CMS Germplasm Innovation And Sterile Mechanism Of Transgenic Upland Cotton With GhbZIP1 Gene

Cotton(Gossypium hirsutum L.)is one of the main natural fiber crops in the world,and also an significant oil crop.The heterosis of cotton is very significant,but cotton cytoplasmic male sterility(CMS)germplasm resources are scarce at present.The CMS lines used are mainly cotton cytoplasmic.Because the cytoplasmic donor is diploid,and the nuclear donor is tetraploid donor,the cytoplasmic nuclear interaction is seriously discordant,and there are few restorer lines.In order to solve the problem of serious cytoplasmic nucleus disharmony caused by nucleus replacement backcross in distant hybridization,it is necessary to breed CMS lines with homologous cytoplasm and nucleus.In this study,based on the transcriptome sequencing results of the genic male sterile line"Dong A"in upland cotton,a GhbZIP1 with differential expression in sterile and fertile plants was screened and transferred into an upland cotton cultivar J4B with excellent characters.After obtaining the transgenic male sterile mutant,the CMS line J4A was successfully developmented by saturated backcrossing with J4B.After that,the morphological and cytological observation,mitochondrial genome,whole transcriptome analysis and biochemical characteristics of J4A were carried out,the main results are as follows:(1)Morphological observation showed that the flowers organ of J4A were smaller,the style was prominent and the anthers were shriveled without dehiscence,and the abortion type of J4A belonged to non-pollen type.(2)Observation of pollen abortion characteristics showed that the anther abortion stage of J4A was from microspore pollen mother cell stage to tetrad stage(meiosis),which showed that tapetum cells did not degrade and no tetrad structure appeared during the whole period of anther development.The ultrastructural observation of anther cells showed that the inner ridges of mitochondria in tapetal cells of J4A were fuzzy at tetrad stage.(3)The mitochondrial genome of three CMS lines(J4A-1,J4A-2 and J4A-3)and their maintainer line J4B were dwarfed,and their similarity was beyond 99%.Four ORFs(orf116b,orf186a-1,orf186a-2 and orf305a)with CMS related gene characteristics were found in the CMS lines J4A-1,J4A-2 and J4A-3,which were identified as CMS related candidate genes.(4)The anthers at abortion stage were sequenced by Illumina Hiseq 4000 sequencing platform.The results of m RNA transcriptome sequencing showed that 4,461 differentially expressed genes(7.16%)were screened out of62,270 genes detected,among which 2,940 genes were up-regulated and 1,521genes were down-regulated.Bioinformatics analysis showed that 489differentially expressed genes were enriched in redox reaction,47 differentially expressed genes were involved in glycolysis pathway,and more down regulated genes were found.By analyzing the regulatory network of the selected differentially expressed genes,the screening scope of cotton CMS key genes was narrowed.(5)Mi RNAs were sequenced by Illumina Hiseq 4000 sequencingplatform,a total of 1,293 miRNAs were obtained,including 734 known miRNAs and 59 new miRNAs,and there were 26 differentially expressed miRNAs.A pair of target genes(ghi-MIR7484-10/MAPKK6)that may be related to the abnormal meiosis of microspore were screened by miRNA and m RNA association analysis.According to the regulatory relationship between miRNA and its target genes,the correlation analysis of m RNA sequencing results and miRNA sequencing results can more accurately screen the key genes involving the regulation of CMS more accurately.(6)Biochemical indexes were determined.The biochemical indexes (SOD,Mn-SOD,POD,CAT,H₂O₂and MDA)related to the metabolism of reactive oxygen species(ROS)were measured at three important stages of anther development:pollen mother cell stage,meiosis stage and monocyte stage.The results showed that the activities of SOD,Mn-SOD,POD and CAT of J4A were lower than those of J4B,and the contents of H₂O₂and MDA were significantly higher than those of J4B at meiosis stage and monocyte stage.It is speculated that the decrease of antioxidant enzyme activities(SOD,Mn-SOD,POD and CAT)during meiosis leads to the accumulation of reactive oxygen species(H₂O₂and MDA)in J4A anthers.Sugar metabolism related biochemical indexes(sucrose,starch,soluble sugar and fructose)were measured at three important stages of anther development:pollen mother cell stage,meiosis stage and mononuclear stage.The results showed that the sucrose content of J4A was higher than that of J4B at meiosis stage and mononuclear stage,and the contents of starch,soluble sugar and fructose were lower than that of J4B at the three important stages of anther development.It was speculated that the abnormal accumulation of sucrose led to the decrease of glucose and fructose,which resulted in the decrease of starch content and soluble sugar content.