Screening of garlic reference genes and analysis of AsACO gene response to salt stress and plant growth-promoting rizhobacteria

【Objective】 To screen stable reference genes in garlic under salt stress, and to analyze their expression characteristics of 1-aminocyclopropane-1-carboxylate oxidase gene(AsACO) when responding to salt stress and plant growth-promoting rizhobacteria, so as to provide theoretical reference for the studies on growth-promoting mechanism and response mechanism under salt stress.【Method】 Ledu purple-skin garlic was used as the test material. Real-time fluorescence quantitative PCR(qRT-PCR) was used to detect to the expression stability of 5 reference genes, ACT, UBC, HIS3, 18S rRNA and TUB under salt stress. GeNorm, NormFinder and BestKeeper were used to screen the most stable reference gene, and effects of Pseudomonas putida UW4 on AsACO expression under salt stress was analyzed.【Result】 The results showed that primers of the 5 selected candidate reference genes were highly specific without primer dimers. GeNorm analysis showed that the stability of the selected candidate reference genes was ranked as 18S rRNA=TUB>HIS3>UBC>ACT. NormFinder and BestKeeper softwares calculated stability ranking of reference genes as 18S rRNA>HIS3> TUB>UBC>ACT and UBC>HIS3>18S rRNA>TUB>ACT, respectively. Comprehensive analysis of geometric mean showed that 18S rRNA was the most stable reference gene and when it was taken as the reference gene, expression of AsACO showed obvious tissue specificity in garlic, and its expression in leaves was greatly higher than that in roots. During treatment process, salt stress greatly induced up-regulated AsACO expression in different stages and tissues of garlic and AsACO expression in roots and leaves reached the highest at 2 d and 12 h, increased by 124.43% and 238.34% compared with normal cultivation(CK), respectively(P<0.05, the same below). Compared with the salt stress treatment, AsACO expressionin roots and leaves in different stages was significantly reduced after salt stress+P. putida UW4 treatment was applied on salt stress, and the maximum decreases of 112.08% and 343.34% were witnessed at 2 d and 12 h.【Conclusion】 18S rRNA is the most stable reference gene of garlic under salt stress. Salt stress can induce up-regulated AsACO expression, indirectly increase ACO and ethylene of garlic in different stages and tissues, thus accelerating cell aging, even death;but P. putida UW4 can inhibit expression AsACO under salt stress to slow the aging and increase stress tolerance of garlic.