The Functional Analysis Of Differentially Expressed Genes ZmAI And ZmAO In Maize CMS-C

Maize is one of the earliest crops to utilize heterosis.Cytoplasmic male sterility（CMS）has become an important tool for maize hybrid production.The study of its abortion and fertility restoration mechanism has very important application value and practical significance.Previously,453 differentially expressed genes were detected between the CMS-C line C48-2 and its maintainer line 48-2 at the pollen mother cell stage and mononuclear stage of tassel.Particularly,at the mononuclear stage of microspore development,gene GRMZM2G074017 encoding an ATPase inhibitor（AI）was up-regulated in C48-2,which was 3.5 times higher than that of 48-2.And gene GRMZM2G139689 encoding a L-aspartate oxidase（AO）was down-regulated in male sterile line C48-2,and the expression level was only 5.47%in maintainer line 48-2.We speculated that the differential expression of these two genes between the two lines may be related to the pollen abortion caused by energy deficit in CMS-C male sterile line of maize,and preliminary study on the function of these two genes was carried out.In this study,we first explored the dynamic changes of m RNA expression level of target genes at different development stages of anthers in CMS-C male sterile lines,homonuclear heterogenous maintainer lines and near-isogenic line transferred into the restorer gene Rf4 by q RT-PCR,in order to validate the results of transcriptome sequencing.Secondly,q RT-PCR analysis of m RNA of target genes in different tissue of maize at different development stages of microspore was performed to reveal the expression patterns.In addition,we cloned,compared and analyzed the protein coding regions of ZmAI and ZmAO genes between sterile line C48-2 and maintainer line 48-2.And subcellular localization and transgenic function verification were carried out.The main results were as follows:1.We used q RT-PCR to observe the transcription levels of ZmAI and ZmAO genes in anther development of C-type cytoplasmic male sterile lines C48-2,C-Huangzaosi（C-HZS）and maintainer lines 48-2,Huangzaosi（HZS）and[（C-HZS×ZJ401）×HZS]BC₄F₁ fertile restoration lines,which were transferred into restorer gene Rf4 were analyzed.And the results showed that ZmAI was significantly up-regulated during the meiotic phase of C-type cytoplasmic male sterile lines.ZmAO was abundantly expressed in the mononuclear stage of the maintainer lines and fertility restorer lines with introduced restorer gene,but was hardly expressed in the sterile lines.It was speculated that the two genes may be related to anther development.The tissue expression analysis of the two genes showed that both of them were expressed in different tissues and there were different degrees of transcription in the anther.2.The coding regions of the two genes were cloned and sequenced by using anther c DNA of sterile line C48-2 and maintainer line 48-2,respectively.The results showed that both genes shared no base difference between 48-2 and C48-2.The CDS region of ZmAI was 291 bp in length and encoded 96 amino acids.The size of predicted protein was 10.6KD without any conserved domain.Multiple subcellular localization predicted that it might be expressed in nucleus or mitochondria.The CDS region of ZmAO was 1995 bp in length.It encoded 664 amino acids with a predicted protein size of 72.9 KD.SMART predicted that it contained two conservative domains,FAD＿binding＿2 and Succ＿DH＿flav＿C.Subcellular Localization predicted that it was likely to be expressed in mitochondria,chloroplasts or peroxidase.In addition,phylogenetic tree results of both genes indicated that maize had a close relationship with sorghum and setaria italica.3.Subcellular localization results showed that ZmAI was expressed in cytoplasm,nucleus and cell membrane,which was not consistent with the predicted results of software.ZmAO was specifically localized in chloroplast,which was consistent with the expression of the homologous gene in Arabidopsis thaliana.It was speculated that ZmAO in maize might be involved in NAD⁺synthesis in similar metabolic pathways.4.Prokaryotic expression vectors pGEX-ZmAI and pGEX-ZmAO were constructed and transformed into Escherichia coli expression strain Rosetta-gami（DE3）p Lys S.The target fusion proteins were successfully induced by IPTG.The OD₆₀₀ value changes showed that the induced expression of AI protein inhibited the growth of Escherichia coli,suggesting that ZmAI may participate in energy metabolism as a negative regulator.5.The over-expression vector p C1300-35S-ZmAI was constructed and transferred into wild-type Arabidopsis thaliana through Agrobacterium tumefaciens.The fertility of different transgenic lines was reduced to different degrees,which indicated that ZmAI gene in maize could effect the seed setting of plants.At the same time,we observed the phenotype of the homologous gene AO/ao mutants in Arabidopsis thaliana.It was found that homozygous mutants were lethal,and the rosette of the heterozygous mutants became significantly smaller,with weaker growth and less fruiting.At present,expression vector p C1300-35S-ZmAO has been constructed and transformed into heterozygous mutants in Arabidopsis thaliana.The next step is to analyze phenotypic changes of transgenic mutants to explore the biological function of ZmAO.