| MS5(male sterile5) gene, also known as POLLENLESS3gene or TDM1gene, is a gene essential for male fertility in Arabidopsis. first discovered in Arabidopsis thaliana. MS5is a gene essential for male fertility in Arabidopsis. MS5mutants were observed male-sterile. Many researches showed that MS5/POLLENLESS3gene encodes a protein that plays a critical role in regulating cell-cycle activity within meiotically-dividing cells in developing anthers. Pollen tetrads undergo an extra round of division after meiosis Ⅱ without chromosome replication, resulting in chromosome abnormalities in MS5mutants. It’s gene product has some similarity to SCP1, a rat synaptonemal complex protein(Chaudhury et al.,1994). Genetic analyses of the MS5mutation confirm that it is a nuclear recessivesporophytic mutation. The MS5gene is a member of a small gene family highly conserved amongst plant species.Here, the CMS line L23A and its maintainer line L23B in Kenaf (Hibiscus Cannabinus L.) that were bred by Zhou Ruiyang etc. were employed by Li gang, based on the characteristics of definite inflorescence with many and large anthers, the proteins were directly extracted from the mitochondria of the anthers respectively during uninucleate and binucleate stage individually, then the proteins were labeled by4-plex iTRAQ (isobaric Tags for Relative and Absolute Quantification), followed by the two Dimensional Liquid Chromatograph separation and tandem mass spectrometry identification, the quantititaion proteomic differeces among them were compared. The significant proteins between the two types of materials during uninucleate stage were analyzed concerning the Gene Ontology, biology processes and pathways.93proteins with significant differences were identified from mitochondria of anthers between CMS line and its maintainer line in Kenaf. Selected No.326proteins blast with oryza sativa gene bank and protein bank. Characterization No.326proteins homology with POLLENLESS3, Synonym with MS5(male sterile5) gene. In this study,we blast all kind of Nucleotide and Proteins database, selected3MS5/POLLENLESS3gene in oryza sativa gene bank were the orthologs of MS5/POLLENLESS3, molecular function unknown, give the name OsMs5-1, OsMs5-2and OsMs5-3and analysis Gene sequence.The fuction of the3rice MS5gene were studied by RNAi-mediated Gene Silencing and overexpressed. We cloned the MS5of Kenaf(Hibiscus Cannabinus L.) and analysis Gene sequence and studied it’s fuction by overexpressed. Characterized MS5Gene function in Rice(oryza sativa) and Kenaf (Hibiscus Cannabinus L.) The results in detail were as fallows:1. Isolation and characterization of OsMs5-1、OsMs5-2and OsMs5-3To investigate the regulation mechanism male fertility in rice, we isolated full length cDNAs of OsMs5-1、OsMs5-2and OsMs5-3from spikelets The full length of OsMs5-1、OsMs5-2and OsMs5-3encoding predicted protein with299、315、515amino acid residues. In its’N terminus, the proteins had TPR domain which containing TPR motif (PS50005). Sequences analysis revealed that OsMs5-1、OsMs5-2and OsMs5-3all belong to the TPR domain family. Sequencing analysis showed that OsMs5-1、OsMs5-2and OsMs5-3was closer to Arabidopsis MS5suggesting that OsMs5-1、OsMs5-2and OsMs5-3were the orthologs of MS5.2. Function analysis of OsMs5-1、OsMs5-2and OsMs5-3in rice. Our important research is in OsMs5-1、OsMs5-2and OsMs5-3gene of rice,The function of the three gene in the rice male sterile revealed by RNA interfence vector RNA interference we also constructed, grobacterium-mediated transfer into rice. Southern hybridization of the thansplant T1nptⅡ as probe most of the the transgenetic plant T-DNA copy were1-3different copy. The transfer were succeed. examined for defects in male-sterile mutants phenotype and Light microscopy and Scanning electron microscopy. The pollen of the transgenetic plant is abnormal as the wild tpye plant.3. Except RNA interference we also constructed overexpression vector, Agrobacterium-mediated transfer into rice. Molecular analysis comfirmed The transfer were succeed. Subcellular Localization of GFP-Tagged Proteins in the positive plant root tip and callus Cells.4. In order to understand the transcriptal characteristic of MS5gene in kenaf, we tried to clone the full mRNA of kenaf MS5gene on the basis of known partial5’-end cDNA sequence by RT-PCR and ’3’RACE methods. The sequencing results showed that two transcript cDNAs of MS5gene were cloned from kenaf anther. The length of two transcripts were972bp and1105bp, respectively, and were named as HcMs5-a and HcMs5-β subsequently. The structure analysis showed that both of them contained a858bp ORF, encoding a protein of285amino acids with predicted molecular weight of32.4kD and a isoelectric point of7.74. The sequencing results showed that5’-end972bp sequence of the two transcripts was identical, but HcMs5-β possessed an additional133bp sequence at3’-end after972bp compared with HcMs5-α. The alternation of transcription termination in the3’UTR of kenaf MS5maybe related to the posttranscriptional regulation of the gene. This is the first report about the multiple transcripts of MS5gene in higher plant.Semi-RT-PCR analysis result indicated HcMS5gene expressed in all kind of organ. The expression of HcMS5was higher in root, petals than other organs, and maintainer line is higher and sterile line. lower expressed in the stem, leaves, anthers. anthers at the monokaryotic stage and dikaryotic stage of sterile line no difference. This result difference from li gang’s research that protein in anthers at the monokaryotic stage and dikaryotic stage of sterile line is significance difference. maybe the function of HcMS5gene related to the posttranscriptional regulation of the gene. Transfer HcMS5gene by Agrobacterium tumefaciens for rice, Subcellular Localization of GFP-Tagged Proteins in the positive plant root tip and callus Cells.5. The enzyme5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase is essential for the biosynthesis of aromatic compounds in prokaryote, fungi, plants and algae. It is the unique target of the herbicide glyphosate. Glyphosate is a broad-spectrum herbicide. Plants can obtain high doses glyphosate tolerance if the enzyme of this gene is overproduced and accumulated or herbicide-insensitive enzymes produced due to some amino acid mutation in active sites. The Application of Gene Engineering on Creating Male Sterility in Plant, Genetic engineering technology opens up a new way to create male sterility line in plant-but how to maintain male sterility is a problem in Genetic engineering technology create male sterility line in plant. the creation of herbicide resistant Male Sterility line, Screening herbicide resistant Male Sterility line by sprayed glyphosate herbicide treatment can solve male sterility line maintain problem. Here, we constructed overexpression vector with Tobacco gene (gene ID M61904). Transfer Tobacco EPSPS Gene by Agrobacterium tumefaciens for rice. Molecular analysis comfirmed The transfer were succeed. Sprayed30mmol/L and35mmol/L glyphosate on the transgenetic plant T1progeny. Result comfirmed the transgenetic plant power glyphosate resistance than wild type. |
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