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Analyses Of The Floral Organ Morphogenesis And The Differentially Expressed Genes Of An Apetalous Flower Mutant Apet33-10 In Brassica Napus

Posted on:2008-02-26Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y T ZhouFull Text:PDF
GTID:1103360242464093Subject:Biochemistry and Molecular Biology
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Brassica napus line Apet33-10 is an apetalous mutant which is controlled by one gene locus. Floral organ morphogenesis was investigated and the result showed that there was no morphological difference between Apet33-10 and its wildtype line Pet33-10 at the stage of floral organs initiation. All the floral organ morphogenesis of Apet33-10 was normal except that the petal primordium was not observed during flower development. Petal loss occurred at the stage of petal primordium initiation.Eighteen genes were determined to be down-regulated in early floral buds (less than 200μm in length) of Apet33-10 at the stage of floral organ initiation by means of suppressive subtraction hybridization and RT-PCR. These genes were involved in petal identity, calcium iron signal transduction, mRNA processing, protein synthesis and degradation, construction of cytoskeleton, hydrogen transportation, nucleic acid binding, alkaloid biosynthesis and unknown function. Most of the down-regulated genes do not seem to be implicated previously in petal formation except for AP3 gene. It was presumed that the mutation of Apet33-10 could cause some genes of master transcription factors in proliferation of the floral meristem, such as AP3 and PI, down-regulated at first, then the reduced expression of the master transcription factors could cause the downstream genes related to petal primordium initiation down-regulated, resulting in the disruption of petal development.Three overall coding region cDNAs of AP3 gene, BnAP3-2, BnAP3-3 and BnAP3-4 were obtained by RT-PCR, respectively. Phylogenetic analysis shows that BnAP3-4 and BrAP3 belong to a same homological group, revealing that BnAP3-4 should originate from B. rapa and be located in AA genome. BnAP3-2 and BnAP3-3 belong to BoilAP3 homological group, revealing that BnAP3-2 and Bn,4P3-3 should derive from B. oleracea and be located in CC genome. Real-time quantitative PCR analysis showed that the expression proportion among BnAP3-2, BnAP3-3 and BnAP3-4 was 3.67: 3.68:1 in early floral buds of wild type Pet33-10. The expression level of BnAP3-2, BnAP3-3 and BnAP3-4 in early floral buds of Apet33-10 was down-regulated to 36.6%, 28.3% and 66.8% with the comparison of that of wild type, respectively, and the overall expression level of AP3 genes in apetalous mutant amounted to 45.0% of that in wild type. The difference of expression level of each AP3 gene in stamen between apetalous and wild type lines was not significant. Yeast two-hybridization analysis showed that there were different affinities between the three AP3 proteins and BnPI-3 protein. The divergences of sequence, expression and interaction with PI might reflect some functional divergences of the three ap3 genes.BnSmD1 belongs to Sm protein family, which may be involved in pre-mRNA splicing and processing. BnSmD1 expression was obviouly down-regulated in early floral buds of Apet33-10. Plant over expression vector pFGC-BnSmD1(+) and antisense expression vector pFGC-BnSmD1(-) were constructed in order to realize the detail founction of BnSmD1. Cotyledon sections of B. napus line 197 were as recipient to be transformed with pFGC-BnSmD1(+) and pFGC-BnSmD1(-) by Agrobacterium-mediated method. Fifty two regenerated plant with resistance to Basta were obtained, among which 43 plants were transformed with pFGC-BnSmD1(+) and 9 plants with pFGC-BnSmD1(+). Some transformed plants with over expression of BnSmD1 were identified by PCR.
Keywords/Search Tags:Brassica napus, Floral development, Subtractive suppression hybridization, Dot bloting, Real-time quantitative PCR, Yeast two hybridization, Transgene
PDF Full Text Request
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