Study On The GhPDC-E1? Gene Cloning And Regulating Gene Expression Patterns In Cotton

Cytoplasmic male sterile line was the main way for the utilization of heterosis in crops.Previous studies have shown that cytoplasmic male sterility was related to energy metabolism,and imbalanced energy metabolism can cause abnormal development of pollen.Pyruvate dehydrogenase(PDC)was important for the energy metabolism of the organisms,because it can link up sugar degradation and the tricarboxylic acid cycle by catalyzing the oxidative decarboxylation of pyruvate(energy source).The pyruvate dehydrogenase El a subunit was a important regulatory site of PDC activity.PDK and PDP can regulate the homeostasis of PDC activity together by the way of hydroxyl group phosphorylation/dephosphorylation of PDC-Ela subunit serine.The cytoplasmic male sterile lines Jin A,Yamian A and their respective homokaryon maintainers were used as the materials to study the effect of energy metabolism on the development of microspore in cotton in this paper,the genomic DNA and cDNA of GhPDC-Ela,GhPDK and GhPDP were cloned,the expression mode of three genes were analyzed,and pyruvate content was determinated during the different development stages of the microspores.The main results were as follows:1.The GhPDC-E1??GhPDK and GhPDP genomic DNA and cDNA sequence were obtained through the homologous cloning.The genomic total DNA length of GhPDC-Ela was different between the male sterile line and the maintainer line,the length from Jin A,Yamian A and maintainer lines were respectively 8655bp,8672bp and 8658bp,there were eight exons and seven introns;The full-length cDNA were 1185 bp,encoding 394 amino acids.There was a single amino acid difference from amino acid sequence between the male sterile line and the maintainer line;GhPDC-Ela was highly conserved in plants,containing a TPP binding domain and having four conserved functional regions with two serine phosphorylation sites by multiple alignment of amino acid sequences.The full length of the GhPDK genomic DNA were the same as 2232bp in the male sterile line and the maintainer line,there were six exons and five introns;The full-length cDNA were 1110bp,encoding 369 amino acids,the similarity degree of GhPDK amino acid sequence was 99%between two male sterile and maintainer lines.GhPDK was highly conserved in plants,containing a histidine-like kinase domain with the trait of autophophorylation.The total genomic DNA length of GhPDP was different between the male sterile line and the maintainer line,the length from Jin A,Yamian A and maintainer lines were respectively 2007bp,2016bp and 2008bp,there were four exons and three introns;The full-length cDNA were 1152bp,encoding 383 amino acids.The gene belonged to the PP2C family of proteins and played the activity depending on Mn2+,its domains were conserved.2.The expression patterns of GhPDC-E1?,GhPDK,and GhPDP genes in the buds during the different period of micrispore were analyzed by real-time quantitative PCR.The result showed that the expression level of GhPDC-Ela from Jin A male sterile line was not difference with the maintainer line,but the expression level of GhPDC-Ela from Jin A male sterile line was significantly lower than that of maintainer line during the stages of abortion and after abortion;the expression level of GhPDK from Jin A male sterile line was significantly higher than that of maintainer line before and after abortion,and the difference was not significant at the time of microsporosis abortion;the expression level of GhPDP from Jin A male sterile line was significantly higher than that of maintainer lines in the whole the development of microspores.This implied the main reason of the pollen abortion from Jin A sterile line was that the over-expression of GhPDP made the mataboliam of the microsatellites from Jin A sterile line strong,result in ROS increasing,and peroxidation of membrane lipids,leading to premature degradation of the tapetum,resulting in pollen abortion.The expression level of GhPDC-Ela from Yamian A male sterile line was not difference with the maintainer line,but the expression level of GhPDC-Ela from Yamian A male sterile line was significantly lower than that of maintainer line during the stages of abortion and after abortion;the expression level of GhPDK from Yamian A male sterile line was significantly lower than that of maintainer,and the difference between the expression levels before and after abortion was extremely significant.There was no difference between the expression level of GhPDP from Yamian A male sterile line and the maintainer line at the pre-abortion period and the abortion period of the microspores,but the expression level of GhPDP from Yamian A male sterile line was significantly lower than that of the maintainer line after abortion.It suspected that the mains the main reason of the pollen abortion from Yamian A male sterile line was that low content of GhPDC-Ela subunit protein in Yamian A male sterile line may inhibit the conversion of pyruvate to acetyl-CoA,resulting in the low energy which can not meet the development of microspores and thus cause pollen abortion.The comprehensive analysis of the expression characteristics from GhPDC-Ela,GhPDK,and GhPDP genes together with our previous studies between the sterile lines and maintainer lines suggested that the mechanism of microsporation abortion was different between the sterile lines,although energy metabolism were abnormal lin the process of microspore development.3.The pyruvate content determination result in the development of sterile line and maintainer line microspores showed that there was no significant difference in the content of pyruvic acid between Jin A male sterile line and the maintainer line during the development of microspores,indicating that the abortion of Jin A pollen was not caused by the differences in energy metabolism substrates,probably due to the enzyme regulation imbalance during the energy metabolism pathway.The content of pyruvic acid in the male sterile line of Yamian A was higher than that of the maintainer line during the development of microspores,and the content of pyruvic acid was significantly higher than that of the maintainer in the critical period of microspore abortion.The accumulation of pyruvate may inhibit the progress of the tricarboxylic acid cycle,resulting in insufficient energy during the development of microspores.