摘要
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。
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.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2010年第1期155-159,共5页
Genomics and Applied Biology
基金
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
关键词
海马齿
盐胁迫
SMART方法
全长CDNA文库
Sesuvium portulacastrum L.
Salt-stress
SMART technique
Full-length cDNA library
