Preliminary Functional Analysis Of The Key Meiosis Gene Spo11-1 In Watermelon(Citrullus Lanatus)

Watermelon(Citrullus lanatus)is a widely cultivated horticultural crop for high economic and nutritional value.Meiosis is the important process during the sexual reproduction and the exchange of chromatids during meiosis not only enhances the genetic diversity of the species,but also provides more resources for creating new germplasm.The raw materials are essential for the reproductive development of watermelon.SPO11-1 is very important for meiosis and plant reproduction.The SPO11-1 protein is homologous to the topoisomerase VIA subunit in Arabidopsis thaliana,which initiates the process of meiotic recombination.The homologous gene of SPO11-1 were identified in arabidopsis,rice,maize and other plants,and its mutation caused defects in the meiotic recombination process and lead to sterility.In this study,the amino acid sequence of At SPO11-1 was compared in watermelon genome database to obtain the watermelon homologous gene(ID: Cla003301)which named Cl SPO11-1.The transient subcellular localization and CRISPR/Cas9 genome editing system were used to characterize the functional validation.Furthermore,protein interaction was performed by yeast two‐hybrid system.The major findings are as following:1.Amino acid and gene sequences of Cl SPO11-1 were obtained by BLAST search from watermelon "97103" whole genome database.Cl SPO11-1 full genomic DNA was cloned in watermelon material "YL".The full-length c DNA was obtained by 5’RACE PCR technology.The full length of c DNA was 1095 bp which contains 15 exons and 14 introns.It codes for 394 amino acids.2.Phylogenetic analysis showed that Cl SPO11-1 is closely related to Arabidopsis thaliana.The TOPRIM＿Topo6A/Spo11 conserved domain in protein confirmed it belongs to topoisomerase gene family.The protein properties demonstrate its hydrophobic nature.The molecular weight and p I values were recorded 42.10 k Da and 8.73 respectively.3.The results of real-time quantitative PCR showed that the Cl SPO11-1 gene was expressed in different tissues of watermelon,and the highest expression was found in meiotic flower bud(1.6-1.8mm).The constructed p CAMBIA1300-Cl SPO11-1-GFP recombinant plasmid was introduced into Agrobacterium GV3101(p Soup-p19)for instantaneous transformation of onion epidermis.Subcellular localization results showed that Cl SPO11-1 was localized in the cell nucleus.4.The successfully constructed CRISPR/Cas9 editing vector with 19 nt dual targets of Cl SPO11-1 gene was introduced into Agrobacterium rhizogenes Ar.Qual to infect the watermelon cotyledon explants,and DNA was extracted after the explants grew lateral roots.PCR amplification was performed on the target sites,and the sequencing results showed that the two targets selected in this experiment were edited at one target site.The plasmid then was used for the stable transformation in watermelon.5.The successfully constructed editing vector verified by the Agrobacterium rhizogenes system was introduced into Agrobacterium tumefaciens EHA105,and 5 regenerative plants were obtained by plant tissue culture technology.Genomic DNA from the leaves was extracted and subjected to PCR amplification.Sequencing results showed that two of the plants had different degrees of editing at target 1,and amino acid sequences showed that the two resistant plants had different degrees of amino acid variation or premature termination of translation.6.The recombinant plasmid p GBKT7-SPO11 and empty vector p GBKT7 were used to convert yeast strain Y2 HGold.The results showed that the bait protein p GBKT7-SPO11 was non-toxic and not self-activated.7.Using watermelon mixed c DNA library,the Yeast Two-Hybrid experiment was used to screen proteins that interact with Cl SPO11-1.The plasmids were extracted from the four positive clones and transformed into E.coli.After PCR detection,the sequencing results showed that 4 single sequences were initially selected,compared and annotated in the watermelon genome database.8.In order to verify whether the four candidate interaction genes have false positives,the Yeast Two-Hybrid was used.The results reveled tha Cla014788 and Cla008661 proteins from watermelon can interact with Cl SPO11-1.Real-time quantitative PCR was used to analyze the expression of Cla014788 and Cla008661 genes in flower buds of different sizes.As a result,the expression of Cla014788 was first decreased and then gradually increased.The expression of Cla008661 was similar to the trend of Cl SPO11-1 expression,and both of which were first increased and then decreased.The results show that Cl SPO11-1 and Cla008661 genes have consistent expression abundance curves,suggesting that the corresponding proteins of the two are likely to be a pair of interacting regulatory proteins in watermelon.