盐胁迫下紫杆柽柳cDNA文库构建及表达序列标签（EST）分析
摘要
柽柳抗旱、耐盐能力强，因此是进行耐盐碱机理研究的良好材料，但目前，有关柽柳耐盐碱的分子机理研究国内外尚未见报道。本文为了研究柽柳的耐盐机理构建了0.4 mmol／L NaHCO_3胁迫处理的柽柳cDNA文库，随机选取重组克隆测序，用表达序列标签(EST)方法研究NaHCO_3胁迫下柽柳基因的表达。
cDNA初始文库滴度为1.2×10~6pfu／mL，蓝白斑检验结果表明，文库重组率为96％，PCR检测表明插入片段的平均长度1 200bp左右。
随机挑选文库中的克隆进行测序。将获得序列去除5’端载体序列、文库接头序列和poly(A)序列。共获得2 328条读序较好的序列。
以获得的2 328条序列建立本地核酸数据库。本地BLAST获得642个与其它序列无同源区的序列，其余的1 686条序列依照同源性聚为281个序列集，对每个序列集通过序列联配获得359个contig。用于网上进行BLAST分析的独立序列共有1 001个。文库的冗余度为57％。
1 001个序列的BLASTX分析表明，有428个与已知功能的基因序列相似，234个与未知功能的基因相似，339个未能发现相似序列。将428个代表独立已知功能基因的EST，按功能分为11个类，其中防御基因、新陈代谢和蛋白合成及分拣的表达丰度最高，分别为25.26％、20.65％和15.37％。对与耐盐有关基因EST分析结果表明，信号元件、转录调控、应激蛋白和活性氧清除剂所包含的表达的基因比较多，这些基因的表达丰度也较高，分别为4.1％、1.9％、3.3％和0.9％，这说明这四种过程在柽柳抗盐胁迫时起到了重要作用。此外，跨膜运输及质子平衡、蛋白质代谢和新陈代谢类所包含的表达的基因种类相对较少，但是其表达丰度也较高分别为0.9％、0.9％和2.3％，可能在盐胁迫中也起到了重要的作用。
以上研究说明，柽柳的抗盐过程是一个复杂的多基因协同作用体系，涉及到防御反应、基因调控、信号传导、离子转运、活性氧清除、渗透调节、应激反应等多方面的综合作用，从而为系统阐明柽柳抗盐分子机理研究奠定坚实基础。
Tamarix spp. is a good material to study the salt-resistance molecular mechanism because it has high ability to resist drought and salt stress. A cDNA library was constructed from the tissue of Tamarix androssowii treated with NaHCOs Expressed sequence tags (EST) analysis was carried out to study the molecular mechanism of salt-resistance in Tamarix androssowii.
The library primary titer was 1.14
×10 pfu/mL, and the rate of recombination was 96%, the titer of amplified library was 4.0
×109 pfu/mL. The average length of inserted fragments in the library was 1.2kb, which was testified by both PCR and sequencing analysis. From this library, 2 328 high quality ESTs were obtained.
Homology analysis by local BLAST and multiple alignment showed that these clones represented 1 001 unique transcripts. Results of comparing with the genes in unigene database of GenBank based on BLASTX algorithm indicated that 428 ESTs were homologous to the genes identified previously by BLAST from the Internet, while other 234 ESTs matched unknown protein coding regions and 339 ESTs were completely novel sequences. Eleven functional categories were carried out from all the tentative unique genes that were identified. Three group transcripts were significantly expressed: Defence, Metabolism and Protein synthesis and destination that respesented 25.26%, 20.65% and 15.37% respectively. The result of genes related to salt stress-tolerance indicated that Signaling components, Transcriptional regulators, Stress protein and Reactive oxygen scavengers contained more genes than the others, 4.1%, 1.9%, 3.3%and 0.9% respectively. So we can say these four processes played important action in the process of salt-resistance for Tamarix androssowii. Transmembrane transport and ion homeostasis, Protein syntheise and Metabolism were less.
From above, we can conclude that salt-resistance of Tamarix androssowii was an complex process involved in signaling conduction, Transcription regulation, Transmembrane transport and ion homeostasis, reactive oxygen scavenging, osmolyte biosynthesis. The cDNA library construction and the acquiring of many ESTs relation to stess-resistance will make a foundation for thoroughly studying the mechanism of stress resistance of Tamarix androssowii.
cDNA初始文库滴度为1.2×10~6pfu／mL，蓝白斑检验结果表明，文库重组率为96％，PCR检测表明插入片段的平均长度1 200bp左右。
随机挑选文库中的克隆进行测序。将获得序列去除5’端载体序列、文库接头序列和poly(A)序列。共获得2 328条读序较好的序列。
以获得的2 328条序列建立本地核酸数据库。本地BLAST获得642个与其它序列无同源区的序列，其余的1 686条序列依照同源性聚为281个序列集，对每个序列集通过序列联配获得359个contig。用于网上进行BLAST分析的独立序列共有1 001个。文库的冗余度为57％。
1 001个序列的BLASTX分析表明，有428个与已知功能的基因序列相似，234个与未知功能的基因相似，339个未能发现相似序列。将428个代表独立已知功能基因的EST，按功能分为11个类，其中防御基因、新陈代谢和蛋白合成及分拣的表达丰度最高，分别为25.26％、20.65％和15.37％。对与耐盐有关基因EST分析结果表明，信号元件、转录调控、应激蛋白和活性氧清除剂所包含的表达的基因比较多，这些基因的表达丰度也较高，分别为4.1％、1.9％、3.3％和0.9％，这说明这四种过程在柽柳抗盐胁迫时起到了重要作用。此外，跨膜运输及质子平衡、蛋白质代谢和新陈代谢类所包含的表达的基因种类相对较少，但是其表达丰度也较高分别为0.9％、0.9％和2.3％，可能在盐胁迫中也起到了重要的作用。
以上研究说明，柽柳的抗盐过程是一个复杂的多基因协同作用体系，涉及到防御反应、基因调控、信号传导、离子转运、活性氧清除、渗透调节、应激反应等多方面的综合作用，从而为系统阐明柽柳抗盐分子机理研究奠定坚实基础。
Tamarix spp. is a good material to study the salt-resistance molecular mechanism because it has high ability to resist drought and salt stress. A cDNA library was constructed from the tissue of Tamarix androssowii treated with NaHCOs Expressed sequence tags (EST) analysis was carried out to study the molecular mechanism of salt-resistance in Tamarix androssowii.
The library primary titer was 1.14
×10 pfu/mL, and the rate of recombination was 96%, the titer of amplified library was 4.0
×109 pfu/mL. The average length of inserted fragments in the library was 1.2kb, which was testified by both PCR and sequencing analysis. From this library, 2 328 high quality ESTs were obtained.
Homology analysis by local BLAST and multiple alignment showed that these clones represented 1 001 unique transcripts. Results of comparing with the genes in unigene database of GenBank based on BLASTX algorithm indicated that 428 ESTs were homologous to the genes identified previously by BLAST from the Internet, while other 234 ESTs matched unknown protein coding regions and 339 ESTs were completely novel sequences. Eleven functional categories were carried out from all the tentative unique genes that were identified. Three group transcripts were significantly expressed: Defence, Metabolism and Protein synthesis and destination that respesented 25.26%, 20.65% and 15.37% respectively. The result of genes related to salt stress-tolerance indicated that Signaling components, Transcriptional regulators, Stress protein and Reactive oxygen scavengers contained more genes than the others, 4.1%, 1.9%, 3.3%and 0.9% respectively. So we can say these four processes played important action in the process of salt-resistance for Tamarix androssowii. Transmembrane transport and ion homeostasis, Protein syntheise and Metabolism were less.
From above, we can conclude that salt-resistance of Tamarix androssowii was an complex process involved in signaling conduction, Transcription regulation, Transmembrane transport and ion homeostasis, reactive oxygen scavenging, osmolyte biosynthesis. The cDNA library construction and the acquiring of many ESTs relation to stess-resistance will make a foundation for thoroughly studying the mechanism of stress resistance of Tamarix androssowii.
引文
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