Construction And Transformation Of Expression Vectors Containing Bacterial ACC Deaminase Genes.and Analyses Of Pan-genomes And ACC Deaminase Genes Of Herbaspirillum Bacteria

1-aminocyclopropane-l-carboxylic acid (ACC) is the immediate precursor of ethylene in plants. ACC deaminase can catalyze the degradation of ACC into a-ketobutyrate and ammonia. Bacteira contaning ACC deaminase can break down the ACC produced by plants and thus reduce stress ethylene levels, leading to alleviation of the plant growth inhibition under abiotic stress and symptoms caused by pathogen. This study cloned seven bacterial ACC deaminase structure genes(acdS) and constructed their expression vectors, and analyzed the pan-genomes and ACC deaminase genes of Herbaspirillum bacteria.Seven acdS from bacterial strains of Burkholderia, Pseudomonas, Herbaspirillum and Mesorhizobium with different ACC deaminase activities were cloned and constructed into the expression vectors pBBRacdS based on the vector pBBR1MCS5, and then introduced into Agrobacterium tumefaciens strain C58C1. The Agrobacterium engineering strains containing acdS from Burkholderia and Herbaspirillum showed ACC deaminase activities corresponding to the activities of the source strains. In contrast, Agrobacterium engineering strains containing acdS from Mesorhizobium and Pseudomonas did not show ACC deaminase activity; A. tumefaciens C58C1may not have the same mechanisms as the source strains to regulate ACC deaminase.This study also obtained the draft genome sequences of five pathogenic Herbaspirillum strains Os34, Os38, Os44, Os45and Os49by high-throughput sequencing, and found some differences to the genome of the plant growth-promoting H. seropedicae strain SmR1. GGDC (Genome-to-Genome Distance Calculator) results showed that the five pathogenic Herbaspirillum Os strains do not belong to H. seropedicae.PGAP (Pan-Genomes Analysis Pipeline) analyses of the genomes of the five Herbaspirillum Os strains, SmR1, GW103, CF444, YR522and P6-12strains revealed that Herbaspirillum has an open pan-gnome and a stable core genome.The ACC deaminase gene cluster is a core gene cluster of Herbaspirillum bacteria. Phylogenetic analyses of the ACC deaminase amino acid sequences showed that the phylogenetic positions of the five Herbaspirillum Os strains, SmR1, GW103and YR522strains did not match with their taxonomic statuses, and distanced from other bacteria. Sigi-HMM and IVOM analyses did not find the ACC deaminase genes in any predicted genomic islands and did not support horizontal transfer of the ACC deaminase genes. These ACC deaminase genes may be derived from mutation of ACC deaminase homologous genes.The obtained Agrobacterium engineering strains with constitutively expressed ACC deaminase may weaken plant defense response and delay explant senescence through reducing ethylene levels and thus increase the efficiency of Agrobacteriuw-mediated transformation. The expression vectors pBBRacdS can be introduced into other plant growth-promoting bacteria to enhance plant tolerance to environment stresses, or rhizobia to enhance nodulation.The ACC dearninases of the five Herbaspirillum Os strains showed unusual features, and need further studies on their structures.functions and regulations. These five pathogenic Herbaspirillum strains may belong to a novel species. Further comparative genomics will find differences in the genomes among the pathogenic and non-pathogenic Herbaspirillum strains, and reveal the genetic mechanisms of their pathogenicity and non-pathogenicity at the genome level.