玉米雄性不育基因MALE STERILE33的克隆和功能分析
发布时间：2018-12-20 06:24
【摘要】：脂质代谢相关基因在高等植物花药孢粉素和角质素合成过程中具有重要作用,这些基因的突变常常导致花粉外壁和花药角质层发育障碍,并最终导致雄配子败育和雄性不育的发生。尽管目前在玉米中发现了几十个雄性不育突变体,然而仅有少数基因被克隆。玉米经典雄性不育突变体ms33被发现于1995年,其不育表型稳定,具有很强的商业应用潜力。然而,导致该突变的基因却尚未被克隆。在本研究中,我们通过图位克隆的策略获得了玉米MS33基因,并采用分子遗传学,细胞生物学,发育生物学以及分析化学手段对相关突变体进行了全面研究。作为在禾本科植物中被克隆的第一个GPAT蛋白编码基因,SA1/MS33在控制花药相关聚酯合成中具有关键作用,参与指导花药角质层和花粉外壁形成。获得的主要结果如下：1.在国家玉米改良中心构建的玉米MuDR突变体库中,发现了一个隐性雄性核不育突变体shrinking anther 1 (sa1),该突变体营养生长阶段正常,花粉完全败育,不育性状不受环境影响。通过细胞学分析表明,sal突变体花药绒毡层在小孢子发育早期发生降解,花粉外壁异常,花药表皮角质层不能正常形成。2.等位测验表明,sa1突变体与一个经典的玉米雄性不育突变体male sterile 33(ms33)等位。我们用sal突变体分别与郑58,B73白交系构建了F2定位群体,将突变位点定位在玉米2号染色体190 Kb的区间内,随后对区间内全部基因进行测序分析,获得了候选基因GRMZM2G070304。测序发现,在sal突变体中,该基因第二外显子存在247 bp片段插入；在ms33-6019等位系中,基因第一外显子缺失479 bp片段；在ms33-6024等位系中,基因编码区第507碱基位置插入2个碱基,发生移码导致翻译提前终止。SA1/MS33基因编码一个甘油醛-3-磷酸酰基转移酶(GPAT),该基因是在禾本科植物中克隆的第一个GPAT基因。3. qPCR和RNA原位杂交分析表明,SA1/MS33主要在根系以及花药绒毡层中表达。通过气相色谱-质谱分析,发现sa1突变体花药聚酯单体含量的显著降低,然而根系中的聚酯单体成分并未发生显著变异。因此,我们推测基因突变导致花药绒毡层内聚酯合成障碍可能是导致花药外壁和花药表皮角质异常的主要原因。综上所述,本研究证明玉米SA1/MS33基因在花药绒毡层中参与花药相关聚酯合成,这些物质进一步参与花药表皮角质和花粉外壁的形成。这一过程的异常导致小孢子发育早期败育,并最终表现雄性不育表型。研究结果对阐释玉米花药发育和脂质代谢过程具有重要意义,并为雄性核不育基因在育种上的应用提供了新的遗传资源。
[Abstract]:Lipid metabolism related genes play an important role in the anther sporopollen and keratin synthesis in higher plants. The mutations of these genes often lead to the development of pollen outer wall and anther cuticle. And finally lead to male gametes abortion and male sterility. Although dozens of male sterile mutants have been found in maize, only a few genes have been cloned. Maize classic male sterile mutant ms33 was discovered in 1995. Its sterility phenotype is stable and has strong commercial application potential. However, the gene that caused the mutation has not yet been cloned. In this study, we obtained the MS33 gene of maize by the strategy of map-cloning, and studied the mutants by molecular genetics, cytobiology, developmental biology and analytical chemistry. As the first GPAT protein encoding gene cloned in Gramineae, SA1/MS33 plays a key role in the control of anther associated polyester synthesis and plays a key role in the formation of anther cuticle and pollen outer wall. The main results are as follows: 1. A recessive male sterile mutant shrinking anther 1 (sa1) was found in the maize MuDR mutants bank constructed by the National Maize improvement Center. The mutant had normal vegetative growth stage, completely aborted pollen, and had no environmental influence on sterile traits. Cytological analysis showed that the anther tapetum of sal mutant was degraded in the early stage of microspore development, the outer wall of pollen was abnormal, and the epidermal cuticle of anther could not be formed normally. 2. Allelic tests showed that the sa1 mutant was allelic to a classic maize male sterile mutant male sterile 33 (ms33). The F2 locus was constructed by using sal mutants and Zheng58 B73 white cross lines respectively. The mutation site was located in the interval of maize chromosome 2 190 Kb. Then all the genes in the region were sequenced and the candidate gene GRMZM2G070304. was obtained. Sequencing showed that there were 247 bp fragments inserted in the second exon of the gene in the sal mutant, 479 bp fragment was absent in the first exon of the gene in the ms33-6019 allele, and 479 bp fragment was deleted in the first exon of the gene in the ms33-6019 allele. In the ms33-6024 allele, two bases were inserted at the 507th base in the coding region of the gene, resulting in the early termination of translation. The SA1/MS33 gene encodes a glyceraldehyde 3-phosphate acyltransferase (GPAT),. The gene is the first GPAT gene cloned in gramineous plants. 3. 3. QPCR and RNA in situ hybridization analysis showed that SA1/MS33 was mainly expressed in roots and anther tapetum. Gas chromatography-mass spectrometry (GC-MS) analysis showed that the content of polyester monomer in the anther of sa1 mutant decreased significantly, but there was no significant variation in the component of polyester monomer in the root system. Therefore, we speculate that the disorder of polyester synthesis in anther tapetum caused by gene mutation may be the main cause of anther outer wall and anther epidermis keratinocyte abnormality. In conclusion, this study demonstrated that maize SA1/MS33 gene was involved in anther related polyester synthesis in anther tapetum, and these substances were further involved in the formation of epidermal keratinocytes and pollen outer walls of anthers. The abnormality of this process resulted in early abortion of microspore development and eventually the phenotype of male sterility. The results are of great significance in elucidating the anther development and lipid metabolism in maize and provide a new genetic resource for the application of male sterile gene in breeding.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S511
本文编号：2387571
[Abstract]:Lipid metabolism related genes play an important role in the anther sporopollen and keratin synthesis in higher plants. The mutations of these genes often lead to the development of pollen outer wall and anther cuticle. And finally lead to male gametes abortion and male sterility. Although dozens of male sterile mutants have been found in maize, only a few genes have been cloned. Maize classic male sterile mutant ms33 was discovered in 1995. Its sterility phenotype is stable and has strong commercial application potential. However, the gene that caused the mutation has not yet been cloned. In this study, we obtained the MS33 gene of maize by the strategy of map-cloning, and studied the mutants by molecular genetics, cytobiology, developmental biology and analytical chemistry. As the first GPAT protein encoding gene cloned in Gramineae, SA1/MS33 plays a key role in the control of anther associated polyester synthesis and plays a key role in the formation of anther cuticle and pollen outer wall. The main results are as follows: 1. A recessive male sterile mutant shrinking anther 1 (sa1) was found in the maize MuDR mutants bank constructed by the National Maize improvement Center. The mutant had normal vegetative growth stage, completely aborted pollen, and had no environmental influence on sterile traits. Cytological analysis showed that the anther tapetum of sal mutant was degraded in the early stage of microspore development, the outer wall of pollen was abnormal, and the epidermal cuticle of anther could not be formed normally. 2. Allelic tests showed that the sa1 mutant was allelic to a classic maize male sterile mutant male sterile 33 (ms33). The F2 locus was constructed by using sal mutants and Zheng58 B73 white cross lines respectively. The mutation site was located in the interval of maize chromosome 2 190 Kb. Then all the genes in the region were sequenced and the candidate gene GRMZM2G070304. was obtained. Sequencing showed that there were 247 bp fragments inserted in the second exon of the gene in the sal mutant, 479 bp fragment was absent in the first exon of the gene in the ms33-6019 allele, and 479 bp fragment was deleted in the first exon of the gene in the ms33-6019 allele. In the ms33-6024 allele, two bases were inserted at the 507th base in the coding region of the gene, resulting in the early termination of translation. The SA1/MS33 gene encodes a glyceraldehyde 3-phosphate acyltransferase (GPAT),. The gene is the first GPAT gene cloned in gramineous plants. 3. 3. QPCR and RNA in situ hybridization analysis showed that SA1/MS33 was mainly expressed in roots and anther tapetum. Gas chromatography-mass spectrometry (GC-MS) analysis showed that the content of polyester monomer in the anther of sa1 mutant decreased significantly, but there was no significant variation in the component of polyester monomer in the root system. Therefore, we speculate that the disorder of polyester synthesis in anther tapetum caused by gene mutation may be the main cause of anther outer wall and anther epidermis keratinocyte abnormality. In conclusion, this study demonstrated that maize SA1/MS33 gene was involved in anther related polyester synthesis in anther tapetum, and these substances were further involved in the formation of epidermal keratinocytes and pollen outer walls of anthers. The abnormality of this process resulted in early abortion of microspore development and eventually the phenotype of male sterility. The results are of great significance in elucidating the anther development and lipid metabolism in maize and provide a new genetic resource for the application of male sterile gene in breeding.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S511
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1 张磊;玉米雄性不育基因MALE STERILE33的克隆和功能分析[D];中国农业大学;2016年
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