| The recessive genetic male sterility (RGMS) system has been considered as an effective and economical pollination control system in hybrid seed production of Brassica napus for its stable and complete sterility, rich sources of cytoplasm and widely spread of restorers. To date, the major materials being utilized are S45AB (double recessive material) and7365ABC (triple recessive material).7365A is more potential for applying since it’s no need to remove50%fertile plants during commercial hybrid seeds production of the F1generation by using7365C. In this research,7365AB, a near isogenic lines (NILs) were chosen as materials. The only difference between them lies in the BnMs3locus which is responsible for the fertility of7365A. Cytology combined with molecular biology techniques were utilized to demonstrate the molecular mechanism of7365A recessive genetic male sterility, and we have first proposed the functional role of BnMs3in anther of B. napus; meanwhile by using the T-DNA mutant resources in Arabidopsis, functions of several differentially expressed genes were analyzed as well. Our chief achievements are as follows.1. Researches on molecular mechanism of7365A recessive genetic male sterility in B. napusThe cytological differences of anther development in the plant7365A which is male sterile and the plant7365B which is male fertile were analyzed by using semi-thin sections, and we found that there was no apparent difference during the early meiosis stage between the two materials. But when it entered the post-meiotic stage, the tapetal cells of7365A (male sterile) kept on enlarging and vacuolating, and could not convert into the secreted type. Subsequently, the release of microspores was blocked, and most microspores couldn’t be released from tetrad, while the few released were not able to form normal pollen exine, which made the pollen abortion more thorough. The following results of cytochemical staining for callose wall in the wild type and the Bnms3mutant with aniline blue suggested that it was the degradation defect of callose around tetrad that caused the microspores could not be separated normally.The library rich in up-regulated genes in7365B (male fertile) was constructed by using suppression subtractive hybridization (SSH) technology. In combination with the reverse Northern blot screening,76differentially expressed unigenes were identified. The validity of seven unigenes randomly picked was proved by Northern blot analysis which also suggested that these seven genes could be divided into two types based on their different expression patterns. Type I genes only expressed in7365B or showed lower expression level at the corresponding stages in the mutant (7365A). Type Ⅱ genes all expressed in buds of NILs, but their expression was delayed or repressed in7365A at the early stage of anther development. According to the annotated gene function information from Arabidopsis, these76unigenes were preliminarily annotated. As a result, eight of them could not be annotated by this means while the other68unigenes were successfully annotated. They were classified into eight functional categories based on the information from MIPS (Munich Information Center for Protein Sequences). The first group of these unigenes mainly encoded enzymes related to metabolism. The second group of unigenes were annotated as one ATP-binding cassette (ABC) transport protein and eight lipid transfer proteins (LTPs) which were involved in lipid/fatty acid transport processes. Compared with the microarray data of the spl/nzz, emsl, and msl mutants of anther development from Arabidopsis, several co-down regulated genes were identified. Moreover, it suggested that these differentially expressed unigenes participated in the processes of tapetal development, microspore separation, and pollen exine formation.As Arabidopsis and B. napus are both members of cruciferae, they share high sequence identity in their open reading frames (ORFs) and have similar gene functions. By utilizing the gene information from regulatory network of anther development in Arabidopsis, and combining with real-time PCR analysis, we found that the initial expression levels of genes(TDF1, AMS, and MYB103) involved in the transition of tapetum to the secretory type were significantly inhibited in the mutant. Furthermore, the expression of five cysteine protease (CP) genes associated with tapetal degradation was also affected in Bnms3. Microspore separation are relevant to callose degradation and the following degeneration of primary wall of PMC (pollen mother cell). A6and MSR66both encoded P-1,3-glucanase which might be involved in callose degradation, and their expression levels were both repressed in the mutant. QRT1, QRT2, and QRT3all participated in the degradation of primary wall of PMC. Their analysis of real-time PCR indicated that the expression levels of QRT1and QRT3were depressed during the postmeiotic stage in Bnms3. Afterwards genes included NEF1, FLP1, CYP703A2, ACOS5, BnCYP704B1, BnCHS1(At4g00040), CHS2(At4g34850) and MS2related to the synthesis of sporopollenin precursors were analyzed by real-time PCR, and the results demonstrated that their expression levels were all influenced in the mutant. Additionally, the expression level of MSR02gene (belonging to ABC transporter protein family) which might be associated with the transmembrane transport of sporopollenin precursors, and that of lipid transfer proteins (LTPs) which might participate in the long distance transport of sporopollenin precursors were both down-regulated in the Bnms3mutant.Based on all the above results, the function model of BnMs3during anther development in B. napus was proposed for the first time.2. Function analysis of genes related to fertilityThree genes (BnMSR02, BnMSR42, and BnMSR53) were selected for further analysis based on gene expression changes in the Bnms3mutant using T-DNA (transferred DNA) insertion mutants of their homologous genes from Arabidopsis to understand functions of them in anther development. As a result, only MSR02encoding an ABC transporter protein which was involved in transport of lipid molecules was required for pollen development. Lipids synthesized in the tapetal cells have two destinies, namely biosynthesis of sporopollenin and anther epidermis cutin. The following results of scanning electron microscopy and transmission electron microscopy illustrated that the development of pollen exine was abnormal in the msrO2mutant, but that of the anther epidermis cutin was unaffected. It means that MSR02is required for the transmembrane transport of lipids to form pollen exine, but not for that to form anther epidermis cutin. The results of RT-PCR, promoter analysis combined with mRNA in-situ hybridization revealed that MSR02merely expressed in anther tapetal cells of B.napus. |
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