【作者】 刘磊；

【导师】 廖明安；

【作者基本信息】 四川农业大学 ， 果树学， 2021， 博士

【摘要】 硒(Se)是自然界中普遍存在的,能够影响动植物生长发育的一种非金属元素。较低浓度的硒能够清除植物体内的自由基,提高植物的抗性,高浓度的硒则会对植物造成毒害。近年来,随着人们对硒的研究不断加深,提高植物硒富集能力的研究逐渐被人们所重视。我国作为典型的缺硒国家,研究富硒水果配套栽培技术具有十分重要的意义。葡萄作为世界四大水果之一,深受广大消费者的喜爱。为此,通过一定的农艺措施,提高葡萄果实的品质和硒含量,不仅可为高品质葡萄的栽培提供参考,也对鲜食葡萄产区差异化发展具有重要的意义。鉴于此,本文通过研究葡萄(品种为‘夏黑’)幼苗的硒富集特性,筛选出对葡萄幼苗产生胁迫的最低硒浓度;在此基础上,施用不同植物生长调节剂,筛选出最适种类和浓度(褪黑素,150μmol/L),并从生理和转录组水平研究了褪黑素与硒互作对葡萄幼苗硒富集的机理;最后通过田间试验,进一步研究褪黑素对葡萄果实品质及其硒富集的影响,以期得到褪黑素对葡萄硒富集的作用机理。该研究对富硒葡萄乃至其它富硒水果的生产具有重要的理论和实践意义。主要研究结果如下:(1)研究了不同浓度的硒(0.00、0.05、0.10、0.25、0.50、1.00、2.00 mg/L)对葡萄幼苗生长及硒富集特性的影响。结果表明,随着外源硒浓度的增加,葡萄幼苗各部位的生物量、抗氧化酶活性、相对电导率、丙二醛和可溶性蛋白含量呈先增后降的趋势。当硒浓度为0.05 mg/L时,葡萄幼苗根系及地上部分的生物量达到最大值。随着外源硒浓度的增加,葡萄幼苗根系及地上部分的硒含量呈增加的趋势。这些结果说明,低浓度的硒(<0.10 mg/L)能够促进葡萄幼苗生长,而高浓度的硒(≥0.10 mg/L)则产生抑制作用。(2)在硒胁迫(0.10 mg/L)条件下,对葡萄幼苗施用5种植物生长调节剂(褪黑素、胺鲜酯、生长素、水杨酸和油菜素内酯),研究了不同植物生长调节剂对葡萄幼苗生长、生理及硒富集的影响。结果表明,在一定浓度范围内,5种植物生长调节剂均不同程度地增加葡萄幼苗的生物量。5种植物生长调节剂均能够增加葡萄幼苗的硒含量。这些结果说明,5种植物生长调节剂均能通过调控葡萄幼苗的生理生态促进葡萄幼苗的生长和硒富集,其中,浓度为150μmol/L的褪黑素对葡萄幼苗的富硒效果最好。(3)在硒胁迫(0.1 mg/L)和无硒胁迫条件下,研究了褪黑素(150μmol/L)与硒互作对葡萄幼苗生长、生理以及硒富集的影响。结果表明,在硒胁迫和无硒胁迫条件下,褪黑素均能增加葡萄幼苗的生物量、光合色素含量和净光合速率,促进了葡萄幼苗的生长。在抗性生理方面,褪黑素均提高了葡萄幼苗的SOD和POD活性,对CAT活性没有显著影响,一定程度上提高了葡萄幼苗的可溶性蛋白可溶性糖和淀粉含量,降低了葡萄幼苗的脯氨酸含量、相对电导率和MDA含量。在激素水平方面,褪黑素均提高了葡萄幼苗的赤霉素、生长素和褪黑素含量,同时降低了脱落酸含量。就硒富集而言,褪黑素均能够显著提高葡萄幼苗的总硒、有机硒和无机硒含量,改善了葡萄幼苗对养分的吸收和分配。这些结果说明,在硒胁迫和无硒胁迫条件下,褪黑素均能通过调控葡萄幼苗生理和激素水平促进葡萄幼苗的生长,促进葡萄幼苗对硒的吸收和转化,并能缓解硒胁迫对葡萄幼苗生长造成的不利影响。(4)通过对褪黑素和硒互作的葡萄幼苗进行转录组测序分析,结果表明,差异基因主要涉及细胞过程、代谢过程、细胞器和催化活性。KEGG富集分析后得出这些差异基因主要集中在氨酰基t RNA的生物合成、剪接体和类黄酮生物合成等作用通路,其中能够注释到通路信息的共有9个,涉及11条代谢通路。对9个差异基因进行实时荧光定量PCR分析表明,有6个基因(VIT＿12s0035g01160、VIT＿06s0004g01430、VIT＿15s0046g01570、VIT＿19s0027g01190、VIT＿19s0093g00220和VIT＿00s0361g00040)的表达量在褪黑素和硒互作处理条件下相对对照都显著上调,其中3个基因(VIT＿12s0035g01160、VIT＿15s0046g01570和VIT＿19s0027g01190)的表达量与葡萄总硒含量呈极显著正相关。(5)田间试验结果表明,褪黑素显著提高了葡萄果实的单粒重、可溶性固形物含量和Vc含量,同时显著降低了葡萄果实的可滴定酸含量。褪黑素也显著增加了葡萄果实的硒含量,与硒处理相比,褪黑素与硒互作的葡萄果实硒含量增加了37.01%。这些结果说明,褪黑素能够改善葡萄果实品质,提高其硒含量。综上,不同的植物生长调节剂均有利于葡萄幼苗的生长,同时能够促进葡萄幼苗对硒的富集。在这些植物生长调节剂中,褪黑素能够通过调节葡萄植株的生理和代谢通路促进葡萄生长,提高葡萄的硒吸收和转化,提高葡萄果实的品质,其中以浓度为150μmol/L的褪黑素效果最佳。更多还原

【Abstract】 Selenium(Se)is a ubiquitous non-metallic element that can affect the growth of animals and plants.Lower concentration of Se can scavenge free radicals in plants and improve their resistance while high-concentration Se can be toxic to plants.In recent years,investigation on improving the Se enrichment capacity of plants has gradually aroused attention.As a typical Se-deficient country,it is of great significance to study cultivation techniques of Se-enriched fruits in China.Grapes are among one of the four major fruits in the world,and are deeply loved by consumers.Improving the quality and Se content of grapes by agronomic measures not only provides a reference for the cultivation of high-quality grapes but also has important significance for the differential development of fresh grape production areas.Based on these facts,this thesis first studied the Se-enrichment characteristics of grape(the variety of ‘Summer Black’)seedlings to screen the minimum Se concentration that would stress grape seedlings.Following this,different plant growth regulators were used to screen the most suitable types of plant growth regulators and concentrations(melatonin,150 μmol/L)and studied the mechanism of the interaction of melatonin and Se on the Se enrichment of grape seedlings on the physiological and transcriptome levels.Finally,this thesis further studied the effect of melatonin on grape fruit quality and its Se enrichment via field experiment to obtain the mechanism of melatonin on grape Se enrichment.This study had important theoretical and practical significance for the production of Se-enriched grapes and other fruits.The main findings are as follows:(1)The effects of different Se concentrations(0.00,0.05,0.10,0.25,0.50,1.00,2.00mg/L)on the growth and Se accumulation characteristics of grape seedlings were studied.With increased exogenous Se,the Se contents in the roots and shoots of grape seedlings showed an increasing trend whilst the biomass,antioxidant enzyme activity,relative conductivity,malondialdehyde and soluble protein content increased first and then decreased.Specifically,the biomass of the roots and shoots of grape seedlings peaked at 0.05 mg/L Se.These results indicated that low-concentration Se(< 0.10 mg/L)promotes the growth of grape seedlings while high-concentration Se(≥ 0.10 mg/L)showed an inhibition impact.(2)Under the condition of Se stress(0.10 mg/L),five plant growth regulators(melatonin,diethyl aminoethyl hexanoate,auxin,salicylic acid and brassinosteroids)were applied to grape seedlings to study their effects on the growth,physiology and Se enrichment of grape seedlings.All five plant growth regulators significantly increased the biomass of grape seedlings to some extent by increasing the Se content in grape seedlings.These results indicated that all five plant growth regulators could promote the growth and Se enrichment of grape seedlings by regulating the physiological ecology of grape seedlings,among which 150 μmol/L melatonin had the best Se enrichment effect on grape seedlings.(3)Under the condition of Se stress(0.1 mg/L)and no Se stress,the interaction of melatonin(150 μmol/L)and Se on the growth,physiology and Se accumulation of grape seedlings were studied.In both scenarios,melatonin promoted the growth of grape seedlings by increasing the biomass,photosynthetic pigment content and net photosynthetic rate of grapes.In terms of resistance physiology,melatonin increased the SOD and POD activities of grape seedlings with no significant effect on CAT activity.Additionally,melatonin increased the soluble protein,soluble sugar and starch content of grape seedlings to a certain extent and reduced the proline content,relative conductivity and MDA content.In terms of hormone levels,melatonin increased the contents of gibberellin,auxin and melatonin in grape seedlings whilst reduced the content of abscisic acid.With regard to Se enrichment,melatonin significantly increased the total Se,organic and inorganic Se contents in grape seedlings and improved the nutrients absorption and distribution by grape seedlings.These results indicated that melatonin could promote the growth of grape seedlings by regulating the physiological and hormone levels of grape seedlings under both the Se stress and the absence of Se stress,promote the absorption and transformation of Se,and alleviate the unfavorable effects of Se stress on the growth of grape seedlings.(4)The results of transcriptome sequencing analysis of grape seedlings interacting with melatonin and Se showed that the differential genes mainly involved cellular processes,metabolic processes,organelles and catalytic activities.After KEGG enrichment analysis,these differential genes were mainly concentrated in the biosynthesis of aminoacyl t RNA,spliceosome and flavonoid biosynthesis and other pathways,among which 11 metabolic pathways were annotated from information of 9pathways.Real-time fluorescence quantitative PCR analysis of 9 differential genes showed that the expression of 6 genes(VIT＿12s0035g01160,VIT＿06s0004g01430,VIT＿15s0046g01570,VIT＿19s0027g01190,VIT＿19s0093g00220 and VIT＿00s0361g00040)were significantly up-regulated when treated with both melatonin and Se.Among them,the expression of 3 genes(VIT＿12s0035g01160,VIT＿15s0046g01570 and VIT＿19s0027g01190)had a very significant positive correlation with the total Se content of grapes.(5)Field experiment showed that melatonin significantly increased the single-grape weight,soluble solid content and Vc content of grape berries whilst significantly reduced titratable acidity.Melatonin also significantly increased the Se content in grape berries.Compared with Se treatment,the interaction of melatonin and Se increased the Se content in grape berries by 37.01%.These results indicated that melatonin could improve the quality of grape fruit and increase its Se content.In summary,different plant growth regulators were all beneficial to the growth of grape seedlings and could promote the accumulation of Se in grape seedlings.Among these different plant growth regulators,melatonin promoted the growth of grapes by regulating the physiological and metabolic pathways of grape plants.Various concentrations of melatonin increased the absorption and transformation of Se in grapes and improved grape quality,with 150 μmol/L exerting the most influential impact.更多还原