G1+HBP and HBP.2.Detection of Cg AP3 and ABCE model protein interactions.The bait vector of ABCE model proteins were tested for self-activation and toxicity and showed that all proteins were non-toxic,while Cg AP2.1-BD,Cg AP2.2-BD,Cg SEP2-BD and Cg AGL6-BD had self-activation.There were 105 combinations of interacting proteins and 22 proteins interacted with Cg AP3 in yeast two-hybrid of ABCE model proteins.Then,firefly luciferase complementary imaging determined Cg AP3.1 interaction with Cg PI.1,Cg PI.2 and Cg SEP3,Cg AP3.2 interaction with Cg SEP3,and Cg AP3.3interaction with Cg PI.1 Cg PI.2 and Cg SEP1.2.3.Genetic transformation and phenotypic observation of AP3.FhAP3 was overexpressed in Arabidopsis thaliana,and 12 T3-generation transgenic lines stably expressed FhAP3.1,two T1-generation FhAP3.2 transgenic lines and three T1-generation FhAP3.3 transgenic lines were obtained.Compared with wild type Arabidopsis plants,the siliques of over-expressed FhAP3.1 positive plants were significantly shorter(p< 0.0001);The over-expressed FhAP3.2 positive plants were short,with clustered stems and leaves,and could differentiate into flower buds,but the flower development was stagnant and could not bloom;The FhAP3.3 over-expression positive plants was extremely short,the height was only 0.3 cm,with only six normal leaves.After knocking out FhAP3 from kumquat,21 positive plants were obtained,and the editing efficiency of 3 positive plants was more than 48%.After seven months of soil culture,the FhAP3.2/3.3-p YAO:Cas9-1 of the double genes knockout plant(the editing efficiency of FhAP3.2 and FhAP3.3 was 55.28% and 100%,respectively)grew slower and shorter than the negative control plants,with only two normal leaves.The kumquat interference line Cg AP3.1-RNAi-1 and its two clonal grafted plants only blossomed but could not set fruit,and the average anther dehiscence rate was only 11.75%;the average pollen fertility rate of Cg AP3.1-RNAi lines was 62.26%,compared with negative control(the average pollen fertility rate of 84.70%),the pollen fertility of Cg AP3.1-RNAi lines decreased significantly(P < 0.01);RT-q PCR detection showed that Cg AP3.1-RNAi-1 line compared with the negative control,the expression of ABC genes(Cg AP2.2,Cg AP3.1,Cg AP3.2,Cg AP3.3,Cg PI.1,Cg PI.2,Cg AG)were significantly decreased.In summary,this study analyzed the differentiation of AP3 gene in citrus and its homologous relationship with other plants;Using male sterile cytoplasmic hybrid G1+HBP and its fertile parent HBP as research materials,RT-q PCR was used to compare the expression of ABC genes in flower buds and anthers;The experiment of protein interaction was used to detect the protein interaction of citrus ABCE model proteins.The multi-target CRISPR/Cas9 vector was constructed to transform kumquat,and the male sterile material of kumquat was created and the phenotype was observed.The function of AP3 gene was analyzed together,which established the basic for dissecting the mechanism of AP3 regulating stamen development and male sterility in citrus. |
