竹丛枝植原体16SrDNA片段克隆与序列分析

Cloning and sequence analysis of 16S ribosomal DNA of phytoplasma associated with witches' broom of bamboo

利用植原体16SrRNA基因序列设计合成的引物,对表现丛枝的竹子植株总DNA进行直接PCR及巢式PCR扩增,得到长1.2kb的目的片段。将此片段与pGEMTEasy载体连接并转化到大肠杆菌DH5α感受态细胞中。通过酶切、PCR鉴定,对筛选得到的重组阳性克隆进行核酸序列测定及同源性比较分析,结果表明其与植原体16SrⅠ组中的西方翠菊黄化植原体(SAY)同源率为99%。依据16SrDNA序列建立了竹子丛枝病植原体株系的系统进化树。对云南竹子丛枝病植原体株系分类鉴定与已报道的结果相似。

With specific primers designed based on phytoplasmal 16S ribosomal RNA gene sequences, a 1.2kb DNA fragment was amplified by direct PCR and nested PCR from total DNA of bamboo showing witches broom symptom. The amplified fragment was ligated into pGEM-T easy vector and then transformed into DH5 strain of E. coli. The clones were verified by restriction enzyme digestion and PCR. Sequence and homology analyses showed that this phytoplasma strain shares high homology (99.0%) with the phytoplasma of Aster Yellows that belongs to 16SrI group. A phylogenetic tree of bamboo witches broom phytoplasma strain was established based on 16SrDNA sequences. The taxonomic status of bamboo witches broom phytoplasma collected in Yunnan province is consistent with that reported before.