利用mRNA差别显示技术分离红树植物许树耐盐相关cDNA

选用适应甜土种植10余年的红树植物许树分株,在室内分别以盐水与自来水进行沙培.分别提取叶片的总RNA.通过oligo（dT）12CA反转录合成cDNA第1链,以此为模板,用4组10核苷酸随机引物与锚引物o1igo（dT）12CA组成的引物对,进行PCR扩增,选择差异表达片段,发现3个稳定的差异cDNA只存在盐水培养下许树叶片中,而自来水培养条件下却没有.这3个差异cDNA片段分别命名为colb1,colb2,colb3.对3个cDNA采用DIG High Prime DNA Labeling and Detection Starter KitI进行RNA杂交检测,只有colb1在盐培许树叶中呈现稳定的显色信号,在自来水浇灌的许树叶中却无显色信号,colb2和colb3均无显色.可见colbl（463 bp）是耐盐相关cDNA.将片段colb1克隆,并进行序列分析,其AT碱基对占全序列的61.74%.将序列提交NCBI进行BLASTN和BLASTX查询,未发现相关同源cDNA.耐盐相关cDNA的获得将为分离全长耐盐基因和其耐盐机制的研究奠定了基础.

Isolation of cDNA Related to Salt-tolerance in Mangrove Clerodendrum inerme by mRNA Differential Display

The 1st strand of cDNA was synthesized from total RNA isolated from the leaves of Clerodendrum inerme respectively under fresh water and saline water condition, which was used as template for polymerase chain reaction with anchored primer oligo(dT)12CA and 10-oligonucleotide arbitrary primer. Three stable differential cDNA fragments, colb1,colb2 and colb3, were found present only in the leaves of C. inerme culturedunder the saline condition. These fragments were detected by RNA hybridization, but only fragment colb1 was identified to be a salt-tolerant cDNA, which was then cloned and sequenced. After searching BLASTN and BLASTX on the NCBI, no reported similar sequence was found.