‘南通小方柿’DkADH1基因遗传转化及功能分析

Genetic transformation of ‘Nantongxiaofangshi' DkADH1 gene and function analysis

[目的]研究‘南通小方柿’(Diospyros kaki Linn.‘Nantongxiaofangshi’)乙醇脱氢酶基因DkADH1的功能,阐明其在柿果脱涩过程中的作用。[方法]构建了DkADH1基因植物双元表达载体,通过农杆菌介导法将该基因转入番茄中。以转基因和非转基因番茄株系的不同组织为材料,钨酸钠-钼酸钠比色法测定可溶性单宁含量,并用实时荧光定量PCR(qRT-PCR)检测原花青素(PA)合成途径相关基因的表达情况。[结果]经PCR和RT-PCR检测,获得了3个转DkADH1基因番茄株系。过量表达DkADH1基因的转基因番茄植株的叶片、花、果实中可溶性单宁含量均显著低于非转基因番茄植株。qRTPCR显示:转基因植株不同组织中PA合成途径相关基因F3'5'H、LAR、MYB4和PAL的表达量与非转基因植株相比均显著下调。[结论]DkADH1能降低植物可溶性单宁的含量并抑制其生物合成途径相关基因的表达,与柿果脱涩密切相关。

[Objectives]The functions of Diospyros kaki Linn.‘Nantongxiaofangshi＇alcohol dehydrogenas gene DkADH1 were further studied to clarify its effect on persimmon deastringenting.[Methods]The DkADH1 gene plant expression vector was constructed and transferred to tomato by agrobacterium mediated transformation.Folin-Ciocalteu assay was employed to measure the tannin content in transgenic and non-transgenic tomato plants.Expression levels of several genes related to the proanthocyanidins pathway were detected using qRT-PCR technology.[Results]PCR and RT-PCR analysis showed that the DkADH1 gene was successfully transferred into tomato,and three transgenic tomato lines were obtained.The Folin-Ciocalteu assay showed that the tannin content in leaves,flowers and fruits of transgenic tomatoes was significantly lower than that of non-transgenic tomatoes.Significant decrease in relative expression of proanthocyanidins pathway related genes F3＇5＇ H,LAR,MYB4 and PAL was revealed by qRT-PCR.[Conclusions]DkADH1 gene plays a crucial role in persimmon deastringenting process by decreasing tannin content in different tissues of plants and inhibiting the biosynthesis of proanthocyanidins.