我国两种栽培紫菜核糖体RNA基因的克隆与应用
发布时间：2018-08-04 21:45
【摘要】：红毛菜科(Bangiaceae)分为红毛菜属(Bangia)和紫菜属(Pyropia)两个属,在全世界范围内皆有分布,是具有巨大经济价值的海藻。本文选取了我国主要栽培的紫菜品种--坛紫菜(Pyropia haitanensis)和条斑紫菜(Pyropia yezoensis)作为实验材料,首次克隆出坛紫菜核糖体RNA(rDNA)的全长序列,并比较了我国九个主要品种的条斑紫菜r DNA序列差异,进而对条斑紫菜种下进行了分类。对采自山东日照单片条斑紫菜不同部位的r DNA序列进行差异分析,为紫菜减数分裂发生在壳孢子萌发时期提供了一定的分子生物学证据。核糖体RNA基因是由18S rDNA(SSU)、内转录间隔区1(ITS1)、5.8S r DNA、内转录间隔区2(ITS2)、28S r DNA(LSU)、和基因内间隔区(IGS)串联而成的基因家族。通过克隆测序法,扩增出福建莆田栽培的坛紫菜核糖体RNA基因全长序列为15528bp,其中SSU rDNA全长为2953bp,通过反转录cDNA为模板进行扩增,发现SSU rDNA序列包含了上游内含子561bp,和下游内含子555bp,LSU r DNA的全长序列为4444bp,同样具有内含子,上游内含子为372bp,下游内含子1052bp,5.8S rDNA的长度为158bp,ITS1序列长度为331bp,ITS2序列长度为673bp,且不同个体ITS长度基本一致。IGS序列结构较为复杂,其测定存在一定难度,共设计了六对引物对该区域进行了扩增,经手动拼接,得到6969bp的全长序列。利用IGS区序列作为区分条斑紫菜种下差异的分子标签,对我国苏通1号、苏通2号、苏通3号、苏通4号、大连1号、大连2号、青岛野生型9302、坛紫菜与条斑紫菜杂交型Y-H001、Y-H002这九个主要栽培品种进行了序列同源性分析,测得IGS部分序列长度为3628bp-3776bp之间,序列共有278个变异位点,约占总序列的7.5%。其中苏通1号、苏通2号以及大连1号与其余品种具有明显的序列差异,而其余六个品种的序列又存在56个单碱基的转换与颠换。将单片条斑紫菜分为包括生殖区、营养区和基部在内的十个片段,用碱煮法分别提取这十个片段的微量DNA,并利用高度可变的IGS区部分序列834bp作为分子标记,对单片完整条斑紫菜的生殖、营养、基部等部位进行了序列相似性分析,发现条斑紫菜的叶状体既有纯合体又有杂合体,这一结果说明部分条斑紫菜叶状体是一个由不同遗传背景细胞构成的嵌合体,该结论为证明条斑紫菜减数分裂发生在壳孢子萌发时期提供了重要的分子生物学证据。
[Abstract]:The family (Bangiaceae) is divided into two genera, (Bangia) and (Pyropia), which are distributed all over the world and have great economic value. In this paper, the full-length RNA (rDNA) sequences of porphyra sinensis ribosomal RNA (rDNA) were cloned for the first time by using (Pyropia haitanensis) and (Pyropia yezoensis), which are the main varieties of porphyra sinensis cultivated in China, as experimental materials. The differences of r DNA sequences of nine main varieties of porphyra striata in China were compared, and then the classification of the species of porphyra yezoensis was carried out. The differential analysis of r DNA sequences from different parts of Rizhao porphyra monocularis in Shandong Province provided some molecular biological evidence for meiosis of porphyra yezoensis during the period of chestospore germination. Ribosomal RNA gene is a gene family composed of 18s rDNA (SSU), internal transcriptional spacer 1 (ITS1) 5.8S r DNA, internal transcriptional spacer 2 (ITS2) 28S r DNA (LSU), and intergenic spacer (IGS). The full-length RNA gene of porphyra chinensis cultivated in Putian, Fujian Province, was amplified by cloning and sequencing. The total length of ribosomal RNA gene was 15528 BP, and the total length of SSU rDNA was 2953 BP, which was amplified by reverse transcription cDNA. It was found that the SSU rDNA sequence contained 561bp upstream intron and the 555 BP DNA downstream intron was 4444bp. it also had intron. The length of the upstream intron is 372bp, and the downstream intron 1052bp 5.8S rDNA is 158bpPU ITS1 sequence length. The length of the ITS1 sequence is 673 BP, and the length of the ITS sequence of different individuals is basically the same. The structure of the ITS sequence is relatively complex, so it is difficult to determine the length of the ITS sequence. Six pairs of primers were designed to amplify the region, and the full-length sequence of 6969bp was obtained by manual splicing. The sequence of IGS region was used as the molecular label to distinguish the differences between species of porphyra yezoensis. The molecular markers of Sutong 1, Sutong 2, Sutong 3, Sutong 4, Dalian 1, Dalian 2, and Dalian 2 were used. The sequence homology analysis of nine main cultivated varieties of wild type 9302 in Qingdao and hybrid type Y-H001 and Y-H002 of porphyra yezoensis showed that the length of partial IGS sequence was between 3628bp-3776bp and there were 278 mutation sites, accounting for about 7.5% of the total sequence. Among them, Sutong 1, Sutong 2 and Dalian 1 had obvious sequence differences with other varieties, while the other six varieties had 56 single base transversion. Single porphyra yezoensis was divided into ten fragments, including reproductive region, vegetative region and base. The microDNAs of the 10 fragments were extracted by alkaline cooking, and the highly variable IGS region partial 834bp was used as molecular marker. Sequence similarity analysis of reproduction, nutrition and base of porphyra yezoensis showed that there were homozygotes and heterozygotes in the striatum of porphyra yezoensis. The results suggest that the striatum of porphyra yezoensis is a chimera composed of cells from different genetic backgrounds. This conclusion provides important molecular biological evidence for the occurrence of meiosis of porphyra yezoensis during the period of choriospore germination.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
本文编号：2165260
[Abstract]:The family (Bangiaceae) is divided into two genera, (Bangia) and (Pyropia), which are distributed all over the world and have great economic value. In this paper, the full-length RNA (rDNA) sequences of porphyra sinensis ribosomal RNA (rDNA) were cloned for the first time by using (Pyropia haitanensis) and (Pyropia yezoensis), which are the main varieties of porphyra sinensis cultivated in China, as experimental materials. The differences of r DNA sequences of nine main varieties of porphyra striata in China were compared, and then the classification of the species of porphyra yezoensis was carried out. The differential analysis of r DNA sequences from different parts of Rizhao porphyra monocularis in Shandong Province provided some molecular biological evidence for meiosis of porphyra yezoensis during the period of chestospore germination. Ribosomal RNA gene is a gene family composed of 18s rDNA (SSU), internal transcriptional spacer 1 (ITS1) 5.8S r DNA, internal transcriptional spacer 2 (ITS2) 28S r DNA (LSU), and intergenic spacer (IGS). The full-length RNA gene of porphyra chinensis cultivated in Putian, Fujian Province, was amplified by cloning and sequencing. The total length of ribosomal RNA gene was 15528 BP, and the total length of SSU rDNA was 2953 BP, which was amplified by reverse transcription cDNA. It was found that the SSU rDNA sequence contained 561bp upstream intron and the 555 BP DNA downstream intron was 4444bp. it also had intron. The length of the upstream intron is 372bp, and the downstream intron 1052bp 5.8S rDNA is 158bpPU ITS1 sequence length. The length of the ITS1 sequence is 673 BP, and the length of the ITS sequence of different individuals is basically the same. The structure of the ITS sequence is relatively complex, so it is difficult to determine the length of the ITS sequence. Six pairs of primers were designed to amplify the region, and the full-length sequence of 6969bp was obtained by manual splicing. The sequence of IGS region was used as the molecular label to distinguish the differences between species of porphyra yezoensis. The molecular markers of Sutong 1, Sutong 2, Sutong 3, Sutong 4, Dalian 1, Dalian 2, and Dalian 2 were used. The sequence homology analysis of nine main cultivated varieties of wild type 9302 in Qingdao and hybrid type Y-H001 and Y-H002 of porphyra yezoensis showed that the length of partial IGS sequence was between 3628bp-3776bp and there were 278 mutation sites, accounting for about 7.5% of the total sequence. Among them, Sutong 1, Sutong 2 and Dalian 1 had obvious sequence differences with other varieties, while the other six varieties had 56 single base transversion. Single porphyra yezoensis was divided into ten fragments, including reproductive region, vegetative region and base. The microDNAs of the 10 fragments were extracted by alkaline cooking, and the highly variable IGS region partial 834bp was used as molecular marker. Sequence similarity analysis of reproduction, nutrition and base of porphyra yezoensis showed that there were homozygotes and heterozygotes in the striatum of porphyra yezoensis. The results suggest that the striatum of porphyra yezoensis is a chimera composed of cells from different genetic backgrounds. This conclusion provides important molecular biological evidence for the occurrence of meiosis of porphyra yezoensis during the period of choriospore germination.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
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