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促分裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK/MPK)级联是植物响应外界环境变化的重要信号传导途径。为了验证番茄促分裂原活化蛋白激酶3(Solanum lycopersicum mitogenactivated protein kinases 3,Sl MPK3)在低温胁迫中的功能,本研究将醋栗番茄(Solanum pimpinellifolium)Sl MPK3基因在栽培番茄(S.lycopersicum)M82中过表达,获得了过表达Sl MPK3的转基因番茄后代。结果表明,低温胁迫下转基因番茄幼苗Sl MPK3基因被迅速诱导表达。低温处理8 h处,Sl MPK3的表达量达到最大值,转基因植株(OE4,OE6和OE7)中Sl MPK3的表达量显著高于野生型(P<0.05)。苗期耐冷性鉴定结果表明,转基因植株耐冷指数(OE4(0.81),OE6(0.78),OE7(0.77))显著高于对照((0.37),P<0.05)。低温胁迫下表型观察发现,4℃低温胁迫24 h,野生型番茄叶片边缘失水萎蔫,叶面出现明显黄色枯斑;转基因番茄植株叶片表现正常。低温胁迫120 h后,野生型植株的相对电解质渗透率为74.66%,而转基因植株的电解质渗透率平均为59.79%,比对照低14.87%;转基因植株叶片中丙二醛(malondialdehyde,MDA)含量为6.76μmol/g FW,比对照(9.77μmol/g FW)低30.81%;野生型植株叶片中的H2O2含量显著高于转基因植株(P<0.05),比转基因植株高22.02%。低温胁迫处理引起了转基因植株和野生型植株的抗氧化酶(antioxidant enzymes,AOEs)活性显著增加(P<0.05),低温处理120 h后,转基因植株中超氧化物歧化酶(super-oxide dismutase,SOD)、过氧化物酶(peroxidase,POD)和过氧化氢酶(catalase,CAT)活性分别比对照高29.40%、24.24%和22.83%。转基因和野生型植株中可溶性蛋白(soluble protein)和可溶性糖(soluble sugar)含量均随低温处理时间的推移逐步增加,4℃处理120 h后转基因植株可溶性蛋白含量为40.02 mg/g FW(OE-4),42.21 mg/g FW(OE-6)和40.33 mg/g FW(OE-7),显著高于对照(36.86 mg/g FW)(P<0.05);转基因植株可溶性糖含量平均为51.87 mmol/g FW,比对照高21.82%。本研究证明,Sl MPK3过表达提高了番茄植株的低温耐受能力,为进一步研究番茄Sl MPK3基因的功能提供依据,同时为耐低温番茄育种提供了新种质。
Mitogen-activated protein kinase (MAPK / MPK) cascade is an important signal transduction pathways in response to environmental changes in plants. In order to verify the function of Sl MPK3 in low temperature stress, we studied the effect of Sl MPK3 gene from Solanum pimpinellifolium on the growth of S. lycopersicum M82 Overexpression of Sl MPK3 overexpression transgenic tomato progeny. The results showed that Sl MPK3 gene of transgenic tomato seedlings was rapidly induced under low temperature stress. The expression level of Sl MPK3 reached the maximum at 8 h after cold treatment. The expression level of Sl MPK3 in transgenic plants (OE4, OE6 and OE7) was significantly higher than that in wild type (P <0.05). The results of cold tolerance identification at seedling stage showed that the cold tolerance index (OE4 (0.81), OE6 (0.78) and OE7 (0.77)) of transgenic plants were significantly higher than that of the control (0.37, P <0.05). The results of phenotypic observation under low temperature stress showed that the leaves of wild-type tomato withered wilt at the edge of the leaves at 24 ℃ for 24 h, and the yellowish blotches appeared on the leaves. The transgenic tomato plants showed normal leaves. The relative electrolyte permeability of wild type plants was 74.66% after 120 h of low temperature stress, while the electrolyte permeability of transgenic plants was 59.79% on average, 14.87% lower than that of the control plants. The content of malondialdehyde (MDA) in transgenic plants was 6.76μmol / g FW, which was 30.81% lower than that of the control (9.77μmol / g FW). H2O2 content in wild-type plants was significantly higher than that in transgenic plants (22.02%). After cold stress for 120 h, the activities of antioxidant enzymes (AOEs) in transgenic plants and wild type plants were significantly increased (P <0.05). After 120 h of low temperature treatment, the activities of superoxide dismutase (SOD) ), Peroxidase (POD) and catalase (CAT) activity were higher than the control by 29.40%, 24.24% and 22.83% respectively. The content of soluble protein and soluble sugar in transgenic and wild type plants increased gradually with the time of low temperature treatment. The content of soluble protein in transgenic plants was 40.02 mg / g FW (OE- 4), 42.21 mg / g FW (OE-6) and 40.33 mg / g FW (OE-7) significantly higher than the control (36.86 mg / g FW) mmol / g FW 21.82% higher than the control. This study demonstrated that Sl MPK3 overexpression increased the tolerance of tomato plants to low temperature and provided a basis for further study on the function of Sl MPK3 gene in tomato and provided new germplasm for low temperature resistant tomato breeding.