白菜花粉发育相关的C2H2型锌指蛋白新基因BcMF20的克隆与功能验证
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摘要
选育雄性不育系是作物杂种优势利用中简化制种程序、降低制种成本的重要手段,而十字花科作物生产一代杂种的理想系统是雄性不育系,花粉败育是植物雄性不育发生的表型体现,弄清花粉发育的分子机理是研究雄性不育的关键所在。本实验室在成功构建了三种共享同一保持系(核不育两用系的可育株系)的白菜(Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis)'矮脚黄’核不育('Aijiaohuang'genic male sterility, ajhGMS)两用系'Bcajh97-01A/B'、'Polima'核质互作不育(polima genic-cytoplasmic male sterility,polG-CMS)系'Bcpol97-05A'以及'Ogura'细胞质不育(Ogura cytoplasmic male sterility, oguCMS)系'Bcogu97-06A'材料体系的基础上,采用拟南芥基因芯片分析其花蕾转录组差异,对花粉基因表达谱进行系统分析,发现在三种不育系花蕾中均下调表达的基因有29个,在这29条基因中,大部分(14条)是功能未知基因,仅有2条是转录因子。其中一条的转录本标签是At1g26610,这是一条C2H2锌指家族的转录因子。At1g26610在三种不同雄性不育遗传模式的材料中表达均下调,提示我们它可能与花粉发育相关。本研究通过同源克隆的方法获得白菜中At1g26610的同源基因的编码序列和DNA全长,分析其序列特征；克隆其在十字花科芸薹属和萝卜属的18份材料中的同源基因,同时通过与近缘基因的比较,构建分子进化树,探讨进化关系；运用半定量PCR和实时定量荧光PCR技术分析该基因在'Bcajh97-01A/B'不育株和可育株花蕾五级、花序、嫩角果、花茎和花叶中的表达情况,同时采用原位杂交方法分析其在组织中的定位,明确其时空表达情况；最后运用反义RNA技术,对拟南芥进行转化,获得这个基因的功能缺失突变体,鉴别该基因在花粉发育过程中的作用。取得的主要结果如下：
(1)依据拟南芥At1g26610序列,采用同源克隆方法扩增获得在可育株'Bcajh97-01B'中高表达的转录本标签At1g26610在白菜中的同源基因,对该基因的cDNA序列和DNA序列分析可知,该基因最大开放阅读框为1359 bp,无内含子和外显子,Blast搜索没有发现相同序列的基因,说明这是一条新基因,我们将其命名为BcMF20 (Brassica campestris male fertility gene 20)。对推导的BcMF20编码氨基酸序列进行结构和功能域的分析表明,BcMF20编码452个氨基酸。编码的假定蛋白质的分子量为50.874 kDa,等电点为8.562,pH 7.0时的电荷为12.241,包含碱性氨基酸(K,R)72个,酸性氨基酸(D,E)63个,疏水氨基酸(A, I, L, F, W, V)105个,极性氨基酸(N, C, Q, S, T, Y) 133个。BcMF20基因是MMB型的3锌指C2H2锌指家族转录因子,其结构与矮牵牛EPF家族的TAZ1(ZPT3-2)基因的相似程度较高。
(2)根据BcMF20的全长序列设计引物,在十字花科芸薹属和萝卜属的18个物种中克隆到BcMF20的同源序列,对十字花科植物BcMF20同源基因DNA序列进行核苷酸同源序列比对,发现BcMF20同源基因在DNA序列上的相似性为86.9%-100%。所有基因均没有内含子,尤其是在CDS的起始区和终止区,序列完全相同。它们所编码的蛋白质同源性高达77.6%-100%,均是3个锌指的C2H2转录因子,且编码的锌指类型与BcMF20一致,均为MMB型。在锌指的保守区域,碱基序列完全相同。在NCBI上对BcMF20进行BLAST搜索查询,发现12个与BcMF20有较高相似性的C2H2锌指转录因子,NJ法构建系统树,结果显示BcMF20与同科的拟南芥的三条C2H2锌指转录因子聚成一类,然后再与矮牵牛中的两条C2H2的4锌指C2H2转录因子聚为一类。
(3)采用半定量PCR和实时定量PCR技术分析BcMF20在'Bcajh97-01A/B'不育株和可育株花蕾五级、花序、嫩角果、花茎和花叶中的表达情况,半定量PCR结果显示BcMF20基因仅在可育系Ⅳ级、V级花蕾中有表达,说明BcMF20基因可能与花粉发育相关；实时定量PCR结果表明BcMF20在不育系'Bajh97-01A'中的表达量极低,BcMF20基因在花蕾Ⅰ级-V级以及开放的花中表达,在角果、花茎、花叶中无表达,并且BcMF20基因在可育系Ⅳ级、V级花蕾中表达量非常大,实时定量PCR的结果证明该基因确实与花粉发育有关,按照Mascarenhas (1990)对花粉表达基因的分类方法,应该被归于“晚期”基因。原位杂交分析发现杂交信号出现在单核小孢子时期的小孢子和绒毡层细胞中,在花粉成熟早期的小孢子和将要降解的绒毡层细胞中继续表达,提示我们BcMF20可能在单核小孢子时期对绒毡层和花粉发育起作用。
(4)构建含有组成型启动子CaMV35S的RNA反义载体,并利用Floral Dip法将构建的反义载体导入拟南芥中,与空载体对照比较,转基因植株营养生长正常,雌蕊正常,花药上花粉量少,最终不能形成正常饱满果荚。转基因拟南芥花粉不萌发,T1代萌发率为13%,T2代为15%。扫描电镜显示,82%的转基因植株的花粉畸形。与正常的椭圆形、三条萌发沟均匀分布的花粉粒相比,转基因植株的花粉呈现各种空洞,凹陷,异常空瘪。透射电镜显示导致转基因植株花粉萌发率降低的主要原因有两个：一是花药内花粉数量减少,二是花粉畸形、空泡化。转基因植株的花粉畸形是由于花粉壁畸形导致的,而花粉壁的畸形很有可能是由于胼胝质或者初生外壁的合成出现缺陷。在单核小孢子时期,转基因植株绒毡层的细胞质充满形态异常的空泡,花粉粒中也出现空泡。我们推测BcMF20可能是绒毡层发育中减数分裂后期TAZ1与MS1调控机制中的一个组件,它们共同作用丁绒毡层细胞的增殖,维持花粉的正常发育。
Brassica crops is a kind of important crops in agricultural production. The male sterile line was widely utilized in practice on produce F1 hybrid seeds. Much attention was paid to research on the plant breeding of the male sterile line and the basis for application in Brassica crops. The research on male sterility mechanism of Brassica contributed to the understanding of microspore and pollen development process and provided theory direction to create man-made male sterile line. In the former research of our lab, we established three different male sterile types,'Aijiaohuang'genic male sterility AB line (aihGMS'Bcajh97-01A/B'),'Poloma'genic-cytoplasmic male sterility (polG-CMS 'Bcpol97-05A') and'Ogura'cytolasmic male sterility (oguCMS'Bcogu97-06A') in Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis. These male sterile lines share a common maintainer line. Then, we conducted profiling comparisons between flower buds of these male sterile lines and their maintainer line using ATH1 genome array analysis. By analyzing the changes in expression pattern of genes acting downstream due to the gene mutation in different male sterile plant, 29 genes were identified. In this study, one C2H2 transcription factor in the 29 genes was selected for further characterization. The corresponding full-length cDNA and DNA was subsequently amplified by homologous gene amplification method. The gene sequence was analyzed and proteins functions were predicted. Spatial and temporal expression patterns were analyzed by reverse transcriptase PCR (RT-PCR), real-time quantitative PCR (RT-qPCR) and in situ hybridization. On this base the plant anti-sense RNA expression vector was constructed regulated by the constitutive promoter CaMV35S. The transgenic plants were obtained successfully using the floral dip method to transform the vector to the Arabidopsis (Columbia). Finally, molecular, morphological and cytological characteristics of the pollen were studied in transgenic plants, in order to elucidate the relation of the gene and pollen development, and understand the systematic regulation and molecular mechanism of pollen development in Brassica. The results obtained are as follows.
(1) We cloned an cDNA of the gene differentially expressed in pollen using homologous expansion named Brassica Campestris Male Fertility 20 (BcMF20). Based on the cDNA of BcMF20, we designed primers to amplify DNA of BcMF20. A comparison of cDNA and DNA sequence showed that BcMF20 was composed of a 1359bp open reading frame, encoding an 50.874 kDa protein of 452 amino acids.The amino acid sequence of BcMF20 possesses the basic feature of C2H2 zinc finger protein, containing three finger, its finger type is MMB.
(2) We isolated the homologue of BcMF20 from 18 cultivars from genera Brassica and Raphanus of family Cruciferae by homology cloning, and then compared these sequences with BcMF20 by multiple alignment. The similarity of DNA from BcMF20 homologous gene is from 86.9%-100%, which indicated that homologues of BcMF20 from family Cruciferae were highly conserved. All the genes are without introns, and totally identical in the CDs region. The encoded proteins are all C2H2 transcription factor contain three Zinc-finger, and the type of the Zinc-finger are MMB style as BcMF20. In the zinc-finger domain, the base sequences are conservation. When Blast BcMF20 according to NCBI database, found 12 Zinc-finger transcription factor genes have high identity to BcMF20, and all these sequences were analyzed by NJ to construct a phylogenetic tree. From the tree, BcMF20 is neighbored with three Arabidopsis thaliana C2H2 Zinc-finger transcription factors, and then become a tuft with four Zinc-fingers C2H2 transcription factor from Petunia.
(3) Spatial and temporal expression patterns were analysed by RT-PCR and real-time quantitative PCR during the different stages of sterile and fertile plant development. The expression signal of BcMF20 was detected from fertile plant flower buds of stage 4 and stage5 by RT-PCR and real-time quantitative PCR. In contrast, little expression signal of BcMF20 was detected in same tissues of the sterile mutant by real-time quantitative PCR, and undetected from germinal siliques, leaves and scapes from sterile and fertile plant. The result indicated BcMF20 was an important gene in pollen development. BcMF20 was a'late'gene according to the classification of pollen expressed gene by Mascarenhas (Mascarenhas,1990). In situ hybridization indicated expression signal of BcMF20 was detected in uninucleate microspore and the tapetum cells, and expression signal was also detected in the maturing pollen and degradating tapetum. These results indicated that BcMF20 may has an effect on the tapetum and microspore development in uninucleate microspore stage.
(4) Construct the anti-sense RNA vector with CaMV35S promoter, and introduced the vector into Arabidopsis thaliana by Floral Dip. Contrast with the empty vector, transgenic plant is normal during vegetable growth and the female organs, but have less pollen grain, and can not form turgid pods. The pollen from transgenic plant has low germination rate,13% and 15% in the T1 and T2 generations, respectively. 82% pollen from transgenic plant shows malformation under SEM. Contrast with normal pollen grain, the transgenic pollen display inanition, hollow and shrunken. Under TEM, we found two reasons to explain the low germination rate from transgenic pollen, one is the less of pollen grain in the pollen sac, and the other is the vacuole in the pollen. The abnormal pollen is likely due to the abnormal callose and the extine formation. The similarity in the expression pattern and the phenotypes resulting from the lack of their respective expression suggest that TAZ1, MS1 and BcMF20 could serve as components of the regulatory mechanism that controls the postmeiotic phase of tapetum development.
(1)依据拟南芥At1g26610序列,采用同源克隆方法扩增获得在可育株'Bcajh97-01B'中高表达的转录本标签At1g26610在白菜中的同源基因,对该基因的cDNA序列和DNA序列分析可知,该基因最大开放阅读框为1359 bp,无内含子和外显子,Blast搜索没有发现相同序列的基因,说明这是一条新基因,我们将其命名为BcMF20 (Brassica campestris male fertility gene 20)。对推导的BcMF20编码氨基酸序列进行结构和功能域的分析表明,BcMF20编码452个氨基酸。编码的假定蛋白质的分子量为50.874 kDa,等电点为8.562,pH 7.0时的电荷为12.241,包含碱性氨基酸(K,R)72个,酸性氨基酸(D,E)63个,疏水氨基酸(A, I, L, F, W, V)105个,极性氨基酸(N, C, Q, S, T, Y) 133个。BcMF20基因是MMB型的3锌指C2H2锌指家族转录因子,其结构与矮牵牛EPF家族的TAZ1(ZPT3-2)基因的相似程度较高。
(2)根据BcMF20的全长序列设计引物,在十字花科芸薹属和萝卜属的18个物种中克隆到BcMF20的同源序列,对十字花科植物BcMF20同源基因DNA序列进行核苷酸同源序列比对,发现BcMF20同源基因在DNA序列上的相似性为86.9%-100%。所有基因均没有内含子,尤其是在CDS的起始区和终止区,序列完全相同。它们所编码的蛋白质同源性高达77.6%-100%,均是3个锌指的C2H2转录因子,且编码的锌指类型与BcMF20一致,均为MMB型。在锌指的保守区域,碱基序列完全相同。在NCBI上对BcMF20进行BLAST搜索查询,发现12个与BcMF20有较高相似性的C2H2锌指转录因子,NJ法构建系统树,结果显示BcMF20与同科的拟南芥的三条C2H2锌指转录因子聚成一类,然后再与矮牵牛中的两条C2H2的4锌指C2H2转录因子聚为一类。
(3)采用半定量PCR和实时定量PCR技术分析BcMF20在'Bcajh97-01A/B'不育株和可育株花蕾五级、花序、嫩角果、花茎和花叶中的表达情况,半定量PCR结果显示BcMF20基因仅在可育系Ⅳ级、V级花蕾中有表达,说明BcMF20基因可能与花粉发育相关；实时定量PCR结果表明BcMF20在不育系'Bajh97-01A'中的表达量极低,BcMF20基因在花蕾Ⅰ级-V级以及开放的花中表达,在角果、花茎、花叶中无表达,并且BcMF20基因在可育系Ⅳ级、V级花蕾中表达量非常大,实时定量PCR的结果证明该基因确实与花粉发育有关,按照Mascarenhas (1990)对花粉表达基因的分类方法,应该被归于“晚期”基因。原位杂交分析发现杂交信号出现在单核小孢子时期的小孢子和绒毡层细胞中,在花粉成熟早期的小孢子和将要降解的绒毡层细胞中继续表达,提示我们BcMF20可能在单核小孢子时期对绒毡层和花粉发育起作用。
(4)构建含有组成型启动子CaMV35S的RNA反义载体,并利用Floral Dip法将构建的反义载体导入拟南芥中,与空载体对照比较,转基因植株营养生长正常,雌蕊正常,花药上花粉量少,最终不能形成正常饱满果荚。转基因拟南芥花粉不萌发,T1代萌发率为13%,T2代为15%。扫描电镜显示,82%的转基因植株的花粉畸形。与正常的椭圆形、三条萌发沟均匀分布的花粉粒相比,转基因植株的花粉呈现各种空洞,凹陷,异常空瘪。透射电镜显示导致转基因植株花粉萌发率降低的主要原因有两个：一是花药内花粉数量减少,二是花粉畸形、空泡化。转基因植株的花粉畸形是由于花粉壁畸形导致的,而花粉壁的畸形很有可能是由于胼胝质或者初生外壁的合成出现缺陷。在单核小孢子时期,转基因植株绒毡层的细胞质充满形态异常的空泡,花粉粒中也出现空泡。我们推测BcMF20可能是绒毡层发育中减数分裂后期TAZ1与MS1调控机制中的一个组件,它们共同作用丁绒毡层细胞的增殖,维持花粉的正常发育。
Brassica crops is a kind of important crops in agricultural production. The male sterile line was widely utilized in practice on produce F1 hybrid seeds. Much attention was paid to research on the plant breeding of the male sterile line and the basis for application in Brassica crops. The research on male sterility mechanism of Brassica contributed to the understanding of microspore and pollen development process and provided theory direction to create man-made male sterile line. In the former research of our lab, we established three different male sterile types,'Aijiaohuang'genic male sterility AB line (aihGMS'Bcajh97-01A/B'),'Poloma'genic-cytoplasmic male sterility (polG-CMS 'Bcpol97-05A') and'Ogura'cytolasmic male sterility (oguCMS'Bcogu97-06A') in Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis. These male sterile lines share a common maintainer line. Then, we conducted profiling comparisons between flower buds of these male sterile lines and their maintainer line using ATH1 genome array analysis. By analyzing the changes in expression pattern of genes acting downstream due to the gene mutation in different male sterile plant, 29 genes were identified. In this study, one C2H2 transcription factor in the 29 genes was selected for further characterization. The corresponding full-length cDNA and DNA was subsequently amplified by homologous gene amplification method. The gene sequence was analyzed and proteins functions were predicted. Spatial and temporal expression patterns were analyzed by reverse transcriptase PCR (RT-PCR), real-time quantitative PCR (RT-qPCR) and in situ hybridization. On this base the plant anti-sense RNA expression vector was constructed regulated by the constitutive promoter CaMV35S. The transgenic plants were obtained successfully using the floral dip method to transform the vector to the Arabidopsis (Columbia). Finally, molecular, morphological and cytological characteristics of the pollen were studied in transgenic plants, in order to elucidate the relation of the gene and pollen development, and understand the systematic regulation and molecular mechanism of pollen development in Brassica. The results obtained are as follows.
(1) We cloned an cDNA of the gene differentially expressed in pollen using homologous expansion named Brassica Campestris Male Fertility 20 (BcMF20). Based on the cDNA of BcMF20, we designed primers to amplify DNA of BcMF20. A comparison of cDNA and DNA sequence showed that BcMF20 was composed of a 1359bp open reading frame, encoding an 50.874 kDa protein of 452 amino acids.The amino acid sequence of BcMF20 possesses the basic feature of C2H2 zinc finger protein, containing three finger, its finger type is MMB.
(2) We isolated the homologue of BcMF20 from 18 cultivars from genera Brassica and Raphanus of family Cruciferae by homology cloning, and then compared these sequences with BcMF20 by multiple alignment. The similarity of DNA from BcMF20 homologous gene is from 86.9%-100%, which indicated that homologues of BcMF20 from family Cruciferae were highly conserved. All the genes are without introns, and totally identical in the CDs region. The encoded proteins are all C2H2 transcription factor contain three Zinc-finger, and the type of the Zinc-finger are MMB style as BcMF20. In the zinc-finger domain, the base sequences are conservation. When Blast BcMF20 according to NCBI database, found 12 Zinc-finger transcription factor genes have high identity to BcMF20, and all these sequences were analyzed by NJ to construct a phylogenetic tree. From the tree, BcMF20 is neighbored with three Arabidopsis thaliana C2H2 Zinc-finger transcription factors, and then become a tuft with four Zinc-fingers C2H2 transcription factor from Petunia.
(3) Spatial and temporal expression patterns were analysed by RT-PCR and real-time quantitative PCR during the different stages of sterile and fertile plant development. The expression signal of BcMF20 was detected from fertile plant flower buds of stage 4 and stage5 by RT-PCR and real-time quantitative PCR. In contrast, little expression signal of BcMF20 was detected in same tissues of the sterile mutant by real-time quantitative PCR, and undetected from germinal siliques, leaves and scapes from sterile and fertile plant. The result indicated BcMF20 was an important gene in pollen development. BcMF20 was a'late'gene according to the classification of pollen expressed gene by Mascarenhas (Mascarenhas,1990). In situ hybridization indicated expression signal of BcMF20 was detected in uninucleate microspore and the tapetum cells, and expression signal was also detected in the maturing pollen and degradating tapetum. These results indicated that BcMF20 may has an effect on the tapetum and microspore development in uninucleate microspore stage.
(4) Construct the anti-sense RNA vector with CaMV35S promoter, and introduced the vector into Arabidopsis thaliana by Floral Dip. Contrast with the empty vector, transgenic plant is normal during vegetable growth and the female organs, but have less pollen grain, and can not form turgid pods. The pollen from transgenic plant has low germination rate,13% and 15% in the T1 and T2 generations, respectively. 82% pollen from transgenic plant shows malformation under SEM. Contrast with normal pollen grain, the transgenic pollen display inanition, hollow and shrunken. Under TEM, we found two reasons to explain the low germination rate from transgenic pollen, one is the less of pollen grain in the pollen sac, and the other is the vacuole in the pollen. The abnormal pollen is likely due to the abnormal callose and the extine formation. The similarity in the expression pattern and the phenotypes resulting from the lack of their respective expression suggest that TAZ1, MS1 and BcMF20 could serve as components of the regulatory mechanism that controls the postmeiotic phase of tapetum development.
引文
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