Cloning And Functional Analysis Of Ghnad5 Gene In Cotton

Plant cytoplasmic male sterility(CMS)is a biological phenomenon of pollen abortion produced by the interaction of nuclear genes and mitochondrial genes,and is the basis for the utilization of crop heterosis.Studies have shown that mitochondrial genome abnormalities have a direct impact on plant fertility,and mitochondrial dysfunction leads to imbalance of energy metabolism can cause pollen sterility.NADH dehydrogenase is the main importer of mitochondrial respiratory chain electron transfer.It has a complex structure and plays an important role in glycolysis,gluconeogenesis and tricarboxylic acid cycle,and plays an important role in the production of ATP and reactive oxygen species in mitochondria.Insufficient supply of ATP and accumulation of reactive oxygen species promote apoptosis and lead to pollen sterility.In this study,cotton CMS Jin A,Yamian A and their maintainerline were used as experimental materials.The genomic DNA and c DNA of Ghnad5 were cloned and analyzed by RNA editing.The gene expression and the activity of NADH dehydrogenase at different developmental stages were determined.The RNAi and overexpression vectors of Ghnad5 were constructed for functional analysis to investigate the relationship between Ghnad5 gene and CMS.The results are as follows: 1.Using homologous cloning,get the full-length c DNA and genomic DNA of the Ghnad5.The full-length DNA of Ghnad5 in the Jin A,Yamian A and its maintainer line was 3178 bp,and the full-length c DNA of Ghnad5 was 1200 bp,encoding 399 amino acids,the predicted protein molecular weight is about 103 k Da.Amino acid alignment revealed that all four materials had a Proton antipo M conserved domain,and the two male sterile lines had differences in individual amino acid residues compared to their respective maintainer materials,but did not cause changes in their key functional domains.Bioinformatics cluster analysis found that the protein encoded by Ghnad5 gene is closely related to Gossypium raimondii,Gossypium arboreum,Arabidopsis and Glycine max,but far from being related to Nicotiana tabacum and Oryza sativa.The analysis of the promoter sequence of Ghnad5 indicated that the expression level of Ghnad5 gene may be mainly in response to light,phytohormone such as auxin,gibberellic acid,methyl jasmonate and changes in stress elements such as low temperature and drought.2.RNA editing analysis was performed on the genomic DNA and c DNA sequences of the Jin A?Yamian A and their maintainer lines.The results showed that : RNA editing was not performed in the Ghnad5 gene in the maintainer system.Two RNA editings were performed in the Ghnad5 gene in JA-CMS and YMA-CMS,respectively.Among them,the U?C RNA editing site at 206 bp from the start codon in YMA-CMS resulted in its 69 th amino acid being changed from a hydrophobic amino acid to a hydrophilic amino acid.Thus,RNA editing makes the hydrophilicity of nad5 protein in YMA-CMS is the one of the reasons that lead to its CMS.3.The Ghnad5 gene expression was measured in the bud of Jin A,Yamian A and its maintainer microspore development at different stages.The results show : In the early stage of microspores abortion,the expression levels of Ghnad5 gene in Jin A and Yamian A were significantly higher than their homonuclear heterogeneous maintainerline,while in the abortive period and post-abortion period,the expression level of the material Ghnad5 gene was significantly lower than that of its maintainerline in the two sterile lines.It is speculated that the decrease of the expression level of Ghnad5 gene in the abortive period of the sterile line may affect the proton transfer in the electron transport chain,resulting in the decrease of ATP synthesis.The decrease in the expression level can also lead to the reverse transfer of electrons,resulting in ROS,causing disorder in the intracellular environment to damage the cell membrane,causing apoptosis,leading to pollen infertility 4.The NADH dehydrogenase activity of Jin A and Yamian A and their respective maintainer microspores were determined by ELISA.The results showed that the activity of NADH dehydrogenase in Yamian A and its maintainerline was basically unchanged,and the NADH dehydrogenase activity in Yamian A was significantly higher than its maintainerline during the development of microspores.The activity of NADH dehydrogenase in Jin A was significantly different from that of its maintainer line.In the early stage of microspores abortion,the activity of NADH dehydrogenase in Jin A was significantly higher than that in its maintainer line,while the microspore abortion and abortion period the activity of NADH dehydrogenase was lower than that of the maintainer line,which reached a very significant difference.The change of NADH dehydrogenase activity in Jin A was found to be the same as that of Ghnad5 gene.The results of previous experimental study showed that in the critical period of abortion,the ROS content in Jin A increased,while decreased in Yamian A.It is speculated that in the critical stage of microspores abortion,the down-regulation of Ghnad5 gene and the accumulation of ROS may lead to a decrease in NADH dehydrogenase activity in Jin A,while NADH dehydrogenase activity and ROS content and maintainer phase in Yamian A.The difference is larger than that of Jin A.It is speculated that it may be caused by different abortion mechanisms.5.The Ghnad5 gene RNAi and overexpression vector were constructed to verify the function of Ghnad5 gene.