Behboodian Bita, Mohd Ali Zainon, Ismail Ismanizan, Zainal Zamri
School of Biosciences and Biotechnology, Faculty Science and Technology, UKM, Selangor, 43600 Bangi, Malaysia.
ScientificWorldJournal. 2012;2012:439870. doi: 10.1100/2012/439870. Epub 2012 Jul 31.
The plant hormone, ethylene, is an important regulator which involved in regulating fruit ripening and flower senescence. In this study, RNA interference (RNAi) technology was employed to silence the genes involved in ethylene biosynthetic pathway. This was achieved by blocking the expression of specific gene encoding the ACC oxidase. Initially, cDNA corresponding to ACO1 of lowland tomato cultivar (MT1), which has high identity with ACO1 of Solanum lycopersicum in GenBank, was cloned through RT-PCR. Using a partial coding region of ACO1, one hpRNAi transformation vector was constructed and expressed ectopically under the 35S promoter. Results showed that transgenic lines harboring the hpRNA-ACO1 construct had lower ethylene production and a longer shelf life of 32 days as compared to 10 days for wild-type fruits. Changes in cell wall degrading enzyme activities were also investigated in cases where the transgenic fruits exhibited reduced rates of firmness loss, which can be associated with a decrease in pectin methylesterase (PME) and polygalacturonase (PG) activities. However, no significant change was detected in both transgenic and wild-type fruits in terms of β-galactosidase (β-Gal) activity and levels of total soluble solid, titratable acid and ascorbic acid.
植物激素乙烯是一种重要的调节剂,参与调控果实成熟和花朵衰老。在本研究中,采用RNA干扰(RNAi)技术使参与乙烯生物合成途径的基因沉默。这是通过阻断编码ACC氧化酶的特定基因的表达来实现的。最初,通过RT-PCR克隆了与低地番茄品种(MT1)的ACO1相对应的cDNA,其在GenBank中与番茄的ACO1具有高度同源性。利用ACO1的部分编码区,构建了一个hpRNAi转化载体,并在35S启动子的调控下异位表达。结果表明,与野生型果实10天的货架期相比,携带hpRNA-ACO1构建体的转基因株系乙烯产量较低,货架期延长了32天。在转基因果实硬度损失率降低的情况下,还研究了细胞壁降解酶活性的变化,这可能与果胶甲酯酶(PME)和多聚半乳糖醛酸酶(PG)活性的降低有关。然而,转基因果实和野生型果实在β-半乳糖苷酶(β-Gal)活性以及总可溶性固形物、可滴定酸和抗坏血酸水平方面均未检测到显著变化。