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为了研究盐生植物耐盐基因表达调控,本实验以海水浇灌的海马齿植株为供试材料,构建了盐胁迫下的全长cDNA文库。构建方法如下:采用改良的CTAB法提取总RNA,SMART法反转录合成cDNA,LD-PCR方法合成双链cDNA。LD-PCR产物经蛋白酶K消化和SfiⅠ酶切后,经CHROMA SPIN+TE-1000分离柱子除去小片段DNA后,回收0.5kb以上的片段,按照适当的比例连接λTripIEX2载体。连接产物利用MaxPlaxTMLambda Packaging Extracts进行体外包装,得到海马齿初级cDNA文库。初始文库的独立克隆数为2.4×106pfu,初级文库滴度大于4.80×106pfu/mL,重组率为93.75%,插入片段为0.5～5kb,扩增文库的滴度为1.21×109pfu/mL,所得文库质量较高。本研究表明该cDNA文库适合于盐生植物海马齿相关基因的克隆和分析。
Abstract：For study the gene regulation of halophytes in response to salt stress,the first full-length cDNA library of the tested material of S. portulacastrum was constructed in this research. The construction method as follows,the total RNA was isolated from the salt-stressed plant by using the improved CTAB,and the mRNA was converted into the first single-strand complementary cDNA by SMART method. The double-strand cDNAs (ds-cDNAs) were amplified by long distance polymerase chain reaction (LD-PCR),then were digested by proteinase K,and nuclease SfiI,After extration through CHROMA SPIN+TE-1000 separated columns. The longer than 0.5 kb ds-cD-NAs were collected and ligated into λTripIEX2. The recombinant bacteriophages were packaged by using Max-PlaxTM Lambda Packaging Extracts. The titer of the first unamplified library is larger than 4.80×106 pfu/mL,the rate of recombinant clones is 93.75%,the inserted cDNA fragments are longer than 0.5 kb. The numbers of the dependent clones of the first library is 2.4×106 pfu. The titer of amplified library was 1.21×109 pfu/mL,and we have got the high quality library. These data indicated that the cDNA library was suitable for analysis and cloning of the related genes of halophytes S. portulacastrum.
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基本信息:
中图分类号:Q943.2
引用信息:
[1]郑善清,段瑞军,郭建春.盐生植物海马齿盐胁迫全长cDNA文库的构建与分析[J].基因组学与应用生物学,2010,29(01):155-159.
基金信息:
国家重点基础研究发展计划(2007CB108903);; 牧草现代农业产业技术体系建设专项资金资助;; 中央级公益性科研院所基本科研项目(ITBBYB072)共同资助
2010-02-28
2010-02-28