Plant Growth Promotion And Salt Stress Relief Of ACC Deaminase-containing Endophytic Bacteria From Halophyte Suaeda Salsa And Cloning Of The ACC Deaminase Gene

Suaeda salsa, a type of salt-resistant halophyte that grows in saline-alkali soil and on sandy beaches, is widely distributed in China, and at present, it is the first-choice species for improving saline soil. Numerous studies have demonstrated that the salt tolerance of S. salsa mainly derives from the expression or regulation of key genes related to salt resistance. However, to date, the influence of endophytes on the growth of S. salsa and their physiological functions have been neglected. Symbiosis is extremely common, and the majority of endophytes have co-evolved with their host plant species, allowing the plants to grow and reproduce in certain circumstances. Studies have shown that plants infected with endophytic fungi have a high capacity for stress tolerance, and some 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing endophytic bacteria are able to promote the growth of host plants and confer resistance to heavy metal stress. The enzyme ACC deaminase cleaves ACC, the immediate precursor of ethylene in plants, to form ammonia andα-ketobutyrate. Thus, bacteria with this enzyme can reduce the deleterious effect of ethylene, ameliorating plant stress and promoting plant growth.For the further research of the ecology action of endophytic bacteria of S. salsa in the high level of saline circumstance, ACC deaminase-containing endophytic bacteria were isolated from root, stalk and leaf of S. salsa and were identified based on morphological, physiological-biochemical properties, API and 16S rRNA sequence analysis. Isolates were evaluated for ACC deaminase production, antifungal activity, protease activity, siderophores production, phytohormone production (including indole-3-acetic acid, gibberellic acid and abscisic acid production), atmospheric nitrogen fixation and phosphate solubilization. Seed germination experiments and pot experiments were performed to evaluate the plant growth promotion and salt stress relief potential of the strain. Finally ACC deaminase gene was cloned and was analyzed by the methods of bioinformatics. The specific results are presented as follows: 1. Four ACC deaminase-containing endophytic bacteria strains named as LP11, SS12, TW1 and TW2 were isolated and identified as Pseudomonas oryzihabitans, Pseudomonas sp., Pantoea agglomerans and Pseudomonas putida respectively based on morphological, physiological-biochemical properties, API and 16S rRNA sequence analysis. All the strains possessed the phosphate-solubilizing ability and could produce siderophores and phytohormones more or less. None of them could fix atmospheric nitrogen or produce protease. Only strain SS12 showed antagonism against two phytopathogenic fungi viz Fusarium oxysporum f. sp. conglutinans and F. oxysporum f. sp. cucumerinum.2. Seed germination experiments and pot experiments were performed to evaluate the effects of plant growth promotion and salt stress relief of strain LP11 on different plants in the presence of different concentrations of salt. The results showed that strain LP11 was able to promote germination and increase the germinal length of S. salsa, cucumber and rape. Treatment with strain LP11 increased the growth of cucumber and maize plantlets in the presence of different concentrations of salt. Significant increases in the shoot height, root length, leaf length and fresh mass compared to the control were observed. Furthermore, strain LP11 was able to increase the chlorophyll, soluble sugar and proline contents of cucumber and maize plantlets to enhance their stress resistance.3. For the further research of the transgenic plants, we have cloned ACC deaminase gene of strain LP11. The partial conserved acdS gene sequence of strain LP11 was amplified using CODEHOP-designed primers, and the 5'and 3'partial non-conserved sequences of the acdS gene were amplified using the genome walking method. Then, the coding sequence (cds) of a 996-bp fragment of the LP11 acdS gene was obtained by PCR using a pair of specific primers designed based on the complete sequence of the LP11 acdS gene. The Blast results and phylogenetic analysis showed that the acdS gene of strain LP11 and the encoded AcdS protein shared 90% identity and 100% query coverage with those of Pseudomonas fluorescens SBW25. Finally, the tertiary structure of the AcdS protein was predicted and displayed using the SWISS-MODEL software. Key words: Suaeda salsa; ACC deaminase; Endophytic bacteria; Plant growth...