毛萼田菁茎瘤菌被种子浸提物诱导表达基因的筛选及其功能

Identification and functional characterization of genes induced by seed exudates in Azorhizobium caulinodans ORS571

摘要:

摘要：【目的】选择毛萼田菁茎瘤菌ORS571为研究对象，筛选毛萼田菁茎瘤菌在菌植互作早期被宿主植物种子浸提物诱导表达的基因。【方法】采用新颖的基于抗性的活体表达筛选技术，以种子浸提物为诱导物，筛选获得能够稳定被种子浸提物诱导的突变株。通过中间片断融合报告基因的方法研究信号分子对相关基因的诱导情况。【结果】随机引物PCR产物测序及网上BLAST结果表明，被诱导表达的基因为LysE家族的成员。而且，对另外3个属根瘤菌中lysE基因的研究发现，它们都可以被相应的宿主植物种子浸提物诱导表达。此外，这四株根瘤菌的lysE突变株都表现对刀豆氨酸极为敏感。本文首次报道了lysE家族在根瘤菌中的生理功能。【结论】我们推测：LysE家族蛋白可能广泛存在于根瘤菌中，并在根瘤菌与宿主植物的早期相互作用中发挥重要作用。

Abstract:

Abstract: [Objective] To identify genes induced by plant seed exudates in Azorhizobium caulinodans ORS571. [Methods] Using promoterless kanamycin resistance gene (Kmr) on transposon as reporter gene and seed exudates as inducers, we screened genes of interest from transposon insertion mutants libraries. We streaked mutants on TY solid medium with Km, and another with Km and seed exudates correspondingly. If Kmr is inserted into a gene that can be induced by plant signals, Kmr will possibly express at the same time. Thus, mutants were selected that can grow on medium with Km and exudates, rather than on medium with Km. [Results] We identified a lysE family gene named asiE in strain Azc0 that can be induced by seed exudates and further analysis indicated that the inducing substance is canavanine(CAN). lacZ transcriptional fusion of asiE confirmed that its expression increased by ten-fold or so under the induction of CAN. Besides, lysE gene in four different species of Rhizobia can be induced by CAN. lysE mutants are all sensitive to CAN treatment whereas wild type are resistant. [Conclusion] The existence of LysE can make rhizobia better survived in the rhizosphere and may play an important role in early stage of interaction between rhizobia and host plant.