根癌农杆菌对感染植原体的泡桐组培苗症状的影响

Effects of Agrobacterium tumefaciens on the Symptoms of Paulownia sp. Plantlet in Vitro Cultured

采用含有激素合成相关基因的根癌农杆菌 ,伤口接种已感染植原体的泡桐丛枝组培苗和健康组培苗 ,结果发现对丛枝苗的致瘤能力明显低于健康对照苗 ,且被接种病苗的丛枝症状缓解。从健苗获得的 T- DNA转化泡桐瘤组织细胞能在无激素培养基上稳定生长和连续继代培养 2年以上 ,说明瘤组织细胞自身已获得了细胞分裂素和生长素合成能力。根据已报道的根癌农杆菌株系p Til5955T- DNA的异戊烯基转移酶基因 ( ipt)的保守序列 ,设计了一对引物 ( CYT和 CYT′) ,用多聚酶链式反应 ( PCR)扩增了我国杨树致瘤农杆菌 ipt基因部分序列 ( 4 2 7bp片段 ) ,也从遗传转化的两个泡桐无性系瘤组织 At- ZH和 At- T35扩增出此特异片段 ,从而进一步肯定了 T- DNA已被整合到泡桐的染色体上 ,表明泡桐易于通过 Ti质粒载体途径进行基因转移操作 ,但用此引物未能从泡桐、甘薯健株和感染植原体的组培病苗扩增出相应的 4 2 7bp特异片段。当用此遗传转化瘤组织嫁接病苗时 ,可减轻丛枝症状的严重度 ,延长病苗的存活时间和诱导病株生根 。

By using Agrobacterium tumefaciens isolated from poplar crown gall disease with the hormone-producing genes in the T-DNA to inoculate healthy and infected Paulownia plantlets with phytoplasma, it is showed that tumorigensis of diseased plantlets dropped apparently and the symptoms of witches' broom suppressed to some extent. The T-DNA was transformed into Paulownia resulting in tumor formation independent of exogenous hormone addition and keeping subculture of tumor tissues for more than 2 years, thus confirming that the tumor tissues gained the ability to synthesize cytokinin and auxin by itself. Based on the conserved sequence of isopentenyl adenosine transferase gene (ipt) of Agrobacterium tumefaciens Opine pTil 5955 strain, a pair of DNA primers (CYT and CYT′) were designed and synthesed. A 427 bp polymerase chain reaction (PCR) product, the same size as the ipt gene fragment of pTil 5955 was amplified in association with Agrobacterium tumefaciens strain causing poplar stem gall disease rather than recombinant plasmid without this gene. The specific fragment of 427 bp was also amplified using the DNA extracts from transformed tumor tissues of two Paulownia clones (AT-ZH and AT-T35) with the Agrobacterium tumefaciens as templates, therefore further verifying the insert of T-DNA to the chromosome DNA of Paulownia and Paulownia can be surely transformed through Agrobacterium tumefaciens Ti plasmid vector. The specific 427 bp product was not amplified by using total DNA extracts of both in vitro cultured healthy and infected paulownia and sweetpotato infected with phytoplasmas as templates. When the transformed tumor tissues were grafted onto the infected in vitro cultured Paulownia plantlets with phytoplasmas, the symptoms of witches' broom reduced apparently, including reduced severity, prolonged survival time and increased rooting ability of the plantlet, which, on other aspect, suggests the involvement of the hormone metabolism in paulownia-phytoplasma interaction.