甘蓝显性细胞核雄性不育相关基因及其启动子序列的克隆与功能分析
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摘要
甘蓝类(Brassica oleracea)作物是世界范围内普遍栽培的重要蔬菜,在我国也有很大的栽培面积,利用雄性不育杂交制种在甘蓝生产上具有重要的意义。但育性作为一个发育性状涉及到雄蕊形态建成及花粉形成中的大量结构与调节基因,任何基因的结构或者表达的变异均会直接影响小孢子的发育而导致雄器败育,因而研究起来非常困难。虽然已经有很多的生理生化、细胞学、分子生物学的研究结果报道,但是要彻底阐明小孢子发育的分子机理尚需要对育性相关基因进行深入广泛的研究,以最终明确各基因间以及基因与雄蕊阶段性发育间的相互关系。
本研究利用与甘蓝显性细胞核雄性不育相关的差异表达片段(TDFs Transcript Derived Fragments),通过电子克隆的策略获得了基因BoRALFL1、BoPMEI和BoDHAR的全长序列,并构建了BoRALFL1和BoPMEI基因的正、反义植物表达载体,经农杆菌介导对菜心(Brassica campestris L.var. Parachinesis)与拟南芥(Arabidopsis thaliana)进行了遗传转化,以探讨其在植株雄蕊发育中的作用;同时克隆了三个基因的5’端非编码区序列,构建了基因BoRALFL1和BoPMEI 5’端序列的植物表达载体,利用瞬时表达系统与稳定表达系统对其启动子活性进行了研究;利用原核表达系统探讨了BoPMEI蛋白的表达、纯化与蛋白活性分析。获得的主要结论如下:
1.快速碱化因子BoRALFL1基因(GenBank序列登录号DQ059310)的cDNA序列全长490 bp,开放阅读框ORF 240bp,编码79个氨基酸。经序列分析,编码蛋白存在长度为28个氨基酸的前导信号肽与多个磷酸化位点,与同源基因RALFL8核酸序列在88 bp上有82%的相似性,推导的氨基酸序列在74个氨基酸上存在56%的相似性;不同植物来源的RALFL蛋白氨基酸序列N-端差异大,C-端序列具有较高的保守性,四个半胱氨酸残基在所有RALF蛋白中位置保守。
果胶甲酯酶抑制蛋白基因BoPMEI(GenBank序列登录号DQ116449)的cDNA序列长750 bp,与同源基因AT1G10770在核酸序列与氨基酸序列上分别存在81%与73.8%的相似性。该基因编码一个含有169个氨基酸的蛋白多肽,分子量18.323 kD。预测分析表明N-端存在信号肽(32个氨基酸)与一个跨膜序列结构;可能参与成熟蛋白正确折叠的4个保守半胱氨酸残基在所有的PMEI氨基酸序列中位置高度保守。
脱氢抗坏血酸还原酶基因BoDHAR的DNA序列长842 bp,含有两个内含子(+59 bp~
Crop of the Cabbage group are worldwide cultivated vegetables. Hybrid seeds are adopted widely in Brassica species for its great contribution on the production enhancement. Male sterility is a very useful alternative strategy in hybrid seeds production in avoiding the labor consuming manual cross-pollination, and it is also advantage on the seed quality assurement. In recent years, although an increasing number of studies have been focused on characterization of microspores and pollen formation from cytological and biochemical aspects, and the description of the abnormal phenomenon in meiosis and microspore development have been reported, knowledge about molecular aspects of pollen abortion is still rather limited. Cloning and characterization of the genes related to male sterility maybe a useful tool in investigating the molecular mechanisms of microsporogenesis.
Three different TDFs (Transcript Derived Fragments) from male sterile and fertile lines of cabbage were used to clone their full length cDNA sequences as querying probes. The sense and anti-sense sequences of genes BoRALFL1 and BoPMEI were used to construct the plant expression vectors, and were transfered into Brassica campestris and Arabidopsis plants by Agrobacterium-mediated method. The functions of these genes in pollen formation were investigated. We also cloned the 5’untranscription regions of genes BoRALFL1 and BoPMEI, constructed two plant expression vectors with 5’sequences as promoters, and analyze the promoter activity of these sequences with transient and stability expression systems. Highly efficient expression, purification of recombinant protein BoPMEI and its activity against enzyme PME was performed. Main conclusions are listed as follows:
1. The cDNA sequence of gene BoRALFL1 (GenBank accession number DQ059310 ) was 490 bp in length, and contain an 240 bp obvious open reading frame (ORF) , which encodes a 79 amino acids peptide. Homologous analysis shows that the amplified cDNA has 82% identity on the nucleotide acid sequence, and 56% identity on the amino acids sequence with Arabidopsis gene RALFL8. Further analysis shows that the pepetide is possibly a preprotein with a signal pepetide and multi-phosphorylation sites, and the C-terminal amino acids of the pepetide are highly conserved among different plant species.
The full-length cDNA of gene BoPMEI (GenBank accession number DQ116449) was
本研究利用与甘蓝显性细胞核雄性不育相关的差异表达片段(TDFs Transcript Derived Fragments),通过电子克隆的策略获得了基因BoRALFL1、BoPMEI和BoDHAR的全长序列,并构建了BoRALFL1和BoPMEI基因的正、反义植物表达载体,经农杆菌介导对菜心(Brassica campestris L.var. Parachinesis)与拟南芥(Arabidopsis thaliana)进行了遗传转化,以探讨其在植株雄蕊发育中的作用;同时克隆了三个基因的5’端非编码区序列,构建了基因BoRALFL1和BoPMEI 5’端序列的植物表达载体,利用瞬时表达系统与稳定表达系统对其启动子活性进行了研究;利用原核表达系统探讨了BoPMEI蛋白的表达、纯化与蛋白活性分析。获得的主要结论如下:
1.快速碱化因子BoRALFL1基因(GenBank序列登录号DQ059310)的cDNA序列全长490 bp,开放阅读框ORF 240bp,编码79个氨基酸。经序列分析,编码蛋白存在长度为28个氨基酸的前导信号肽与多个磷酸化位点,与同源基因RALFL8核酸序列在88 bp上有82%的相似性,推导的氨基酸序列在74个氨基酸上存在56%的相似性;不同植物来源的RALFL蛋白氨基酸序列N-端差异大,C-端序列具有较高的保守性,四个半胱氨酸残基在所有RALF蛋白中位置保守。
果胶甲酯酶抑制蛋白基因BoPMEI(GenBank序列登录号DQ116449)的cDNA序列长750 bp,与同源基因AT1G10770在核酸序列与氨基酸序列上分别存在81%与73.8%的相似性。该基因编码一个含有169个氨基酸的蛋白多肽,分子量18.323 kD。预测分析表明N-端存在信号肽(32个氨基酸)与一个跨膜序列结构;可能参与成熟蛋白正确折叠的4个保守半胱氨酸残基在所有的PMEI氨基酸序列中位置高度保守。
脱氢抗坏血酸还原酶基因BoDHAR的DNA序列长842 bp,含有两个内含子(+59 bp~
Crop of the Cabbage group are worldwide cultivated vegetables. Hybrid seeds are adopted widely in Brassica species for its great contribution on the production enhancement. Male sterility is a very useful alternative strategy in hybrid seeds production in avoiding the labor consuming manual cross-pollination, and it is also advantage on the seed quality assurement. In recent years, although an increasing number of studies have been focused on characterization of microspores and pollen formation from cytological and biochemical aspects, and the description of the abnormal phenomenon in meiosis and microspore development have been reported, knowledge about molecular aspects of pollen abortion is still rather limited. Cloning and characterization of the genes related to male sterility maybe a useful tool in investigating the molecular mechanisms of microsporogenesis.
Three different TDFs (Transcript Derived Fragments) from male sterile and fertile lines of cabbage were used to clone their full length cDNA sequences as querying probes. The sense and anti-sense sequences of genes BoRALFL1 and BoPMEI were used to construct the plant expression vectors, and were transfered into Brassica campestris and Arabidopsis plants by Agrobacterium-mediated method. The functions of these genes in pollen formation were investigated. We also cloned the 5’untranscription regions of genes BoRALFL1 and BoPMEI, constructed two plant expression vectors with 5’sequences as promoters, and analyze the promoter activity of these sequences with transient and stability expression systems. Highly efficient expression, purification of recombinant protein BoPMEI and its activity against enzyme PME was performed. Main conclusions are listed as follows:
1. The cDNA sequence of gene BoRALFL1 (GenBank accession number DQ059310 ) was 490 bp in length, and contain an 240 bp obvious open reading frame (ORF) , which encodes a 79 amino acids peptide. Homologous analysis shows that the amplified cDNA has 82% identity on the nucleotide acid sequence, and 56% identity on the amino acids sequence with Arabidopsis gene RALFL8. Further analysis shows that the pepetide is possibly a preprotein with a signal pepetide and multi-phosphorylation sites, and the C-terminal amino acids of the pepetide are highly conserved among different plant species.
The full-length cDNA of gene BoPMEI (GenBank accession number DQ116449) was
引文
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