Cloning And Analysis Of Male Sterile Genes HcMS1 And HcAMs In Kenaf

Kenaf（Hibiscus cannabinus L.）is a fast-growing fiber crop of Hibiscus.Kenaf has obvious heterosis.Cytoplasmic male sterile line can save artificial emasculation and improve hybrid purity,which is an effective way to utilize heterosis.The cytological reason of CMS line abortion was that the tapetum degenerated seriously in mononuclear stage,which was obviously earlier than the maintainer line.In this experiment,the excellent kenaf CMS line LC0301A and maintainer line LC0301B were used as materials.The microspore was abnormal and could not form normal pollen.To further explore the molecular biological causes of male sterility,transcriptome sequencing and metabonomic analysis were carried out on male sterile lines and maintainer lines to explore the genes,metabolites and key metabolic pathways involved in the regulation of kenaf male sterility,and the sterility related genes HcMS1 and HcAMS were cloned and transformed to clarify the gene function.The main results are as follows:1.A total of 3651 differentially expressed genes,1816 up-regulated genes and 1835 down regulated genes were detected between kenaf male sterile line and maintainer line.GO and KEGG enrichment analysis showed that oxidation-reduction process,regulation of transcription,DNA-templated and protein phosphorylation were significantly enriched,and a large number of genes were differentially expressed in these biological processes;plant hormone signal transduction,starch and sucrose metabolism,phenylpropanoid biosynthesis,pentose and glucuronate interconversions were significantly enriched in KEGG.These metabolic pathways play an important role in kenaf male sterility.In addition,14 sterility related genes,including MS1 and AMS homologous genes,were found in NR database.2.Analysing metabonomics of male sterile line and maintainer line kenaf buds at the developmental stage before and after mononuclear stage,410 differential metabolites were detected in A1-B1 group（before uninucleate stage）,including 257 up-regulated metabolites and 153 down-regulated metabolites;913 differential metabolites were detected in A3-B3 group（after uninucleate stage）,including 391 up-regulated metabolites and 522 down-regulated metabolites.A large number of metabolites were down-regulated in male sterile lines,mainly glycerophosphate,organic oxides,flavonoids,carboxylic acids and their derivatives.KEGG enrichment analysis indicated that oxidative phosphorylation,tyrosine metabolism,citric acid cycle pathway were significantly enriched in A1-B1group;flavonoids,flavonols biosynthesis,amino t RNA biosynthesis,flavonoid biosynthesis pathway were enriched in A3-B3 group.Total 703 differential metabolites were found in A3-A1 group,containing 374 up-regulated metabolites and 329 down-regulated metabolites;Total 365 differential metabolites were detected in B3-B1 group,including 221 up-regulated metabolites and 144 down-regulated metabolites.The metabolism of alanine,aspartic acid and glutamic acid,linoleic acid and citric acid cycle pathway were enriched in A3-A1 group;the metabolism of purine,pentose phosphate pathway and aminoacyl t RNA biosynthesis pathway were essentially enriched in B3-B1 group.3.The HcMS1 and HcAMS genes were cloned and identified to have typical PHD finger domain and b HLH domain by bioinformatics analysis respectively.The results of q RT-PCR presented that the expression level of HcMS1 in kenaf flower buds increased firstly and then decreased,and the expression level of HcMS1 in maintainer lines was significantly higher than that in male sterile lines;the expression level of HcAMS in maintainer lines increased firstly and then decreased,while decreased all the time in male sterile lines,and the expression level of HcMS1 in maintainer lines was much higher than that in male sterile lines.HcMS1 and HcAMS overexpression Arabidopsis and HcMS1overexpression tobacco were obtained by transgenic experiment.The results manifested that the phenotype,pollen grains and seed setting of transgenic Arabidopsis were normal;transgenic tobacco plants were dwarfed,calyx deformed,corolla tube shortened and unable to reproduce seeds under weak light source.Tobacco corolla tube and calyx shape returned to normal under strong light source,anthers cracked normally and pollen appeared,but still could not bred seeds normally.The results of I₂-KI staining showed that the pollen grains of transgenic tobacco were full and vigorous.Scanning electron microscope showed that the appearance of pollen grains of transgenic tobacco was different from that of wild type,and the germinal pore was sunken on one side,which might be the cause of transgenic tobacco sterility.