【作者】 苏桐；

【导师】 魏小红；

【作者基本信息】 甘肃农业大学 ， 作物遗传育种， 2008， 硕士

【摘要】 本实验以番茄(Lycopersicon esculentum Mill)为材料,运用盆栽土培试验,结合生物化学和分子生物学的方法,分别用不同浓度的蔗糖(Sucrose)、外源NO供体硝普钠(Sodium Nitropresside, SNP)及其组合(V:V=1:1)处理材料,对盐胁迫下NO与蔗糖诱导番茄抗逆基因和幼苗生长及抗盐性的作用机制开展了较为深入的研究。并获得以下主要结果:1.用0.1、0.5和1.0 mmol/L的SNP处理番茄幼苗,均能缓解盐胁迫对番茄幼苗造成的氧化损伤,提高超氧化物酶歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性,其中0.1 mmol/LSNP处理效果最好,尤其能显著诱导APX和POD活性的增高;不同浓度的蔗糖对番茄体内的保护酶也具有不同的诱导作用,和SNP诱导一样具有剂量效应,其中3.0 mmol/L的蔗糖能显著提高酶活性,胁迫24 h各种酶活性均达到最大值,随着胁迫时间的延长开始下降,表明蔗糖对酶活性的诱导也有一定的调控范围。2.盐分胁迫下,番茄种子的萌发受到了抑制,72 h才开始萌发,而使用NO、蔗糖及其互作处理番茄种子的发芽率、发芽势、发芽指数及活力指数都显著高于100 mmol/LNaCl单独处理,NO、蔗糖及其互作处理番茄种子的最终发芽率分别为68.33 %、66.67 %,81.67 %,其中组合处理缓解盐胁迫的效果显著,与单盐处理之间差异显著。3.组合处理与SNP和蔗糖单独处理相比,对缓解盐胁迫下番茄幼苗的氧化损伤存在正协同效应,主要表现在进一步增强了番茄幼苗抗氧化酶的活性;提高脯氨酸(Pro)的含量,同时膜脂过氧化产物丙二醛(MDA)含量显著降低。采用聚丙烯酰胺凝胶电泳对盐胁迫24 h和48 h材料的POD同功酶研究表明,当NaCl单独处理时,番茄幼苗叶片POD同功酶第V条带缺失,其它谱带酶量减少,抑制了POD同功酶的表达;SNP和蔗糖单独处理能够保护盐胁迫(24、48 h)所导致的POD同功酶条带的完整;而NO、蔗糖及组合处理均能诱导番茄叶片POD同功酶活性,尤其组合处理对POD同功酶诱导效果最好,胁迫48 h各处理POD同功酶表达量较24 h减少。说明SNP和蔗糖组合处理能够更有效地缓解盐胁迫对番茄幼苗植株造成的氧化损伤。4.利用实时荧光定量RT-PCR技术,检测了抗逆相关基因NP24和PR-5在mRNA水平上的表达量。胁迫12 h SNP、蔗糖及组合处理均诱导了幼苗中NP24基因的表达量,与单盐处理相比分别提高了38.44﹪、24.48﹪和74.51﹪,其中组合处理中NP24基因的表达量最高。24 h单盐处理的幼苗PR-5基因达到最大值,而SNP、蔗糖及两者组合处理在胁迫36 h均达到各自的最大值,与单盐处理苗相比,PR-5基因的表达量分别提高了46.53﹪,43.20﹪和65.02﹪,其中组合处理的诱导效果最明显。更多还原

【Abstract】 Mainly by using pot culture methodology and started with the systematic analysis of plant biology and physiological responses on salt stress, studies on regulation of different concentrations of exogenous nitric oxide and sucrose to tomato seedlings resistance genes and physiological characteristics were carried out in present thesis, including mechanism of plant salt resistance under salt stress, regulation effects of exogenous nitric oxide and sucrose induced expressing their preexistent genetic information for tolerance under salt stress in seedlings. The major results are presented as follows:1. Under 100 mmol/L NaCl stress condition, the different concentrations at 0.1, 0.5 and 1.0 mmol/L SNP could alleviation of NaCl stress damage and increase activities of antioxidant enzymes (including SOD, CAT, POD and APX) in leaves, the best effect was observed in the treatment of 0.1 mmol/L SNP. The same dosage effect of sucrose existed on the alleviation of NaCl stress in tomato seedlings were observed, 3.0 mmol/L increase activities of antioxidant enzymes at 24 h significantly, with the tolerance time, the activities of antioxidant enzymes were decreased.2. Germination of tomato seed is reduced at relatively low NaCl concentrations and lengthening the time needed to complete germination. During the germination process in tomato, Applying the 0.1 mmol/L SNP, 1.0 mmol/Lsucrose and the mix-solution of SNP and sucrose increased the seed germination rate、germination potential、germination index and activity index of tomato, the mix solution of SNP and sucrose could largely promote germination rate, which was 81.67%, the germination rate was 56.67% under single salt stress.3. The results indicated that there were protective function to tomato seedling leaves from NaCl oxidative damage when applications of SNP, sucrose and their mix solutions. However, their mix solutions showed a much better protective impact than the treatments of SNP and sucrose as it could significantly increase activities of antioxidant enzymes and content of proline,while largely decrease MDA content in tomato seedling leaves under salt stress. Moreover, the effect of salt stress on the peroxidase enzyme expression in tomato leaves was further analyzed by using polyacrylamide concentration gradient gel electrophoresis technique. The results showed that salt stress could inhibit the expression of small molecule POD isoenzymes. The mix of SNP and sucrose could largely promote POD isoenzyme activity and protective them from hydrolyzation than single application of either SNP or sucrose solutions. Irrespective of solution, the POD isoenzymes expression was higher at 24 h than 48 h salt stress. The mix solution of SNP and sucrose could have a big potential for reduction of oxidative damage to tomato seedling leaves under salt stress. 4. The resistance gene NP24 and PR-5 expression were assayed by QRT-PCR at mRNA level in tomato plants treated with SNP, sucrose and the mix solution of SNP under salt stress. They could induce NP24 gene expression at 12 h. compared to single stress, NP24 gene relative expression level increased respectively 38.44﹪、24.48﹪and 74.51﹪.There has the maximum expression of PR-5 under single salt stress at 12 h, the expression level decreased along with the time extending. Compared to single salt stress, PR-5 gene relative expression level increased respectively 46.53﹪, 43.20﹪and 65.02﹪. It was suggested that the mix solution of SNP and sucrose treatment induced the strong expression of NP24 and PR-5.更多还原