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以番茄(Lycopersicum esculentum)LA1996(Aft基因型,紫色花青素积累品系)c DNA为模板,对Lemi R828前体基因和预测到的靶基因Le TAS4(Trans-Acting Si RNA Gene 4)进行克隆,采用RT-PCR技术检测其在番茄果实发育中的差异表达,并用RLM 5′RACE技术验证Lemi R828对预测靶基因Le TAS4m RNA的剪切作用及靶作用位点。结果表明,Lemi R828的前体序列含有完整的茎环结构;在果实发育成熟的过程中,Lemi R828及Le TAS4在番茄中的表达量存在差异。在绿色果阶段的表达量较低且Lemi R828对Le TAS4的负调控关系不明显;而在成熟果阶段,Lemi R828及Le TAS4的表达量均升高,且存在明显的负调控关系。Lemi R828能够靶作用于Le TAS4的转录本上调控其降解,其剪切位点为Lemi R828序列5′端的第10位碱基。因此,在LA1996番茄果实发育过程中,Lemi R828和Le TAS4参与果实发育的调控,并且Lemi R828负调控其靶基因Le TAS4的表达。
The Lemi R828 precursor gene and the predicted target gene Le TAS4 (Trans-Acting Si RNA Gene 4) were cloned using c DNA of Lycopersicum esculentum LA1996 (Aft genotype, purple anthocyanin accumulation strain) as a template, The differential expression of Le TAS4m RNA in Leymus chinensis was detected by RT-PCR and RLM 5’RACE was used to confirm the cleavage and target sites of Le TAS4m RNA. The results showed that the precursor sequence of Lemi R828 contained a complete stem-loop structure. There were differences in the expression of Lemi R828 and Le TAS4 in tomato during fruit maturation. The expression level of Lemi R828 and Le TAS4 was low in the green stage and Lemi R828 had no negative relationship with Le TAS4. Lemi R828 is able to target its transcription on Le TAS4 to regulate its degradation at its cleavage site at the 5 ’end of the Lemi R828 sequence. Therefore, during LA1996 tomato fruit development, Lemi R828 and Le TAS4 are involved in the regulation of fruit development, and Lemi R828 negatively regulates the expression of its target gene Le TAS4.