条斑紫菜 SSU 中内含子分析 李星辰, 徐佳杰, 林中姮, 沈颂东 1 材料和方法 1.1 材料和主要试剂 RNA. , 13000ha, RNA DNA(rDNA) DNA, rdna , 9 (P1-P9), 4. , Oliverira [8]

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1 条斑紫菜 SSU 中内含子分析 李星辰, 徐佳杰, 林中姮, 沈颂东 (,, ) 摘要 : 根据 GenBank 中已知的条斑紫菜 (Porphyra yezoensis)ssu 和 LSU rrna 基因序列分别设计引物, 分别以混合个体和单个个体的基因组 DNA 以及 cdna 为模板进行扩增, 测序分析后推测 SSU rrna 基因中的内含子转录后加工可能仍被保留, 而在 LSU rrna 基因上游序列中没有发现内含子 长 511bp 的内含子位于 SSU rrna 基因序列的上游, 其插入位点为 TCTGGTG-CCAGCAGCC, 内含子 5 和 3 端的核苷酸序列分别为 AAC- 和 -ATGG 该内含子的剪接位点以及保守区 P, Q, R, S 与 I 型内含子是不同的 研究发现, 在同种紫菜中, 不同个体 SSU 基因中内含子的有无 分布及结构特征是不同的, 说明内含子对于分类是不太适合的 但是内含子的长度以及核苷酸序列在种间又具有一定的特异性, 可以为红藻种间以及种内亲缘关系较远的亚种和不同地理群的区分和鉴定提供参考 关键词 : 条斑紫菜 (Porphyra yezoensis); rrna 基因 ; 内含子 ; 同质性 ; 系统发育中图分类号 : Q 文献标识码 : A 文章编号 : (2015) doi: /hykx RNA DNA(rDNA) DNA, rdna,,,, I RNA [1], ( ), rrna, I [2] [3] [4, 5] RNA (rdna) [6] I, 9 (P1-P9), 4 P Q R S, 5 -U G-3 Stiller [7] SSU rrna, Oliverira [8] (Porphyra yezoensis) (Rhodophyta) (Rhodophyphyta) (Bangiales) (Bangiaceae) (Porphyra),,, 13000ha, ( ) ( ),,,, [9] PCR TA, RNA, 1 材料和方法 1.1 材料和主要试剂, RNA RT-PCR : ; : : 国家自然科学基金资助项目 ( ) : (1989-),,,, lixingchen2222@126.com; 12 / 2015 / 39 / 3

2 Recombinant DNase I, peasy-t3, DEPC 1.2 DNA 提取 CTAB [10] DNA DNA TE (ph = 8.0), 1% DNA 1.3 RNA 提取及基因组 DNA 的去除 RNA RNA RNA, 80 Recombinant DNase I DNA, RNA 1.4 cdna 的合成 PrimeScript RT-PCR Kit, cdna 引物和 PCR GenBank SSU rrna (GeneBank accession number: JN104582), SF (5 CGACTCACGAAGGGTGGTAT TT-3 ), SR (5 -GGACCATTCAATCGGTAGGAGC-3 ), 2008 bp; LSU rrna (GeneBank accession number: JN104580), LF (5 -CGGGTTGTATAGTAAAGTGGTGTC-3 ), LR (5 - ACCAGTTCT AATCCGTTCGTTG-3 ), 2083bp DNA cdna PCR, 50 L, : DNA 4 L, dntp Mixture (2.5mM each) 5 L, (20 mol/l) 1 L, 10 LA PCR Buffer II (Mg 2+ plus) 5 L, Ta- KaRa LA Taq (5U/ L) 0.5 L, dh 2 O 50 L TaKaRa PCR, PCR 95 3 min, s, s, 72 2 min, 72 7 min PCR 1%, 1.6 PCR 产物的克隆及测序 peasy-t ~15min, Trans1-T1, IPTG, X-gal, Amp + LB PCR 生物信息学分析 BLAST, cdna, DNAMAN Porphyra yezoensis (GeneBank accession number: AB013177) MEGA5, (Maximum Likelihood, ML) NCBI SSU rdna 2 结果 2.1 以混合个体及单个体基因组 DNA 为模板的 PCR 扩增 DNA, SSU LSU rdna PCR 1 DNA SSU rdna, 500bp; LSU 1 DNA PCR SSU LSU rdna Fig.1 The electrophoresis image of SSU and LSU rdna fragments amplified by PCR. Genomic DNA of mixed Porphyra yezoensis was used as template M. DNA DL-5000; 1~2.PCR SSU rdna; 3~4.PCR LSU rdna M shows the DNA molecular weight marker DL-5000; Lane 1 and lane 2 are the PCR products of SSU rdna; Lane 3 and lane 4 are the PCR products of LSU rdna Marine Sciences / Vol. 39, No. 3 /

3 rdna, 2000bp, 3 DNA SSU LSU rdna PCR, 2, SSU rdna PCR,, DNA SSU rdna ; LSU rdna PCR 1, LSU rrna, 1 4, 3 cdna SSU LSU rrna 2 DNA PCR SSU LSU rdna Fig.2 The electrophoresis image of SSU and LSU rdna fragments amplified by PCR. Genomic DNA of Porphyra yezoensis individual was used as template M. DNA DL-5000; 1~3. SSU rdna PCR ; 4~6. LSU rdna PCR M shows the DNA molecular weight marker DL-5000; Lanes 1 to 3 are the SSU rdna PCR products of three individuals, respectively; Lanes 4 to 6 are the LSU rdna PCR products of three individuals, respectively. 2.2 混合个体及单个体 cdna 的 PCR 扩增 cdna, SSU LSU rrna PCR, 3, 4 3 cdna SSU rrna 3 cdna PCR SSU LSU rrna Fig.3 The electrophoresis image of SSU and LSU rrna fragments amplified by PCR. cdna of mixed Porphyra yezoensis was used as template M. DNA DL-5000; 1~3. SSU rrna PCR ; 4~6. LSU rrna PCR M shows the DNA molecular weight marker DL-5000; Lanes 1 to 3 are the SSU rrna PCR products; Lanes 4 to 6 are the LSU rrna PCR products. 4 cdna PCR SSU LSU rrna Fig.4 The electrophoresis image of SSU and LSU rrna fragments amplified by PCR. cdna of Porphyra yezoensis individual was used as template M. DNA DL-5000; 1~3. SSU rrna PCR ; 4~6. LSU rrna PCR M shows the DNA molecular weight marker DL-5000; Lanes 1 to 3 are the SSU rrna PCR products of three individuals, respectively; Lanes 4 to 6 are the LSU rrna PCR products of three individuals, respectively. 2.3 序列分析, SSU rdna SSU rrna (GenBank accession number: KF or KF501434), LSU rdna LSU rrna (GenBank accession number: KF501435) BLAST SSU rdna,, 511bp, 5 3 AAC- -ATGG, TCTGGTG-CCAGCAGCC, 5 511bp P. yezoensis (GenBank accession number: AB013177) 517bp, 6,, 5% 511bp NCBI P.yezoensis (GenBank accession number: AB013177) P. suborbiculata (GenBank accession number: AB013180) P. dentata (GenBank accession number: AB013183) P. katadae 14 / 2015 / 39 / 3

4 5 SSU rdna Fig.5 The schematic diagram of intron in SSU rdna sequence 6 SSU rdna Fig.6 Alignment of upstream intron sequences of SSU rdna. The four conserved elements P, Q, R and S are marked by boxes (GenBank accession number: AB013184) P. tenera (GenBank accession number: AB013175) P. pseudolinearis (GenBank accession number: AB013185) Porphyra sp. (GenBank accession number: AB013182) SSU rdna,, 7 SSU rdna, 8 3 讨论 3.1 核糖体基因簇的同质性 DNA SSU rdna, DNA, Marine Sciences / Vol. 39, No. 3 /

5 7 SSU rdna ML Fig.7 The maximum likelihood tree constructed based on the upstream introns of SSU rdna, 50% The number in each node represents bootstrap support value. Only bootstrap values greater than 50% are shown cdna SSU rrna,, : (1) RNA,, SSU rrna ; (2), RNA cdna, PCR NCBI,, 3 4,, LSU rdna 5, 3.3 SSU rrna 内含子分析 8 SSU rdna Fig.8 The maximum likelihood tree constructed based on the SSU rdna exons, 50% The number in each node represents bootstrap support value. Only bootstrap values greater than 50% are shown. DNA [11-12] [13-15],,, 3.2 条斑紫菜核糖体具有多样性, SSU rrna,, RNA RNA,, RNA,,,,,,,, I I 9 (P1-P9) P, Q, R, S [16] I, RNA [17] SSU rrna Kunimoto [18] SSU rrna, 5 3 AAC- -ATGG, TCTGGTG-CCAGCAGCC,,, I P1 - U-G 3 G 5 I U G, Q I, P R S,,, 3.4 利用内含子进行系统进化研究的分析, SSU rbcl [19, 20],, 8 P. dentata, 7 P. dentata P. katadae I,, SSU 16 / 2015 / 39 / 3

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7 [21] Brian W T, Andrew W, Anita S K, et al. Distribution and evolution of variable group-i introns in the small ribosomal subunit of North Atlantic Porphyra (Bangiales, Rhodophyta)[J]. Eur J Phycol, 2009, 125: [22] Eun-Jeong P, Satoru F, Hirotoshi E, et al. Genetic polymorphism within Porphyra yezoensis (Bangiales, Rhodophyta) and related species from Japan and Korea detected by cleaved amplified polymorphic sequence analysis[j]. Eur J Phycol, 2007, 42: [23] Kirsten M M, Jamie J, Cannone R R, et al. A structural and phylogenetic analysis of the group IC1 introns in the order Bangiales (Rhodophyta)[J]. Mol Biol Evol, 2001, 18: Analysis of intron in SSU of Porphyra yezoensis LI Xing-chen, XU Jia-jie, LIN Zhong-heng, SHEN Song-dong (Cell Biology Department, School of Biology and Basic Medical Sciences, Medical College of Soochow University, Suzhou City, Jiangsu Province, Suzhou , China) Received: May, 11, 2014 Key words: Porphyra yezoensis; ribosomal DNA; introns; homogeneity; phylogenetic development Abstract: Fragments were amplified using primers designed on the basis of SSU and LSU rrna gene sequences of Porphyra yezoensis, respectively published in GenBank. Genomic DNA and cdna of mixed or single Porphyra yezoensis thallus were used as templates. After sequencing and analyzing these fragments, it is presumed that the intron in SSU rrna gene is not excised in the succedent processing after transcription, and intron was not discovered in LSU rrna gene. The intron which locates upstream in SSU rrna gene is 511 bp. The location of intron is the TCTGGTG-CCAGCAGCC exon sequence, and the 5 -end and 3 -end of the intron are AAC- and -ATGG, respectively. The splicing site and conserved regions P, R and S of it are different from type I intron. Moreover, there are differences in characteristics such as presence, location and structural feature of introns between individuals of Porphyra yezoensis thallus. So it is not appropriate to use introns for classification between intra-species. However, the length and nucleotide sequence of introns are specific in various species, which may provide references for discrimination of red algae between inter-species and intra-species with distant relationship or different biogeography. ( 本文编辑 : 梁德海 ) 18 / 2015 / 39 / 3