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摘要:龙须菜(Gracilaria lemaneiformis Bory)是重要的产琼胶海藻。本文采用抑制性消减杂交(SSH)技术构建了龙须菜高温胁迫差异表达cDNA文库,筛选到360个阳性克隆。测序结果经比对初步确定功能的基因56个,按功能分为:细胞防御;细胞信号转导;能量传递;蛋白质转录、翻译;代谢相关等几大类。研究结果为进一步研究龙须菜响应高温胁迫的分子机制提供了基础。
关键词:抑制性消减杂交;龙须菜;高温;cDNA
Construction of Suppression subtractive hybridization library of Gracilaria lemaneiformis under heat stress
Zang Xiaonan*, Zhang Xuan, Gu Yinghui, Lu Ning, Li Guangqi, Zhang Xuecheng, Zhang Lu, Tan Yanmiao, Yan Aiting
(College of Marine Life Sciences, Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, Qingdao 266003,China)
Gracilaria lemaneiformis is one of the most important economical marine algae in China. In order to gain a mechanism of responce of G. lemaneiformis to heat stress, the suppression subtractive hybridization (SSH) between the cDNA library of the wild type that lives in common condition and under the heat-stress is used to make up the cDNA discrepancy library. In the discrepancy library, there are 360 positive clones are selected by differential screening. By sequencing and BLAST, 56 differential genes that response to the heat stress are selected. The function of the differential genes can be divided into Cell defense, Cellular signal transduction, Energy transfer, Protein transcription and translation, Metabolism. The results provide bases for further study the molecular mechanism of Gracilaria lemaneiformis response to high temperature stress.
Key words:Suppression subtractive hybridization;Gracilaria lemaneiformis;heat stress;cDNA
龙须菜(Gracilaria Lemaneiformis)是红藻门(Rhodophyta)、江蓠属(Gracilaria)的一种重要产琼胶海藻。野生型龙须菜适温范围窄(11° C~23° C),生长期短,而在野生龙须菜基础上驯化并筛选的优良品系981龙须菜,使龙须菜的耐高温性能从23° C提高到了26° C,延长了栽培期,提高了产量[1]。但是目前产业化需要进一步提高龙须菜的耐高温性能,以适应夏季的高温。研究龙须菜高温胁迫响应机制,将为促进龙须菜耐高温新品系的选育起到有利的支撑作用。
自1992年Liang和Pardee首次提出了mRNA差异显示技术以来,已发展了多种分离和鉴定差别表达基因的技术[2]。抑制性消减杂交(SSH)是一种极为有效的差减杂交技术,它可以在抑制非目的DNA片段扩增的同时,选择性的扩增差异表达目的cDNA片段[3]。SSH技术在藻类中应用的并不多,但近年来利用该技术研究藻类的报道也在逐年增多,主要集中在发育相关基因、胁迫条件下特异表达基因及其它方面基因的分离。关于藻类胁迫条件下特异表达基因的筛查方面, Pearson等 (2001) 研究了墨角藻在失水60-70% 条件下的特异基因的表达[4]。Zhang等 (2002) 在研究盐生杜氏藻在盐胁迫条件下特异基因的表达时,发现长期胁迫条件下有2个特异表达的基因,短期有3个特异表达的基因[5]。Happe等 (2002) 构建了衣藻在氧缺乏时特异表达基因的消减文库[6]。Chung等 (2003) 研究了东海束毛藻营养缺乏条件下特异表达的基因 [7]。
本文构建了野生型龙须菜高温胁迫差异表达的抑制性消减杂交(SSH)文库,筛查龙须菜在高温胁迫条件下的差异表达基因,为进一步研究龙须菜高温胁迫响应机制提供分子基础。
1 材料与方法
1.1实验材料
野生型龙须菜:于山东省青岛沿海采集后经实验室预培养再用于实验研究。预培养条件为:光强50-60 mol photon/m2·s1,光暗周期12L:12D,溫度22 1C,每周更换新鲜的f/2培养基。热处理藻体用f/2培养基培养,放入35° C光照培养箱,光强50-60 mol photon/m2·s1,光暗周期12L:12D,培养24h。 1.2 实验方法
1.2.1总 RNA 的抽提
分别提取龙须菜热处理藻体RNA(简写为WH)和正常培养的龙须菜藻体RNA(简写为W)。提取RNA前,藻体都在无菌水中清洗,擦干藻体表面水后,立即用于RNA的提取。总RNA提取采用天根生物技术有限公司的RNAprep 植物总RNA提取试剂盒。
1.2.2 抑制性消减杂交文库的构建
为检测龙须菜在高温胁迫下上调表达和下调表达的cDNA,同时构建正向消减杂交文库和反向消减杂交文库。其中正向消减杂交文库以高温胁迫龙须菜cDNA为tester(简写为WHT),正常生长龙须菜cDNA为driver(简写为W);反向消减杂交文库以正常生长龙须菜cDNA为tester(简写为WT),高温胁迫龙须菜cDNA为driver(简写为WH)。
抑制性消减杂交文库的构建采用Super SMART? cDNA 合成试剂盒(Clontech)和Clontech PCR-Select? cDNA消减杂交试剂盒(Clontech)。
2 实验结果和分析
2.1 总RNA提取
正常生长龙须菜(W)和高温胁迫龙须菜(WH)总RNA的电泳结果见图1。高温胁迫龙须菜(WH)的RNA提取结果中可见2条明显的亮带,其中28S rRNA带约为18S rRNA带亮度的1.5-2倍,最前面的小分子量RNA的含量较少,说明没有降解的18S或28S rRNA的掺入。正常生长龙须菜(W)的RNA提取结果中可见4条明显的亮带,从大到小依次是Genome DNA,28S rRNA ,18S rRNA和5S rRNA,为避免DNA的干扰,后续实验均采用DNA酶消化。测定WH和W的RNA A260/A280均大于1.8,说明纯度较高,RNA提取质量较好。
图1:龙须菜总RNA提取结果
M:Marker DL2000;W:正常生长龙须菜总RNA提取结果;WH:高温胁迫龙须菜总RNA提取结果
Fig.1 Total RNA of Gracilaria lemaneiformis
M:Marker DL2000;W:Total RNA of Gracilaria lemaneiformis under common condition; WH: Total RNA of Gracilaria lemaneiformis under heat shock
2.2差减结果
差减产物第二轮PCR鉴定结果见图2。各差减组均有较弥散条带,说明各差减组均有差异性的PCR产物,且通过Hoefer DyNA Quant 200进行PCR产物浓度微量测定,测定时以标准浓度的 DNA为定量标准,测定显示产物浓度均在60ng/μL以上,足够进行SSH文库斑点杂交筛选差异表达产物。
图2. 差减产物第二轮PCR电泳结果
1:正向文库差减杂交PCR结果;2:反向文库差减杂交PCR结果;
M:Marker DL2000
Fig.2 The second round PCR result of subtractive hybridization library
1: PCR result of forward subtractive hybridization library; 2: PCR result of reverse subtractive hybridization library; M:Marker DL2000
2.3 斑点杂交差示筛选分析
从正向、反向库中各选取384个符合要求的克隆的PCR产物,以消减杂交后两轮抑制性PCR的产物为探针进行斑点杂交筛选。抑制性消减杂交文库斑点杂交结果见图3。
1 2
图3 抑制性消减杂交文库斑点杂交结果
1:龙须菜高温胁迫的抑制性消减杂交文库(含正、反向文库所有克隆)以WHT- W为探针斑点杂交的结果;
2:龙须菜高温胁迫的抑制性消减杂交文库(含正、反向文库所有克隆)以WT- WH为探针斑点杂交的结果
Fig.3 Dot blot result of Suppression subtractive hybridization library
1:Dot blot with WHT- W as the probe;2:Dot blot with WT- WH as the probe
龙须菜高温胁迫的抑制性消减杂交文库(含正、反向文库所有克隆)的探针斑点杂交结果的归一化系数为0.5855,归一化后正向文庫(WHT- W)中每个克隆信号比值Ration (F/R) >2,反向文库(WH- WT)中每个克隆信号比值Ration (F/R) <0.5均为阳性克隆。结果显示:龙须菜高温胁迫的抑制性消减杂交正向文库(WHT-W)中阳性克隆144个,反向文库(WT-WH)中阳性克隆216个,即龙须菜高温诱导后与正常生长龙须菜的差异表达基因共360个,阳性克隆比例为46.88%。
2.4 测序结果分析
将龙须菜高温胁迫抑制性消减杂交文库中的360个阳性克隆全部测序。测序结果经BLAST比对初步确定功能的基因56个,其中24个基因表达上调,32个基因表达下调。
在表达上调的基因中,主要包括hsp90、hsp70及其他帮助蛋白质折叠的分子伴侣、抗氧化酶基因(Superoxide dismutase,Peroxidase,Bromoperoxidase等)、泛素基因(Polyubiquitin,Ubiquitin-acting enzyme,Ubiquitin-conjugating enzyme)等与细胞防御相关的基因;细胞信号传导(Calmodulin、Receptor of activated protein kinase等)和能量传递的基因;及代谢相关的基因等。 在表达下调的基因中,主要包括糖代谢途径的基因;藻胆体和光系统等光能捕获相关的基因;能量传递、细胞运输相关的基因;蛋白质转录、翻译及代谢相关等的基因。
表1. 龙须菜高温胁迫抑制性消减杂交文库中表达上调的主要基因
Table 1. The major upregulated genes of suppression subtractive hybridization library of Gracilaria lemaneiformis under heat stress
克隆号 基因功能注释 Genbank No. 相似性 E值
8-2-18,8-2-21,82-52,8-2-84,8-3-30,8-3-33,8-3-63,8-4-3,8-4-19,8-4-44,8-4-95,8-5-68,8-5-82 Hsp90 EU095964.1 79% 3e-104
8-1-19,8-5-8 hsp70 AC210918.1 78% 4e-35
8-4-15 mitochondrial chaperone XM_003068698.1 79% 2e-07
8-4-60 FKBP-type peptidyl-prolyl cis-trans isomerase AP009552.1 75% 3e-09
8-1-84 Mn-superoxide dismutase DQ057354.1 74% 1e-40
8-2-33 Peroxidase
CP000781.1 82% 5e-08
8-4-39 vanadium-dependent bromoperoxidase AF218810.1 67% 3e-14
8-5-28 glutathione-disulfide reductase CP000926.1 74% 3e-16
8-4-12 Polyubiquitin AY957885.1 99% 0.0
8-3-41 ubiquitin-acting enzyme BT000094.1 72% 0.022
8-2-31,8-4-15 ubiquitin-conjugating enzyme AF517850.1 79% 7e-102
8-4-67 Calmodulin AY656700.1 72% 1e-39
8-3-62 Receptor of activated protein kinase XM_001422821.1 71% 7e-65
8-1-33 Na-K ATPase NM_001124630.1 73% 2e-09
8-3-79 GTP-binding protein NM_001032362.1 74% 4e-29
8-1-8 ATP binding domain XM_001745297.1 65% 1e-15
8-1-73 ATP-dependent RNA helicase XM_002902982.1 68% 9e-13
8-1-17 Nitrilase NM_001091940.1 70% 2e-11
8-1-22 sugar nucleotide epimerase XM_001689445.1 76% 4e-34
8-1-87 glucose-6-phosphate isomerase DQ812899.1 76% 1e-82
8-2-26,8-2-65 2-isopropylmalate synthase BA000039.2 71% 7e-32
8-4-22 gamma-glutamyltransferase NM_001006723.1 70% 0.004
8-5-41 GlcNAc-1-P transferase BC003943.1 71% 2e-16
8-1-66 DEAH (Asp-Glu-Ala-His) box polypeptide XR_026284.1 72% 4e-13
表2. 龙须菜高温胁迫抑制性消减杂交文库中表达下调的主要基因
Table 2. The major downregulated genes of suppression subtractive hybridization library of Gracilaria lemaneiformis under heat stress
克隆號 基因功能注释 Genbank No. 相似性 E值
7-1-36,7-1-95,7-2-48,
7-2-89, 7-3-48, 7-3-96, 7-4-66, 7-4-83, 7-5-41 Agll Y18737.1 99% 0.0
7-2-7 fructose-bisphosphate aldolase AM384903.1 71% 1e-06
7-2-99 D-xylose reductase XM_652935.1 76% 4e-08
7-4-86 starch-branching enzyme AF042842.1 73% 2e-10
7-1-55, 7-5-65 Light-harvest protein AF517881.1 70% 4e-15
7-3-40, 7-4-2 Light-harvesting complex I AY124024.1 69% 2e-22 7-2-50, 7-2-82 chloroplast AY673996.1 87% 0.0
7-4-28 R-phycoerythrin gamma-subunit L13695.1 80% 1e-04
7-4-33 Phycobilisome 31.8kD linker polypeptide AF517883.1 78% 4e-13
7-4-56 phycobilisome 7.8 kDa linker polypeptide AF542025.1 79% 7e-12
7-3-87 T-complex protein 1 (theta subunit) XM_001908042.1 69% 2e-19
7-3-78 cytochrome c oxidase subunit AF542022.1 80% 7e-28
7-4-10 cytochrome b-5 reductase XM_001194747.1 75% 8e-08
7-3-98 plastid oxygen-evolving enhancer AY856512.1 72% 1e-34
7-1-61 adenylate cyclase-associated protein (CAP) XR_027078.1 74% 7e-07
7-1-42 CTP synthase XM_001697557.1 80% 2e-31
7-1-80 ADP-ribosylation factor XM_001682205.1 82% 2e-05
7-3-83, 7-4-34 ATP-sulfurylase NM_123745.5 72% 7e-48
7-4-26 Plastid ATP-ADP transport protein AJ251356.1 79% 2e-18
7-2-22 methionine aminopeptidase XM_969765.1 79% 1e-13
7-2-36 alternative oxidase NM_113678.2 87% 1e-08
7-2-72 glutamate-1-semialdehyde aminomutase AP009552.1 77% 2e-09
7-3-16 chaperonin containing TCP1 eta subunit NW_001263736.1 72% 4e-13
7-4-22 prohibitin XM_001941783.1 72% 2e-13
7-4-48 protein transport protein sec61 XM_001378028.1 75% 2e-45
7-5-20, 7-5-54 cell division cycle protein XM_001776891.1 76% 6e-101
7-5-43 COP9 signalosome subunit AY133536.1 69% 6e-65
7-4-96 TATA-binding-protein-associated factor XM_001646431.1 73% 2e-36
7-4-7, 7-4-91, 7-4-95 putative translation initiation factor 5A DQ234392.1 74% 1e-24
7-2-40 translation initiation factor 3 NM_001046968.1 68% 5e-26
7-5-52 translation initiation factor 4 XM_655444.1 76% 4e-150
7-4-61 translation elongation factor 2 CP000319.1 73% 1e-16
3、结论
本实验通过构建龙须菜高温胁迫的抑制性消减杂交文库,筛选到龙须菜在高温胁迫下的差异表达克隆360个,经BLAST比对初步确定功能的基因56个,其中24个表达上调,32个表达下调。在表达上调的基因中,多是与细胞防御、细胞信号传导(Calmodulin等)和能量传递相关的基因。在表达下调的基因中,多是与糖代谢、光能捕获、能量传递及蛋白质转录、翻译等相关的基因。此外还有近60%的基因功能未知(不计算重复克隆),在大量未知功能的基因中,可能蕴含着响应高温胁迫的重要基因,仍需深入研究。
龙须菜高温胁迫的抑制性消减杂交文库的构建为进一步研究龙须菜响应高温胁迫的分子机制提供了基础。
参考文献
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[3] 王力华,戴晓枫. 抑制性减法杂交技术及其应用[J]. 分子植物育种. 2003, 1(4): 551-554.
[4] Pearson G, Serrao E A, Cancela M L. Suppression subtractive hybridization for studying gene expression during aerial exposure and desiccation in fucoid algae[J]. European Journal of Phycology, 2001, 36: 359-366.
[5] Zhang X N, Qu Z C, Wan Y Z,et al. Application of suppression subtraction hybridization (SSH) to cloning differentially expressed cDNA in Dunaliella Salina (chlorophyta) under hyperosmotic shock[J]. Plant Molecular Biology Reporter, 2002, 20: 49–57.
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[7] Chung C C, Hwang S P, Chang J. Identification of a high-affinity phosphate transporter gene in a Prasinophyte Alga, Tetraselmis chui, and its expression under nutrient limitation[J]. Applied and Environmental Microbiology, 2003, 69: 754–759.
通訊联系人: 臧晓南(1978-), 女, 教授, 现从事藻类环境胁迫分子机理研究
基金项目: 高等学校博士学科点专项科研基金资助课题(20070423011); 国家863计划海水养殖种子工程重大项目(2006AA10A413); 广东省汕头市科技计划项目(2007-44) ; 经济海藻良种产业化技术体系研究与示范(200803052)
关键词:抑制性消减杂交;龙须菜;高温;cDNA
Construction of Suppression subtractive hybridization library of Gracilaria lemaneiformis under heat stress
Zang Xiaonan*, Zhang Xuan, Gu Yinghui, Lu Ning, Li Guangqi, Zhang Xuecheng, Zhang Lu, Tan Yanmiao, Yan Aiting
(College of Marine Life Sciences, Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, Qingdao 266003,China)
Gracilaria lemaneiformis is one of the most important economical marine algae in China. In order to gain a mechanism of responce of G. lemaneiformis to heat stress, the suppression subtractive hybridization (SSH) between the cDNA library of the wild type that lives in common condition and under the heat-stress is used to make up the cDNA discrepancy library. In the discrepancy library, there are 360 positive clones are selected by differential screening. By sequencing and BLAST, 56 differential genes that response to the heat stress are selected. The function of the differential genes can be divided into Cell defense, Cellular signal transduction, Energy transfer, Protein transcription and translation, Metabolism. The results provide bases for further study the molecular mechanism of Gracilaria lemaneiformis response to high temperature stress.
Key words:Suppression subtractive hybridization;Gracilaria lemaneiformis;heat stress;cDNA
龙须菜(Gracilaria Lemaneiformis)是红藻门(Rhodophyta)、江蓠属(Gracilaria)的一种重要产琼胶海藻。野生型龙须菜适温范围窄(11° C~23° C),生长期短,而在野生龙须菜基础上驯化并筛选的优良品系981龙须菜,使龙须菜的耐高温性能从23° C提高到了26° C,延长了栽培期,提高了产量[1]。但是目前产业化需要进一步提高龙须菜的耐高温性能,以适应夏季的高温。研究龙须菜高温胁迫响应机制,将为促进龙须菜耐高温新品系的选育起到有利的支撑作用。
自1992年Liang和Pardee首次提出了mRNA差异显示技术以来,已发展了多种分离和鉴定差别表达基因的技术[2]。抑制性消减杂交(SSH)是一种极为有效的差减杂交技术,它可以在抑制非目的DNA片段扩增的同时,选择性的扩增差异表达目的cDNA片段[3]。SSH技术在藻类中应用的并不多,但近年来利用该技术研究藻类的报道也在逐年增多,主要集中在发育相关基因、胁迫条件下特异表达基因及其它方面基因的分离。关于藻类胁迫条件下特异表达基因的筛查方面, Pearson等 (2001) 研究了墨角藻在失水60-70% 条件下的特异基因的表达[4]。Zhang等 (2002) 在研究盐生杜氏藻在盐胁迫条件下特异基因的表达时,发现长期胁迫条件下有2个特异表达的基因,短期有3个特异表达的基因[5]。Happe等 (2002) 构建了衣藻在氧缺乏时特异表达基因的消减文库[6]。Chung等 (2003) 研究了东海束毛藻营养缺乏条件下特异表达的基因 [7]。
本文构建了野生型龙须菜高温胁迫差异表达的抑制性消减杂交(SSH)文库,筛查龙须菜在高温胁迫条件下的差异表达基因,为进一步研究龙须菜高温胁迫响应机制提供分子基础。
1 材料与方法
1.1实验材料
野生型龙须菜:于山东省青岛沿海采集后经实验室预培养再用于实验研究。预培养条件为:光强50-60 mol photon/m2·s1,光暗周期12L:12D,溫度22 1C,每周更换新鲜的f/2培养基。热处理藻体用f/2培养基培养,放入35° C光照培养箱,光强50-60 mol photon/m2·s1,光暗周期12L:12D,培养24h。 1.2 实验方法
1.2.1总 RNA 的抽提
分别提取龙须菜热处理藻体RNA(简写为WH)和正常培养的龙须菜藻体RNA(简写为W)。提取RNA前,藻体都在无菌水中清洗,擦干藻体表面水后,立即用于RNA的提取。总RNA提取采用天根生物技术有限公司的RNAprep 植物总RNA提取试剂盒。
1.2.2 抑制性消减杂交文库的构建
为检测龙须菜在高温胁迫下上调表达和下调表达的cDNA,同时构建正向消减杂交文库和反向消减杂交文库。其中正向消减杂交文库以高温胁迫龙须菜cDNA为tester(简写为WHT),正常生长龙须菜cDNA为driver(简写为W);反向消减杂交文库以正常生长龙须菜cDNA为tester(简写为WT),高温胁迫龙须菜cDNA为driver(简写为WH)。
抑制性消减杂交文库的构建采用Super SMART? cDNA 合成试剂盒(Clontech)和Clontech PCR-Select? cDNA消减杂交试剂盒(Clontech)。
2 实验结果和分析
2.1 总RNA提取
正常生长龙须菜(W)和高温胁迫龙须菜(WH)总RNA的电泳结果见图1。高温胁迫龙须菜(WH)的RNA提取结果中可见2条明显的亮带,其中28S rRNA带约为18S rRNA带亮度的1.5-2倍,最前面的小分子量RNA的含量较少,说明没有降解的18S或28S rRNA的掺入。正常生长龙须菜(W)的RNA提取结果中可见4条明显的亮带,从大到小依次是Genome DNA,28S rRNA ,18S rRNA和5S rRNA,为避免DNA的干扰,后续实验均采用DNA酶消化。测定WH和W的RNA A260/A280均大于1.8,说明纯度较高,RNA提取质量较好。
图1:龙须菜总RNA提取结果
M:Marker DL2000;W:正常生长龙须菜总RNA提取结果;WH:高温胁迫龙须菜总RNA提取结果
Fig.1 Total RNA of Gracilaria lemaneiformis
M:Marker DL2000;W:Total RNA of Gracilaria lemaneiformis under common condition; WH: Total RNA of Gracilaria lemaneiformis under heat shock
2.2差减结果
差减产物第二轮PCR鉴定结果见图2。各差减组均有较弥散条带,说明各差减组均有差异性的PCR产物,且通过Hoefer DyNA Quant 200进行PCR产物浓度微量测定,测定时以标准浓度的 DNA为定量标准,测定显示产物浓度均在60ng/μL以上,足够进行SSH文库斑点杂交筛选差异表达产物。
图2. 差减产物第二轮PCR电泳结果
1:正向文库差减杂交PCR结果;2:反向文库差减杂交PCR结果;
M:Marker DL2000
Fig.2 The second round PCR result of subtractive hybridization library
1: PCR result of forward subtractive hybridization library; 2: PCR result of reverse subtractive hybridization library; M:Marker DL2000
2.3 斑点杂交差示筛选分析
从正向、反向库中各选取384个符合要求的克隆的PCR产物,以消减杂交后两轮抑制性PCR的产物为探针进行斑点杂交筛选。抑制性消减杂交文库斑点杂交结果见图3。
1 2
图3 抑制性消减杂交文库斑点杂交结果
1:龙须菜高温胁迫的抑制性消减杂交文库(含正、反向文库所有克隆)以WHT- W为探针斑点杂交的结果;
2:龙须菜高温胁迫的抑制性消减杂交文库(含正、反向文库所有克隆)以WT- WH为探针斑点杂交的结果
Fig.3 Dot blot result of Suppression subtractive hybridization library
1:Dot blot with WHT- W as the probe;2:Dot blot with WT- WH as the probe
龙须菜高温胁迫的抑制性消减杂交文库(含正、反向文库所有克隆)的探针斑点杂交结果的归一化系数为0.5855,归一化后正向文庫(WHT- W)中每个克隆信号比值Ration (F/R) >2,反向文库(WH- WT)中每个克隆信号比值Ration (F/R) <0.5均为阳性克隆。结果显示:龙须菜高温胁迫的抑制性消减杂交正向文库(WHT-W)中阳性克隆144个,反向文库(WT-WH)中阳性克隆216个,即龙须菜高温诱导后与正常生长龙须菜的差异表达基因共360个,阳性克隆比例为46.88%。
2.4 测序结果分析
将龙须菜高温胁迫抑制性消减杂交文库中的360个阳性克隆全部测序。测序结果经BLAST比对初步确定功能的基因56个,其中24个基因表达上调,32个基因表达下调。
在表达上调的基因中,主要包括hsp90、hsp70及其他帮助蛋白质折叠的分子伴侣、抗氧化酶基因(Superoxide dismutase,Peroxidase,Bromoperoxidase等)、泛素基因(Polyubiquitin,Ubiquitin-acting enzyme,Ubiquitin-conjugating enzyme)等与细胞防御相关的基因;细胞信号传导(Calmodulin、Receptor of activated protein kinase等)和能量传递的基因;及代谢相关的基因等。 在表达下调的基因中,主要包括糖代谢途径的基因;藻胆体和光系统等光能捕获相关的基因;能量传递、细胞运输相关的基因;蛋白质转录、翻译及代谢相关等的基因。
表1. 龙须菜高温胁迫抑制性消减杂交文库中表达上调的主要基因
Table 1. The major upregulated genes of suppression subtractive hybridization library of Gracilaria lemaneiformis under heat stress
克隆号 基因功能注释 Genbank No. 相似性 E值
8-2-18,8-2-21,82-52,8-2-84,8-3-30,8-3-33,8-3-63,8-4-3,8-4-19,8-4-44,8-4-95,8-5-68,8-5-82 Hsp90 EU095964.1 79% 3e-104
8-1-19,8-5-8 hsp70 AC210918.1 78% 4e-35
8-4-15 mitochondrial chaperone XM_003068698.1 79% 2e-07
8-4-60 FKBP-type peptidyl-prolyl cis-trans isomerase AP009552.1 75% 3e-09
8-1-84 Mn-superoxide dismutase DQ057354.1 74% 1e-40
8-2-33 Peroxidase
CP000781.1 82% 5e-08
8-4-39 vanadium-dependent bromoperoxidase AF218810.1 67% 3e-14
8-5-28 glutathione-disulfide reductase CP000926.1 74% 3e-16
8-4-12 Polyubiquitin AY957885.1 99% 0.0
8-3-41 ubiquitin-acting enzyme BT000094.1 72% 0.022
8-2-31,8-4-15 ubiquitin-conjugating enzyme AF517850.1 79% 7e-102
8-4-67 Calmodulin AY656700.1 72% 1e-39
8-3-62 Receptor of activated protein kinase XM_001422821.1 71% 7e-65
8-1-33 Na-K ATPase NM_001124630.1 73% 2e-09
8-3-79 GTP-binding protein NM_001032362.1 74% 4e-29
8-1-8 ATP binding domain XM_001745297.1 65% 1e-15
8-1-73 ATP-dependent RNA helicase XM_002902982.1 68% 9e-13
8-1-17 Nitrilase NM_001091940.1 70% 2e-11
8-1-22 sugar nucleotide epimerase XM_001689445.1 76% 4e-34
8-1-87 glucose-6-phosphate isomerase DQ812899.1 76% 1e-82
8-2-26,8-2-65 2-isopropylmalate synthase BA000039.2 71% 7e-32
8-4-22 gamma-glutamyltransferase NM_001006723.1 70% 0.004
8-5-41 GlcNAc-1-P transferase BC003943.1 71% 2e-16
8-1-66 DEAH (Asp-Glu-Ala-His) box polypeptide XR_026284.1 72% 4e-13
表2. 龙须菜高温胁迫抑制性消减杂交文库中表达下调的主要基因
Table 2. The major downregulated genes of suppression subtractive hybridization library of Gracilaria lemaneiformis under heat stress
克隆號 基因功能注释 Genbank No. 相似性 E值
7-1-36,7-1-95,7-2-48,
7-2-89, 7-3-48, 7-3-96, 7-4-66, 7-4-83, 7-5-41 Agll Y18737.1 99% 0.0
7-2-7 fructose-bisphosphate aldolase AM384903.1 71% 1e-06
7-2-99 D-xylose reductase XM_652935.1 76% 4e-08
7-4-86 starch-branching enzyme AF042842.1 73% 2e-10
7-1-55, 7-5-65 Light-harvest protein AF517881.1 70% 4e-15
7-3-40, 7-4-2 Light-harvesting complex I AY124024.1 69% 2e-22 7-2-50, 7-2-82 chloroplast AY673996.1 87% 0.0
7-4-28 R-phycoerythrin gamma-subunit L13695.1 80% 1e-04
7-4-33 Phycobilisome 31.8kD linker polypeptide AF517883.1 78% 4e-13
7-4-56 phycobilisome 7.8 kDa linker polypeptide AF542025.1 79% 7e-12
7-3-87 T-complex protein 1 (theta subunit) XM_001908042.1 69% 2e-19
7-3-78 cytochrome c oxidase subunit AF542022.1 80% 7e-28
7-4-10 cytochrome b-5 reductase XM_001194747.1 75% 8e-08
7-3-98 plastid oxygen-evolving enhancer AY856512.1 72% 1e-34
7-1-61 adenylate cyclase-associated protein (CAP) XR_027078.1 74% 7e-07
7-1-42 CTP synthase XM_001697557.1 80% 2e-31
7-1-80 ADP-ribosylation factor XM_001682205.1 82% 2e-05
7-3-83, 7-4-34 ATP-sulfurylase NM_123745.5 72% 7e-48
7-4-26 Plastid ATP-ADP transport protein AJ251356.1 79% 2e-18
7-2-22 methionine aminopeptidase XM_969765.1 79% 1e-13
7-2-36 alternative oxidase NM_113678.2 87% 1e-08
7-2-72 glutamate-1-semialdehyde aminomutase AP009552.1 77% 2e-09
7-3-16 chaperonin containing TCP1 eta subunit NW_001263736.1 72% 4e-13
7-4-22 prohibitin XM_001941783.1 72% 2e-13
7-4-48 protein transport protein sec61 XM_001378028.1 75% 2e-45
7-5-20, 7-5-54 cell division cycle protein XM_001776891.1 76% 6e-101
7-5-43 COP9 signalosome subunit AY133536.1 69% 6e-65
7-4-96 TATA-binding-protein-associated factor XM_001646431.1 73% 2e-36
7-4-7, 7-4-91, 7-4-95 putative translation initiation factor 5A DQ234392.1 74% 1e-24
7-2-40 translation initiation factor 3 NM_001046968.1 68% 5e-26
7-5-52 translation initiation factor 4 XM_655444.1 76% 4e-150
7-4-61 translation elongation factor 2 CP000319.1 73% 1e-16
3、结论
本实验通过构建龙须菜高温胁迫的抑制性消减杂交文库,筛选到龙须菜在高温胁迫下的差异表达克隆360个,经BLAST比对初步确定功能的基因56个,其中24个表达上调,32个表达下调。在表达上调的基因中,多是与细胞防御、细胞信号传导(Calmodulin等)和能量传递相关的基因。在表达下调的基因中,多是与糖代谢、光能捕获、能量传递及蛋白质转录、翻译等相关的基因。此外还有近60%的基因功能未知(不计算重复克隆),在大量未知功能的基因中,可能蕴含着响应高温胁迫的重要基因,仍需深入研究。
龙须菜高温胁迫的抑制性消减杂交文库的构建为进一步研究龙须菜响应高温胁迫的分子机制提供了基础。
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通訊联系人: 臧晓南(1978-), 女, 教授, 现从事藻类环境胁迫分子机理研究
基金项目: 高等学校博士学科点专项科研基金资助课题(20070423011); 国家863计划海水养殖种子工程重大项目(2006AA10A413); 广东省汕头市科技计划项目(2007-44) ; 经济海藻良种产业化技术体系研究与示范(200803052)