盐碱胁迫下星星草（Puccinellia tenuiflora）根部转录应答的初步分析
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摘要
星星草是一种具有较强耐盐碱能力的盐生植物。为了了解星星草的耐盐碱分子机制,分离重要的耐盐碱基因,本研究测定了星星草在盐碱胁迫条件下的生理指标,分别构建了盐碱地胁迫条件下和300 mM NaCl胁迫条件下的星星草根部的抑制性消减杂交文库,挑取插入片段大于500 bp的克隆进行了序列测定,对文库中的表达序列标签进行了序列对比、功能注释和功能分类等生物信息学分析,主要研究结果如下:
1.本研究对星星草在盐碱胁迫条件下的根长、株高、含水量、游离脯氨酸含量和花青素含量等生理指标进行了测定,发现在300 mM NaCl和150 mM Na_2CO_3短时间胁迫下星星草的生长受到轻微抑制,但仍呈现出积极的应答反应。
2.对盐碱地胁迫条件下星星草根部抑制性消减杂交文库测序311条,获得289个高质量的ESTs,用phrap软件对ESTs序列进行组装。得到共199条有意义的Unigene序列。用Blastn和Blastx进行分析,有141条序列(70.85%)与已知基因有较高相似性(Blastn E-valuee~(-10), Blastx E-value>e~(-5)),可视为新基因。
3.盐碱地胁迫条件下星星草根部抑制性消减杂交文库中的ESTs序列经GO功能分类将序列分为生物学途径、分子功能和细胞组件三个层次。经COG功能分类分为九大类:翻译、核糖体结构和生物发生;氨基酸转运和代谢;碳水化合物转运和代谢;能量产生和转化;细胞壁/膜/被膜发生;功能未知;翻译后修饰、蛋白质周转和分子伴侣;转录和信号传导机制。
4.在盐碱地胁迫条件下的星星草根部消减文库中筛选到一些与植物耐盐碱性相关的序列,包括WD-40重复蛋白、锌指蛋白、半胱氨酸蛋白酶、半胱氨酸蛋白酶抑制剂、蔗糖合成酶、液泡H+-ATP酶、甘油醛- 3-磷酸-脱氢酶和普遍应激蛋白等基因的片段。这些ESTs的获得为进一步克隆全长基因的cDNA以及探索星星草耐盐碱机制奠定了重要基础。
5.对NaCl胁迫条件下星星草根部的抑制性消减杂交文库测序68条,对68个序列与数据库中的序列比对,有40条序列相似性较高(Blastx E-valuee~(-5)),可视为新基因。
6.在NaCl胁迫条件下星星草根部抑制性消减杂交文库中筛选到有丝分裂原活化蛋白激酶、翻译延伸因子1A和半胱氨酸蛋白酶抑制剂等与植物耐盐性相关的基因的片段。这些ESTs的获得为进一步克隆全长基因的cDNA以及探索星星草耐盐机制奠定了重要基础。
Puccinellia tenuiflora is one of the halophytes with high capacity of saline and alkaline resistance. In order to understand saline-alkali resistance mechanism of Puccinellia tenuiflora and isolate important genes associated with saline-alkali resistance of Puccinellia tenuiflora, in this study, physiological index of Puccinellia tenuiflora under saline-alkali stressed conditions had been determined, and suppressed subtractive hybridization libraries of Puccinellia tenuiflora roots under saline-alkali soil and 300 mM NaCl stressed conditions had been constructed respectively. Recombinant clones with the insert fragment larger than 500 bp were selected randomly and sequenced, bioinformatic analysis including sequence alignment, functional annotation and functional classification of expressed sequence tags in libraries was performed. The main results are as follows:
1. Physiological index including root length, stem length, water content, proline content and anthocyanin content of Puccinellia tenuiflora was determined under 300 mM NaCl and 150 mM Na_2CO_3 stressed conditions during short time courses respectively. The results indicated that the growth of Puccinellia tenuiflora seedlings was slightly suppressed under the above stressed conditons, but seedlings still actively responded to stress.
2. From the suppressed subtractive hybridization libray of Puccinellia tenuiflora roots under saline-alkali soil stressed conditon, 311 ESTs were sequenced, 289 of them were high-quality ESTs, sequences were assembled using phrap software to get a total of 199 meaningful unigene sequences. Blastn and Blastx analysis respectively showed that 141 (70.85%) unigene fragments were homologous (Blastn E-valuee~(-10), Blastx E-value>e~(-5)) with the sequences in database, which could be regarded as new genes.
3. In the suppressed subtractive hybridization library of Puccinellia tenuiflora roots under saline-alkali soil stressed condition, ESTs were divided into three aspects including Biological Process, Molecular function and Cell component based on GO analysis. In COG functional classification, ESTs can be divided in to 9 categories: Translation, ribosomal structure and biogenesis; Amino acid transport and metabolism; Carbohydrate transport and metabolism; Energy production and conversion; cell wall/membrane/envelope biogenesis; Function unknown; Posttranslational modification, protein turnover, chaperones; Transcription; Signal transduction mechanisms.
4. In the suppressed subtractive hybridization library of Puccinellia tenuiflora roots under saline-alkali soil conditions, several ESTs associated with saline-alkali resistance of plants were screened, such as WD-40 repeat protein, Zn~+ finger protein, cysteine proteinase, cysteine proteinase inhibitor, sucrose synthase, vacuolar ATPase, glyceraldehyde-3-phosphate dehydrogenase and universe stress protein genes. The gain of the ESTs will lay an important foundation for cloning the full-length cDNA genes and exploring the mechanism of Puccinellia tenuiflora under saline and alkaline stress.
5. In the suppressed subtractive hybridization libray of Puccinellia tenuiflora roots under NaCl stressed conditions, 68 ESTs were sequenced randomly, Blastx analysis showed that 40 ESTs were homologous (Blastx E-value 6. In the suppressed subtractive hybridization libray of Puccinellia tenuiflora roots under NaCl stressed conditions, several ESTs associated with salt resistance of plants were screened, such as MAPK, EF1A and CPI. The gain of the ESTs will lay an important foundation for cloning the full-length cDNA genes and exploring the mechanism of Puccinellia tenuiflora under salt stress.
1.本研究对星星草在盐碱胁迫条件下的根长、株高、含水量、游离脯氨酸含量和花青素含量等生理指标进行了测定,发现在300 mM NaCl和150 mM Na_2CO_3短时间胁迫下星星草的生长受到轻微抑制,但仍呈现出积极的应答反应。
2.对盐碱地胁迫条件下星星草根部抑制性消减杂交文库测序311条,获得289个高质量的ESTs,用phrap软件对ESTs序列进行组装。得到共199条有意义的Unigene序列。用Blastn和Blastx进行分析,有141条序列(70.85%)与已知基因有较高相似性(Blastn E-value
3.盐碱地胁迫条件下星星草根部抑制性消减杂交文库中的ESTs序列经GO功能分类将序列分为生物学途径、分子功能和细胞组件三个层次。经COG功能分类分为九大类:翻译、核糖体结构和生物发生;氨基酸转运和代谢;碳水化合物转运和代谢;能量产生和转化;细胞壁/膜/被膜发生;功能未知;翻译后修饰、蛋白质周转和分子伴侣;转录和信号传导机制。
4.在盐碱地胁迫条件下的星星草根部消减文库中筛选到一些与植物耐盐碱性相关的序列,包括WD-40重复蛋白、锌指蛋白、半胱氨酸蛋白酶、半胱氨酸蛋白酶抑制剂、蔗糖合成酶、液泡H+-ATP酶、甘油醛- 3-磷酸-脱氢酶和普遍应激蛋白等基因的片段。这些ESTs的获得为进一步克隆全长基因的cDNA以及探索星星草耐盐碱机制奠定了重要基础。
5.对NaCl胁迫条件下星星草根部的抑制性消减杂交文库测序68条,对68个序列与数据库中的序列比对,有40条序列相似性较高(Blastx E-value
6.在NaCl胁迫条件下星星草根部抑制性消减杂交文库中筛选到有丝分裂原活化蛋白激酶、翻译延伸因子1A和半胱氨酸蛋白酶抑制剂等与植物耐盐性相关的基因的片段。这些ESTs的获得为进一步克隆全长基因的cDNA以及探索星星草耐盐机制奠定了重要基础。
Puccinellia tenuiflora is one of the halophytes with high capacity of saline and alkaline resistance. In order to understand saline-alkali resistance mechanism of Puccinellia tenuiflora and isolate important genes associated with saline-alkali resistance of Puccinellia tenuiflora, in this study, physiological index of Puccinellia tenuiflora under saline-alkali stressed conditions had been determined, and suppressed subtractive hybridization libraries of Puccinellia tenuiflora roots under saline-alkali soil and 300 mM NaCl stressed conditions had been constructed respectively. Recombinant clones with the insert fragment larger than 500 bp were selected randomly and sequenced, bioinformatic analysis including sequence alignment, functional annotation and functional classification of expressed sequence tags in libraries was performed. The main results are as follows:
1. Physiological index including root length, stem length, water content, proline content and anthocyanin content of Puccinellia tenuiflora was determined under 300 mM NaCl and 150 mM Na_2CO_3 stressed conditions during short time courses respectively. The results indicated that the growth of Puccinellia tenuiflora seedlings was slightly suppressed under the above stressed conditons, but seedlings still actively responded to stress.
2. From the suppressed subtractive hybridization libray of Puccinellia tenuiflora roots under saline-alkali soil stressed conditon, 311 ESTs were sequenced, 289 of them were high-quality ESTs, sequences were assembled using phrap software to get a total of 199 meaningful unigene sequences. Blastn and Blastx analysis respectively showed that 141 (70.85%) unigene fragments were homologous (Blastn E-value
3. In the suppressed subtractive hybridization library of Puccinellia tenuiflora roots under saline-alkali soil stressed condition, ESTs were divided into three aspects including Biological Process, Molecular function and Cell component based on GO analysis. In COG functional classification, ESTs can be divided in to 9 categories: Translation, ribosomal structure and biogenesis; Amino acid transport and metabolism; Carbohydrate transport and metabolism; Energy production and conversion; cell wall/membrane/envelope biogenesis; Function unknown; Posttranslational modification, protein turnover, chaperones; Transcription; Signal transduction mechanisms.
4. In the suppressed subtractive hybridization library of Puccinellia tenuiflora roots under saline-alkali soil conditions, several ESTs associated with saline-alkali resistance of plants were screened, such as WD-40 repeat protein, Zn~+ finger protein, cysteine proteinase, cysteine proteinase inhibitor, sucrose synthase, vacuolar ATPase, glyceraldehyde-3-phosphate dehydrogenase and universe stress protein genes. The gain of the ESTs will lay an important foundation for cloning the full-length cDNA genes and exploring the mechanism of Puccinellia tenuiflora under saline and alkaline stress.
5. In the suppressed subtractive hybridization libray of Puccinellia tenuiflora roots under NaCl stressed conditions, 68 ESTs were sequenced randomly, Blastx analysis showed that 40 ESTs were homologous (Blastx E-value
引文
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