玉米雄性不育基因MALE STERILE33的克隆和功能分析
發(fā)布時間：2018-12-20 06:24
【摘要】：脂質(zhì)代謝相關(guān)基因在高等植物花藥孢粉素和角質(zhì)素合成過程中具有重要作用,這些基因的突變常常導(dǎo)致花粉外壁和花藥角質(zhì)層發(fā)育障礙,并最終導(dǎo)致雄配子敗育和雄性不育的發(fā)生。盡管目前在玉米中發(fā)現(xiàn)了幾十個雄性不育突變體,然而僅有少數(shù)基因被克隆。玉米經(jīng)典雄性不育突變體ms33被發(fā)現(xiàn)于1995年,其不育表型穩(wěn)定,具有很強(qiáng)的商業(yè)應(yīng)用潛力。然而,導(dǎo)致該突變的基因卻尚未被克隆。在本研究中,我們通過圖位克隆的策略獲得了玉米MS33基因,并采用分子遺傳學(xué),細(xì)胞生物學(xué),發(fā)育生物學(xué)以及分析化學(xué)手段對相關(guān)突變體進(jìn)行了全面研究。作為在禾本科植物中被克隆的第一個GPAT蛋白編碼基因,SA1/MS33在控制花藥相關(guān)聚酯合成中具有關(guān)鍵作用,參與指導(dǎo)花藥角質(zhì)層和花粉外壁形成。獲得的主要結(jié)果如下：1.在國家玉米改良中心構(gòu)建的玉米MuDR突變體庫中,發(fā)現(xiàn)了一個隱性雄性核不育突變體shrinking anther 1 (sa1),該突變體營養(yǎng)生長階段正常,花粉完全敗育,不育性狀不受環(huán)境影響。通過細(xì)胞學(xué)分析表明,sal突變體花藥絨氈層在小孢子發(fā)育早期發(fā)生降解,花粉外壁異常,花藥表皮角質(zhì)層不能正常形成。2.等位測驗(yàn)表明,sa1突變體與一個經(jīng)典的玉米雄性不育突變體male sterile 33(ms33)等位。我們用sal突變體分別與鄭58,B73白交系構(gòu)建了F2定位群體,將突變位點(diǎn)定位在玉米2號染色體190 Kb的區(qū)間內(nèi),隨后對區(qū)間內(nèi)全部基因進(jìn)行測序分析,獲得了候選基因GRMZM2G070304。測序發(fā)現(xiàn),在sal突變體中,該基因第二外顯子存在247 bp片段插入；在ms33-6019等位系中,基因第一外顯子缺失479 bp片段；在ms33-6024等位系中,基因編碼區(qū)第507堿基位置插入2個堿基,發(fā)生移碼導(dǎo)致翻譯提前終止。SA1/MS33基因編碼一個甘油醛-3-磷酸�；D(zhuǎn)移酶(GPAT),該基因是在禾本科植物中克隆的第一個GPAT基因。3. qPCR和RNA原位雜交分析表明,SA1/MS33主要在根系以及花藥絨氈層中表達(dá)。通過氣相色譜-質(zhì)譜分析,發(fā)現(xiàn)sa1突變體花藥聚酯單體含量的顯著降低,然而根系中的聚酯單體成分并未發(fā)生顯著變異。因此,我們推測基因突變導(dǎo)致花藥絨氈層內(nèi)聚酯合成障礙可能是導(dǎo)致花藥外壁和花藥表皮角質(zhì)異常的主要原因。綜上所述,本研究證明玉米SA1/MS33基因在花藥絨氈層中參與花藥相關(guān)聚酯合成,這些物質(zhì)進(jìn)一步參與花藥表皮角質(zhì)和花粉外壁的形成。這一過程的異常導(dǎo)致小孢子發(fā)育早期敗育,并最終表現(xiàn)雄性不育表型。研究結(jié)果對闡釋玉米花藥發(fā)育和脂質(zhì)代謝過程具有重要意義,并為雄性核不育基因在育種上的應(yīng)用提供了新的遺傳資源。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S511
本文編號：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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S511
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1 張磊;玉米雄性不育基因MALE STERILE33的克隆和功能分析[D];中國農(nóng)業(yè)大學(xué);2016年
，本文編號：2387571
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