我國兩種栽培紫菜核糖體RNA基因的克隆與應(yīng)用
發(fā)布時間：2018-08-04 21:45
【摘要】：紅毛菜科(Bangiaceae)分為紅毛菜屬(Bangia)和紫菜屬(Pyropia)兩個屬,在全世界范圍內(nèi)皆有分布,是具有巨大經(jīng)濟價值的海藻。本文選取了我國主要栽培的紫菜品種--壇紫菜(Pyropia haitanensis)和條斑紫菜(Pyropia yezoensis)作為實驗材料,首次克隆出壇紫菜核糖體RNA(rDNA)的全長序列,并比較了我國九個主要品種的條斑紫菜r DNA序列差異,進(jìn)而對條斑紫菜種下進(jìn)行了分類。對采自山東日照單片條斑紫菜不同部位的r DNA序列進(jìn)行差異分析,為紫菜減數(shù)分裂發(fā)生在殼孢子萌發(fā)時期提供了一定的分子生物學(xué)證據(jù)。核糖體RNA基因是由18S rDNA(SSU)、內(nèi)轉(zhuǎn)錄間隔區(qū)1(ITS1)、5.8S r DNA、內(nèi)轉(zhuǎn)錄間隔區(qū)2(ITS2)、28S r DNA(LSU)、和基因內(nèi)間隔區(qū)(IGS)串聯(lián)而成的基因家族。通過克隆測序法,擴增出福建莆田栽培的壇紫菜核糖體RNA基因全長序列為15528bp,其中SSU rDNA全長為2953bp,通過反轉(zhuǎn)錄cDNA為模板進(jìn)行擴增,發(fā)現(xiàn)SSU rDNA序列包含了上游內(nèi)含子561bp,和下游內(nèi)含子555bp,LSU r DNA的全長序列為4444bp,同樣具有內(nèi)含子,上游內(nèi)含子為372bp,下游內(nèi)含子1052bp,5.8S rDNA的長度為158bp,ITS1序列長度為331bp,ITS2序列長度為673bp,且不同個體ITS長度基本一致。IGS序列結(jié)構(gòu)較為復(fù)雜,其測定存在一定難度,共設(shè)計了六對引物對該區(qū)域進(jìn)行了擴增,經(jīng)手動拼接,得到6969bp的全長序列。利用IGS區(qū)序列作為區(qū)分條斑紫菜種下差異的分子標(biāo)簽,對我國蘇通1號、蘇通2號、蘇通3號、蘇通4號、大連1號、大連2號、青島野生型9302、壇紫菜與條斑紫菜雜交型Y-H001、Y-H002這九個主要栽培品種進(jìn)行了序列同源性分析,測得IGS部分序列長度為3628bp-3776bp之間,序列共有278個變異位點,約占總序列的7.5%。其中蘇通1號、蘇通2號以及大連1號與其余品種具有明顯的序列差異,而其余六個品種的序列又存在56個單堿基的轉(zhuǎn)換與顛換。將單片條斑紫菜分為包括生殖區(qū)、營養(yǎng)區(qū)和基部在內(nèi)的十個片段,用堿煮法分別提取這十個片段的微量DNA,并利用高度可變的IGS區(qū)部分序列834bp作為分子標(biāo)記,對單片完整條斑紫菜的生殖、營養(yǎng)、基部等部位進(jìn)行了序列相似性分析,發(fā)現(xiàn)條斑紫菜的葉狀體既有純合體又有雜合體,這一結(jié)果說明部分條斑紫菜葉狀體是一個由不同遺傳背景細(xì)胞構(gòu)成的嵌合體,該結(jié)論為證明條斑紫菜減數(shù)分裂發(fā)生在殼孢子萌發(fā)時期提供了重要的分子生物學(xué)證據(jù)。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2
本文編號：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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2
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