龙眼LEAFY基因克隆与功能研究
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摘要
龙眼(Dimocarpus longan Lour.)属于无患子科(Sapindaceae)龙眼属,是我国重要而有特色的一种亚热带木本果树,也是福建省主要种植的果树之一。LEAFY(简称LFY)同源基因是控制花分生组织形成的基因之一,是决定营养生长向生殖生长过渡的一个关键基因,且LFY基因被认为与启动花芽发育的遗传控制有关。本试验对龙眼LFY同源基因进行克隆,并对龙眼LFY同源基因在花芽分化及“冲梢”过程中的功能进行研究,以探索龙眼花芽分化及成花逆转的分子机理。主要研究结果如下:
1.龙眼基因组DNA提取方法的优化。本试验参考了有关果树基因组DNA常用的提取方法,在此基础上结合龙眼本身的特点进行改进,采用改良STE法、改良SDS—KAc法、试剂盒法从龙眼叶片中提取基因组DNA。最终证明改良SDS—KAc法能够有效地去除龙眼叶片中的蛋白质、多糖等次生物质,获得纯净且产率较高的基因组DNA样品。
2.龙眼LFY基因5′端序列的克隆。以龙眼基因组DNA为模板,采用染色体步移技术得到长为1516bp的龙眼LFY基因5′端序列。经分析发现该序列与其他物种LFY基因有很高的同源性,确定了龙眼LFY基因编码区的起始位置。
3.龙眼LFY基因cDNA全长序列的克隆及基因组全长序列的获得。根据龙眼LFY基因5′端序列和3′端序列设计特异性引物,RT-PCR扩增获得龙眼LFY同源基因cDNA全长序列(LLFY)。经分析该序列与其他其他物种LFY基因核苷酸序列有很高的同源性。该cDNA序列全长1184bp,包含一个1167bp的开放阅读框,编码388个氨基酸。将得到的龙眼LFY基因5′端基因组序列与与之前得到的龙眼LFY基因组部分DNA序列进行拼接,得到龙眼LFY基因组全长序列,大小为3395bp。经分析龙眼LFY基因组序列中包含2个内含子,大小分别为536bp和1391bp。
4.龙眼LFY基因的功能研究。采用半定量RT—PCR技术检测花序分化过程、“冲梢”过程中LLFY基因的表达以及比较LLFY基因在“红核子”和“四季蜜”嫁接后新梢中的表达量。结果表明龙眼LFY基因的表达量在花序分化过程有逐渐升高的趋势;在“冲梢”过程则随着变态叶的展开而下降;在“红核子”龙眼嫁接后新梢中没有表达,而在“四季蜜”龙眼接后新梢中有微弱表达。说明LLFY基因与龙眼花序分化及发育有关。
5.龙眼LFY基因植物表达载体的构建。将龙眼LFY基因代替载体pC1301PMI—GUS上的GUS基因,构建成pC1301PMI—LLFY载体。
Longan(Dimocarpus longan Lour.)is a member of the family Sapindaceae,and one of the most important subtropical orchard fruit.LEAFY(LFY)homologous gene is one of genes which control the formation of flower meristem,it plays a major role in the process from vegetative growth to reproductive growth,and LFY gene is also considered to relate with the dominating of flower buds growth.In order to explore the molecular machanism of flower buds differentiation and floral reversion in longan,further studies including longan LFY homologous gene cloning,the funcion research of longan LFY homologous gene in the process of flower buds differentiation and floral reversion were performed,and the main results showed as follows:
1.The optimizing of longan genomic DNA isolation.The study consulted the isolation methods of other fruit trees,and improved them with combining the specialty of longan.We adopted modified STE method,modified SDS-KAc method and the KIT method to extract genomic DNA from longan leaves.The result showed that modified SDS-KAc method can effectively remove secondary substances including protein,amylose etc.in longan leaves,and obtain genomic DNA with high qualiy and yield.
2.Cloning the 5′terminal sequence and the genome sequence of longan LFY gene.Using longan genomic DNA as template,we adopted genome walking to obtain the 5′terminal sequence, it was 1516bp in length.The sequence had high homology with other plant LFY homologous gene, we confirmed the starting position of longan LFY coding region.
3.cDNA cloning of longan LFY gene.On the basis of the 5′terminal sequence and 3′sequence,we designed specific primers and cloned the cDNA full sequence of longan LFY gene(LLFY)by RT-PCR.The sequence had high homology with other plant LFY homologous gene, the full sequence was 1184bp in length,containing a 1167-nucleotide of open reading frame (ORF),encoding a putative protein of 388 amino acid residues.We got the genome sequence by spliced the 5′terminal sequence and the sequence obtained by WANG Qing-lian etc.The genome sequence was 3395bp in length,it contained two introns,one was 536bp in length,another was 1391bp.
4.The function research of longan LFY gene.Expression patterns of longan LFY gene in the process of inflorescence differentiation and floral reversion revealed by semi-quantitative RT-PCR analysis,and compared expression patterns of the grafting shoot of "honghezi" and "sijimi".The result showed that the expression of longan LFY gene increased gradually in process of inflorescence differentiation,whereas it decreased in process of floral reversion.In addtion,there was no expression of longan LFY gene in the "honghezi" grafting shoot,while in the "sijimi" grafting shoot it showed a feeble expression,it showed LLFY related to inflorescence differentiation and growth.
5.Construction of longan LFY gene plant expression vector.Longan LFY gene was cloned into expression vector pC1301PMI-GUS which GUS gene was digested,then we gained plant expression vector of pC1301PMI-LLFY.
1.龙眼基因组DNA提取方法的优化。本试验参考了有关果树基因组DNA常用的提取方法,在此基础上结合龙眼本身的特点进行改进,采用改良STE法、改良SDS—KAc法、试剂盒法从龙眼叶片中提取基因组DNA。最终证明改良SDS—KAc法能够有效地去除龙眼叶片中的蛋白质、多糖等次生物质,获得纯净且产率较高的基因组DNA样品。
2.龙眼LFY基因5′端序列的克隆。以龙眼基因组DNA为模板,采用染色体步移技术得到长为1516bp的龙眼LFY基因5′端序列。经分析发现该序列与其他物种LFY基因有很高的同源性,确定了龙眼LFY基因编码区的起始位置。
3.龙眼LFY基因cDNA全长序列的克隆及基因组全长序列的获得。根据龙眼LFY基因5′端序列和3′端序列设计特异性引物,RT-PCR扩增获得龙眼LFY同源基因cDNA全长序列(LLFY)。经分析该序列与其他其他物种LFY基因核苷酸序列有很高的同源性。该cDNA序列全长1184bp,包含一个1167bp的开放阅读框,编码388个氨基酸。将得到的龙眼LFY基因5′端基因组序列与与之前得到的龙眼LFY基因组部分DNA序列进行拼接,得到龙眼LFY基因组全长序列,大小为3395bp。经分析龙眼LFY基因组序列中包含2个内含子,大小分别为536bp和1391bp。
4.龙眼LFY基因的功能研究。采用半定量RT—PCR技术检测花序分化过程、“冲梢”过程中LLFY基因的表达以及比较LLFY基因在“红核子”和“四季蜜”嫁接后新梢中的表达量。结果表明龙眼LFY基因的表达量在花序分化过程有逐渐升高的趋势;在“冲梢”过程则随着变态叶的展开而下降;在“红核子”龙眼嫁接后新梢中没有表达,而在“四季蜜”龙眼接后新梢中有微弱表达。说明LLFY基因与龙眼花序分化及发育有关。
5.龙眼LFY基因植物表达载体的构建。将龙眼LFY基因代替载体pC1301PMI—GUS上的GUS基因,构建成pC1301PMI—LLFY载体。
Longan(Dimocarpus longan Lour.)is a member of the family Sapindaceae,and one of the most important subtropical orchard fruit.LEAFY(LFY)homologous gene is one of genes which control the formation of flower meristem,it plays a major role in the process from vegetative growth to reproductive growth,and LFY gene is also considered to relate with the dominating of flower buds growth.In order to explore the molecular machanism of flower buds differentiation and floral reversion in longan,further studies including longan LFY homologous gene cloning,the funcion research of longan LFY homologous gene in the process of flower buds differentiation and floral reversion were performed,and the main results showed as follows:
1.The optimizing of longan genomic DNA isolation.The study consulted the isolation methods of other fruit trees,and improved them with combining the specialty of longan.We adopted modified STE method,modified SDS-KAc method and the KIT method to extract genomic DNA from longan leaves.The result showed that modified SDS-KAc method can effectively remove secondary substances including protein,amylose etc.in longan leaves,and obtain genomic DNA with high qualiy and yield.
2.Cloning the 5′terminal sequence and the genome sequence of longan LFY gene.Using longan genomic DNA as template,we adopted genome walking to obtain the 5′terminal sequence, it was 1516bp in length.The sequence had high homology with other plant LFY homologous gene, we confirmed the starting position of longan LFY coding region.
3.cDNA cloning of longan LFY gene.On the basis of the 5′terminal sequence and 3′sequence,we designed specific primers and cloned the cDNA full sequence of longan LFY gene(LLFY)by RT-PCR.The sequence had high homology with other plant LFY homologous gene, the full sequence was 1184bp in length,containing a 1167-nucleotide of open reading frame (ORF),encoding a putative protein of 388 amino acid residues.We got the genome sequence by spliced the 5′terminal sequence and the sequence obtained by WANG Qing-lian etc.The genome sequence was 3395bp in length,it contained two introns,one was 536bp in length,another was 1391bp.
4.The function research of longan LFY gene.Expression patterns of longan LFY gene in the process of inflorescence differentiation and floral reversion revealed by semi-quantitative RT-PCR analysis,and compared expression patterns of the grafting shoot of "honghezi" and "sijimi".The result showed that the expression of longan LFY gene increased gradually in process of inflorescence differentiation,whereas it decreased in process of floral reversion.In addtion,there was no expression of longan LFY gene in the "honghezi" grafting shoot,while in the "sijimi" grafting shoot it showed a feeble expression,it showed LLFY related to inflorescence differentiation and growth.
5.Construction of longan LFY gene plant expression vector.Longan LFY gene was cloned into expression vector pC1301PMI-GUS which GUS gene was digested,then we gained plant expression vector of pC1301PMI-LLFY.
引文
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