大蒜内参基因筛选及AsACO基因对盐胁迫和促生菌的响应分析

【目的】筛选大蒜在盐胁迫下的稳定内参基因，并分析1-氨基环丙烷-1-羧酸氧化酶基因（AsACO）对盐胁迫和促生菌的响应表达特性，为深入探究促生菌的促生机制及大蒜对盐胁迫的响应机制研究提供理论参考。【方法】以乐都紫皮大蒜为试材，采用实时荧光定量PCR（qRT-PCR）检测5个内参基因ACT、UBC、HIS3、18S rRNA、TUB在盐胁迫下的表达稳定性，结合GeNorm、NormFinder和BestKeeper筛选出最稳定的内参基因，并分析恶臭假单胞菌UW4对盐胁迫下AsACO基因表达的影响。【结果】 5个候选内参基因引物的特异性强、无引物二聚体。GeNorm分析得出候选内参基因稳定性排名为18S rRNA=TUB>HIS3>UBC>ACT，NormFinder分析得出内参基因稳定性排名为18S rRNA>HIS3>TUB>UBC>ACT，BestKeeper分析得出内参基因稳定性排名为UBC>HIS3>18S rRNA>TUB>ACT，最终以几何平均数综合分析得出18S rRNA为最稳定的内参基因。以18S rRNA为内参基因，检测出AsACO基因表达具有明显的组织特异性，在叶片中的相对表达量明显高于根。整个处理过程中，盐胁迫明显诱导AsACO基因在不同处理时间及不同组织中表达上调，根和叶片中AsACO基因的相对表达量分别在2 d和12 h时达最高，较正常培养（CK）分别显著升高124.43%和238.34%（P<0.05，下同），与盐胁迫处理相比，盐胁迫+浇施恶臭假单胞菌UW4处理显著降低AsACO基因在不同处理时间根和叶片中的相对表达量，其中在2 d和12 h时降幅较大，分别降低了112.08%和343.34%。【结论】18S rRNA是盐胁迫下大蒜中最稳定的内参基因。盐胁迫可诱导大蒜根和叶片中AsACO基因的上调表达，从而间接促进ACO和乙烯水平升高，加速由乙烯调控的细胞衰老，甚至死亡，但盐胁迫下恶臭假单胞UW4具有抑制AsACO基因表达的作用，以减少ACO和乙烯的合成，从而延缓大蒜细胞衰老，提高逆境耐受性。

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.