Cloning And Functional Analysis Of GhSPO11-3 Gene In Gossypium Hirsutum

Cotton(Gossypium hirsutum)is an important fiber and oil crop around the world,and its growth and yield are affected by salt stress conditions.Related reports showed that the SPO11-3 gene is associated with plant salt stress and endoreduplication.The SPO11-3 gene was one of eukaryotic Topoisomerase VIA(Topo VIA)homologues involved in modulating several important biological processes,such as growth,development and stress responses in plants.The SPO11-3 protein is widely found in higher plants.However,few researches of cotton SPO11-3 gene have been functionally characterized in cotton.In this study,the BLAST search was carried out in the upland cotton(Gossypium hirsutum)database using the sequence of the AtSPO11-3 gene of Arabidopsis thaliana.The most similar sequences were obtained and primers were designed.Here we identified a homolog of Topo VIA in economically important crop upland cotton designated as GhSPO11-3.For further study the function of GhSPO11-3 gene.The study constructs cotton GhSPO11-3 gene and GFP gene fused expression vector and transformated to onion epidermis to transient express by Agrobacterium-mediated transformation.The target gene-silencing vector was constructed.Agrobacterium-mediated virus-mediated gene silencing(VIGS)experiment was used to silence corresponding gene.The physiological and biochemical experiments of seedlings under salt stress were measured.The main results of this research are as follows:1.The gene of GhSPO11-3 were cloned from the upland cotton “Zhong mian suo79”.The open reading frame of GhSPO11-3 gene is 1254 bp,encoding a protein of417 amino acids.The GhSPO11-3 gene is relatively conserved in evolution.GhSPO11-3 protein also contains N-terminal TP6A_N domain and conserved catalytic domain Toprim.Amino acid sequence alignment analysis showed that the GhSPO11-3 was highly identitied with Theobroma cacao,Arabidopsis thaliana and Oryza sativa.We detected the gene is the GhSPO11-3 gene of Gossypium hirsutum.Cluster analysis also suggests that GhSPO11-3 has closed phylogenetic relationship with Theobroma cacao,Arabidopsis thaliana and Oryza sativa.2.The analysis of GhSPO11-3 expression by real-time quantitative PCR in different cotton tissues displays that,GhSPO11-3 is a constitutive gene but most preferentially expressed in plant leaves.Using GFP as a reporter gene,we built fusion expression vector and injected it to the onion epidermis cells,observed the green fluorescenceunder the confocal microscopy.The results showed that GhSPO11-3 protein was predominantly localized in the cell nucleus.3.The plasmids of pYL-156-GhSPO11-3 were introduced into Agrobacterium tumefaciens GV3101 to obtain the bacteria of VIGS.Inoculating bacteria into the cotyledons of cotton seeding without ture leaves.When the negative control leaves of plants with CLA1 gene silence appeared white spot,the leaves from the plants with target gene silence and control were collected for RNA extraction.The results showed that the expression level of target gene mRNA was suppressed in VIGS plants.The plants with target gene silence exhibits a dwarf phenotype compared with the empty vector-treated cotton(CK)plants.Our flow cytometric analysis indicates that the mechanistic faults in the endocycle progression in VIGS plants.Moreover,our fluorescence microscopy revealed that nuclear size is significantly smaller in many cell types of the plants with target gene silence.4.The analysis of salt-stress treatment showed that the growth of VIGS plants seedings exhibited severe wilting,however,the CK plants seedings exhibited delaying damage symptom.Salt treatment under 400 mmol·L-1 NaCl for cotton physiological indexes analysis.Phenotypically,the down-regulation of GhSPO11-3 led the transgenic plants to more sensitivity in physiological alternations such as increased MDA metabolites,and reduced chlorophyll and proline contents after salt stress.The above results suggested that VIGS plants have lower salt tolerance than the CK plants.In this study,we got a GhSPO11-3 gene from Gossypium hirsutum.The structure,expression pattern and the roles in the progress of endoreduplication and salt stress were analyzed.Our findings suggest that silencing of GhSPO11-3 gene may reduce tolerance to salt stress by decreasing the levels of ploidies,due to a failure of endoreduplication.This study provided theoretical instruction for deeper study of the GhSPO11-3 gene.The research may provide a useful cue for improving salt stress tolerance of upland cotton by genetic manipulation.