油菜单显性细胞核雄性不育分子机理研究
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摘要
本研究以单显性细胞核雄性不育油菜分离群体为材料,在对单显性核不育(MDGMS)油菜的育性、花器性状进行观察的基础上,对MDGMS及其等位可育系的小孢子发育过程及败育方式和特征进行了比较研究;利用集群分离法(BSA)分子标记策略,找到了与单显性核不育基因连锁的RAPD标记,并将其转化为稳定可靠的SCAR标记。通过比较分析分子标记DNA序列的同源序列,设计引物在MDGMS及其等位可育油菜中克隆到油菜输入蛋白α基因的genomic DNA和cDNA编码区全长序列。根据克隆的油菜输入蛋白α基因cDNA序列,构建RNAi和Antisense RNA载体并转化油菜;通过这种基因敲除的反向遗传学方法研究输入蛋白α基因在油菜生长发育中的功能。主要结果如下:
1.MDGMS油菜不育株与可育株相比,雄蕊严重退化,花丝不伸长或略有伸长,花药萎缩呈丝状尖三角,花粉囊中空干瘪,无花粉,自交不能结实。而雌蕊具有正常的形态,少部分的柱头有一定的弯曲。用正常花粉给不育花授粉,能正常结实,表明不育花雌蕊是正常可育的。
通过无性繁殖对应植株在温室和大田育性比较研究表明,该不育系完全由遗传效应决定,不受环境因素的影响。
2.对MDGMS育性分离群体中可育株和不育株小孢子发育过程进行石蜡切片比较观察发现,不育系小孢子败育发生在减数分裂前,并且不能形成四分体,小孢子解体于绒毡层解体之前,最后花药成为干瘪的空壳,表明MDGMS雄性不育不是由于绒毡层细胞给小孢子营养供应不良造成,而是小孢子功能缺陷,自身不能继续发育引起的。
3.对同一时期MDGMS不育及等位可育系的花蕾蛋白质表达差异分析表明,可育系花蕾中表达的蛋白质点数要高于不育系。可育系中表达的特有蛋白质分子量主要分布在60kD以下小分子区域,30kD尤为丰富;不育系中表达的特有蛋白按分子量分布相对均匀。两者表达特有蛋白都相对集中在pI6.0-7.5之间。
4.利用集群分离法(BSA)对MDGMS不育基因进行了RAPD分析,找到了与MDGMS不育基因连锁的RAPD标记OPU-031500。将RAPD标记OPU-031500 DNA片段克隆、测序并进行Blast分析表明,该标记DNA序列与拟南芥基因核输入蛋白α高度同源,根据同源的拟南芥序列及其该基因在物种间的保守性,设计特异性引物SCP1/SCP2,在MDGMS不育及等位可育系中都扩增到2.3 kb的单一特异片段。根据突变位点设计引物SCP3/SCP4,在等位可育系中扩增到约1.5kb的单一特异片段,但在不育系中未扩增到任何带,从而将RAPD标记转化为稳定可靠的SCAR标记。
5.在油菜MDGMS不育和等位可育系中克隆到包括起始密码子ATG和终止密码子TGA在内的油菜核输入蛋白α基因编码区全长genomic DNA和cDNA。二者genomic DNA全长分别为2620bp和2614bp。不育和可育系genomic DNA序列之间多个位点存在突变差异。cDNA全长分别为1629bp和1623bp,不育和可育系cDNA序列之间只有2个位点存在突变差异。genomic DNA和cDNA比较表明,油菜输入蛋白α基因(BIMP a)包含10个外显子和9个内含子。
6.根据克隆到的MDGMS不育和可育系输入蛋白αcDNA序列推导出二者对应的氨基酸序列。油菜输入蛋白α由542个氨基酸组成,而突变不育系只有540氨基酸。比较发现不育系由于碱基缺失突变,导致其肽链氨基酸序列组成上有1个位点缺少2个谷氨酰胺(QQ),并进一步导致该蛋白二级结构的变化:α-螺旋和β-转角在突变不育系MDGMS中明显减少。
对推导的氨基酸序列进行结构域分析表明,油菜输入蛋白α结构域包括1个与输入蛋白β结合的N-末端结构域(IBB)、8个ARM重复单元(ARM repeat)和2个HEAT重复单元(HEAT repeat),组成完全与输入蛋白α结构域一致,从而将其命名为BIMP a(Brassica-napus Importinα, BIMP a)。
7.通过构建油菜输入蛋白αRNAi和Antisense RNA载体并转化油菜表明,油菜输入蛋白α不仅控制雄配子的发育,也控制雌配子的发育,同时与植株的长势及抗病性相关。转基因植株长势较差,不能结实且易感病。
A novel genic male sterile (GMS) line in Brassica napus L., which was identified by Hybrid Rapeseeed Research Center of Shaanxi Province in 1999, was found to be controlled by a monogenic dominant gene which we have designated as MDGMS. The F1 fertility from any fertile lines crossed with MDGMS segregated and the ratio was close to 1:1. Its fertility, floral organ character were investigated. Course of microsporegenesis and the difference of protein expressing in bud were compared and analyzed between MDGMS and its allelic fertile. RAPD marker linked to Ms gene in MDGMS was found using BSA and has been converted into sequence characterized amplified region (SCAR) marker to aid identification of male-fertility genotypes in segregating progenies of MDGMS in marker-assisted selection (MAS) breeding programs. After RAPD marker sequenced and blast, a homologous gene importinαwas found and its complete genomic DNA and cDNA of ORF was screened out from Brassica napus L. The RNAi vector and antisense RNA vector of importinαgene of rapeseed were constructed and were transformed into rapeseed. The primary results are as follow:
1. The MDGMS flowers were characterized by reduced anthers with little withered or no pollen grains and no seed set was observed on selfing. The most prominent change in the male-sterile flowers was a distinct reduction in the length of the stamen filament. However, the gynoecium and nectary did not show any differences in sterile lines compared with that of allelic fertile ones. It can normally set seeds when the male-sterile pistil was pollinated with other fertile pollen, suggesting that the gynoecium of male-sterile was fertile.
Expression of male sterility/fertility in two clones derived from a single plant was generally similar in field and greenhouse. These observations based on 48 cloned genotypes suggested that this GMS sterility was very likely controlled by a single dominant nuclear gene without environment effect.
2. The microspore development course was observed by paraffin slices, autolysis of tapetal layer took place around the tetrads stage, therefore the nutrition was supplied for the development of the microspore. While in the MDGMS microspores abortion was found before the pollen mother cells meiosis stage, and the tetrads couldn’t be formed, then the contents of microspore leaked out, microspore lysis followed after, at last, sterility occurred. These results indicated that the MDGMS male sterility was caused by its development rather than autolysis of tapetal layer.
3. The differences of protein expressing in buds between MDGMS and its allelic fertile line was detected at the same stage. The results showed that the quantity of detected protein dots in fertile line is more than that in MDGMS. The molecular weight of special proteins in fertile line is mainly below 60kD, especially below 30kD. While the ones in MDGMS are relative uniform. What the same between MDGMS and allelic fertile line is that the isoelectronic points (pI) of respectively special proteins are mainly in 6.0-7.5.
4. Bulked segregation analysis (BSA) was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the Ms gene in MDGMS. Only one RAPD marker OPU031500 was found to closely link to the MDGMS locus in rapeseed. This RAPD marker OPU-031500 was cloned into a T-easy vector and sequenced. The sequence here obtained was highly homologous to one of the Arabidopsis DNA sequences. According to this DNA conserved region in different species, we designed a pair of specific primers SCP1/SCP2 and amplified only one specific 2.3kb DNA fragment in each bulk. There are some mutant loci between the two DNA fragments after sequencing. Both sequences are highly homologous to Arabidopsis Importinαgene. We designed another pair of specific primers SCP3 /SCP4 according to the DNA sequence at the mutant loci. A specific DNA segment was amplified only in the fertile line but not in the sterile line using the primers SCP3 and SCP4. Therefore the RAPD marker was converted into SCAR marker.
5. The genomic DNA and cDNA of Importinαgene were cloned by PCR in MDGMS and allelic fertile line in Brassica napus L., including initiation codon ATG and termination codon TGA. The genomic DNA is 2620bp and 2614bp respectively, and cDNA is 1629bp and 1620bp respectively. There are some mutant loci in genomic DNA between MDGMS and allelic fertile line, but only two mutant loci in cDNA. Comparing on the genomic DNA and cDNA, Importinαgene of rapeseed (BIMP a) includes 10 exons and 9 introns.
6. Comparing on the induced protein sequence by cDNA sequence between MDGMS and allelic fertile line, Importinαof rapeseed is composed of 542 amino acids, but 540 amino acids in MDGMS. Andαhelix andβturns of Importinαsecondary structure are less in MDGMS than that in allelic fertile line because of two QQ amino acid absence in MDGMS.
Protein structure analysis (DNAstar) showed that rapeseed Importinαis consist of a flexible N-terminal Importin-β-binding (IBB) domain and a highly structured domain composed of eight armadillo (ARM) repeats and two HEAT repeats. ARM repeats are the Importinαclassical structural characters and HEAT repeats are Importinβclassical structural characters. So we designated it as BIMP a (Brassica napus Importinα, BIMP a).
7. Rapeseed Importinαgene regulates not only oogamete development but also androgamete development. Gene knocking-down plants by RNAi and antisense RNA can’t set seeds on either selfing or crossing, indicating that both oogamete and androgamete are sterile. And the gene knocking-down plants are susceptible to downy mildew and virus diseases in comparison to wild type.
1.MDGMS油菜不育株与可育株相比,雄蕊严重退化,花丝不伸长或略有伸长,花药萎缩呈丝状尖三角,花粉囊中空干瘪,无花粉,自交不能结实。而雌蕊具有正常的形态,少部分的柱头有一定的弯曲。用正常花粉给不育花授粉,能正常结实,表明不育花雌蕊是正常可育的。
通过无性繁殖对应植株在温室和大田育性比较研究表明,该不育系完全由遗传效应决定,不受环境因素的影响。
2.对MDGMS育性分离群体中可育株和不育株小孢子发育过程进行石蜡切片比较观察发现,不育系小孢子败育发生在减数分裂前,并且不能形成四分体,小孢子解体于绒毡层解体之前,最后花药成为干瘪的空壳,表明MDGMS雄性不育不是由于绒毡层细胞给小孢子营养供应不良造成,而是小孢子功能缺陷,自身不能继续发育引起的。
3.对同一时期MDGMS不育及等位可育系的花蕾蛋白质表达差异分析表明,可育系花蕾中表达的蛋白质点数要高于不育系。可育系中表达的特有蛋白质分子量主要分布在60kD以下小分子区域,30kD尤为丰富;不育系中表达的特有蛋白按分子量分布相对均匀。两者表达特有蛋白都相对集中在pI6.0-7.5之间。
4.利用集群分离法(BSA)对MDGMS不育基因进行了RAPD分析,找到了与MDGMS不育基因连锁的RAPD标记OPU-031500。将RAPD标记OPU-031500 DNA片段克隆、测序并进行Blast分析表明,该标记DNA序列与拟南芥基因核输入蛋白α高度同源,根据同源的拟南芥序列及其该基因在物种间的保守性,设计特异性引物SCP1/SCP2,在MDGMS不育及等位可育系中都扩增到2.3 kb的单一特异片段。根据突变位点设计引物SCP3/SCP4,在等位可育系中扩增到约1.5kb的单一特异片段,但在不育系中未扩增到任何带,从而将RAPD标记转化为稳定可靠的SCAR标记。
5.在油菜MDGMS不育和等位可育系中克隆到包括起始密码子ATG和终止密码子TGA在内的油菜核输入蛋白α基因编码区全长genomic DNA和cDNA。二者genomic DNA全长分别为2620bp和2614bp。不育和可育系genomic DNA序列之间多个位点存在突变差异。cDNA全长分别为1629bp和1623bp,不育和可育系cDNA序列之间只有2个位点存在突变差异。genomic DNA和cDNA比较表明,油菜输入蛋白α基因(BIMP a)包含10个外显子和9个内含子。
6.根据克隆到的MDGMS不育和可育系输入蛋白αcDNA序列推导出二者对应的氨基酸序列。油菜输入蛋白α由542个氨基酸组成,而突变不育系只有540氨基酸。比较发现不育系由于碱基缺失突变,导致其肽链氨基酸序列组成上有1个位点缺少2个谷氨酰胺(QQ),并进一步导致该蛋白二级结构的变化:α-螺旋和β-转角在突变不育系MDGMS中明显减少。
对推导的氨基酸序列进行结构域分析表明,油菜输入蛋白α结构域包括1个与输入蛋白β结合的N-末端结构域(IBB)、8个ARM重复单元(ARM repeat)和2个HEAT重复单元(HEAT repeat),组成完全与输入蛋白α结构域一致,从而将其命名为BIMP a(Brassica-napus Importinα, BIMP a)。
7.通过构建油菜输入蛋白αRNAi和Antisense RNA载体并转化油菜表明,油菜输入蛋白α不仅控制雄配子的发育,也控制雌配子的发育,同时与植株的长势及抗病性相关。转基因植株长势较差,不能结实且易感病。
A novel genic male sterile (GMS) line in Brassica napus L., which was identified by Hybrid Rapeseeed Research Center of Shaanxi Province in 1999, was found to be controlled by a monogenic dominant gene which we have designated as MDGMS. The F1 fertility from any fertile lines crossed with MDGMS segregated and the ratio was close to 1:1. Its fertility, floral organ character were investigated. Course of microsporegenesis and the difference of protein expressing in bud were compared and analyzed between MDGMS and its allelic fertile. RAPD marker linked to Ms gene in MDGMS was found using BSA and has been converted into sequence characterized amplified region (SCAR) marker to aid identification of male-fertility genotypes in segregating progenies of MDGMS in marker-assisted selection (MAS) breeding programs. After RAPD marker sequenced and blast, a homologous gene importinαwas found and its complete genomic DNA and cDNA of ORF was screened out from Brassica napus L. The RNAi vector and antisense RNA vector of importinαgene of rapeseed were constructed and were transformed into rapeseed. The primary results are as follow:
1. The MDGMS flowers were characterized by reduced anthers with little withered or no pollen grains and no seed set was observed on selfing. The most prominent change in the male-sterile flowers was a distinct reduction in the length of the stamen filament. However, the gynoecium and nectary did not show any differences in sterile lines compared with that of allelic fertile ones. It can normally set seeds when the male-sterile pistil was pollinated with other fertile pollen, suggesting that the gynoecium of male-sterile was fertile.
Expression of male sterility/fertility in two clones derived from a single plant was generally similar in field and greenhouse. These observations based on 48 cloned genotypes suggested that this GMS sterility was very likely controlled by a single dominant nuclear gene without environment effect.
2. The microspore development course was observed by paraffin slices, autolysis of tapetal layer took place around the tetrads stage, therefore the nutrition was supplied for the development of the microspore. While in the MDGMS microspores abortion was found before the pollen mother cells meiosis stage, and the tetrads couldn’t be formed, then the contents of microspore leaked out, microspore lysis followed after, at last, sterility occurred. These results indicated that the MDGMS male sterility was caused by its development rather than autolysis of tapetal layer.
3. The differences of protein expressing in buds between MDGMS and its allelic fertile line was detected at the same stage. The results showed that the quantity of detected protein dots in fertile line is more than that in MDGMS. The molecular weight of special proteins in fertile line is mainly below 60kD, especially below 30kD. While the ones in MDGMS are relative uniform. What the same between MDGMS and allelic fertile line is that the isoelectronic points (pI) of respectively special proteins are mainly in 6.0-7.5.
4. Bulked segregation analysis (BSA) was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the Ms gene in MDGMS. Only one RAPD marker OPU031500 was found to closely link to the MDGMS locus in rapeseed. This RAPD marker OPU-031500 was cloned into a T-easy vector and sequenced. The sequence here obtained was highly homologous to one of the Arabidopsis DNA sequences. According to this DNA conserved region in different species, we designed a pair of specific primers SCP1/SCP2 and amplified only one specific 2.3kb DNA fragment in each bulk. There are some mutant loci between the two DNA fragments after sequencing. Both sequences are highly homologous to Arabidopsis Importinαgene. We designed another pair of specific primers SCP3 /SCP4 according to the DNA sequence at the mutant loci. A specific DNA segment was amplified only in the fertile line but not in the sterile line using the primers SCP3 and SCP4. Therefore the RAPD marker was converted into SCAR marker.
5. The genomic DNA and cDNA of Importinαgene were cloned by PCR in MDGMS and allelic fertile line in Brassica napus L., including initiation codon ATG and termination codon TGA. The genomic DNA is 2620bp and 2614bp respectively, and cDNA is 1629bp and 1620bp respectively. There are some mutant loci in genomic DNA between MDGMS and allelic fertile line, but only two mutant loci in cDNA. Comparing on the genomic DNA and cDNA, Importinαgene of rapeseed (BIMP a) includes 10 exons and 9 introns.
6. Comparing on the induced protein sequence by cDNA sequence between MDGMS and allelic fertile line, Importinαof rapeseed is composed of 542 amino acids, but 540 amino acids in MDGMS. Andαhelix andβturns of Importinαsecondary structure are less in MDGMS than that in allelic fertile line because of two QQ amino acid absence in MDGMS.
Protein structure analysis (DNAstar) showed that rapeseed Importinαis consist of a flexible N-terminal Importin-β-binding (IBB) domain and a highly structured domain composed of eight armadillo (ARM) repeats and two HEAT repeats. ARM repeats are the Importinαclassical structural characters and HEAT repeats are Importinβclassical structural characters. So we designated it as BIMP a (Brassica napus Importinα, BIMP a).
7. Rapeseed Importinαgene regulates not only oogamete development but also androgamete development. Gene knocking-down plants by RNAi and antisense RNA can’t set seeds on either selfing or crossing, indicating that both oogamete and androgamete are sterile. And the gene knocking-down plants are susceptible to downy mildew and virus diseases in comparison to wild type.
引文
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