【作者】 雷波；

【导师】 别之龙；

【作者基本信息】 华中农业大学 ， 设施园艺， 2013， 博士

【摘要】 近年来,随着设施栽培的迅速发展,设施内土壤盐渍化日趋严重,成为限制设施栽培发展的主要障碍。黄瓜是设施内栽培的主要蔬菜之一,其耐盐性较弱；但南瓜是相对耐盐的蔬菜作物,本课题组之前的研究表明,耐盐南瓜砧木嫁接可以提高黄瓜的耐盐性,其根本原因在于耐盐砧木具有较强的限制Na+从地下部向地上部运输的能力,但是其调节Na+吸收和运输的机制尚不清楚。本研究以盐敏感黄瓜、耐盐南瓜砧木、黄瓜自嫁苗和嫁接苗等为材料,采用X-射线微区分析、非损伤微测以及基因表达分析等技术,研究了NaCl处理下耐盐砧木嫁接植株和自嫁植株Na+吸收和转运的差异,以期为阐明耐盐砧木嫁接提高黄瓜耐盐性提供理论依据。本研究-主要结果如下：1.以耐NaCl胁迫能力弱的津春2号(Cucumis sativus L.)黄瓜为接穗,耐NaCl胁迫能力强的超级拳王(Cucurbita moschata Duch.)南瓜为砧木,黄瓜和南瓜自根苗为对照,利用X-射线微区分析技术,研究了黄瓜和南瓜以及嫁接苗在0和90mMNaCl处理时不同组织中离子的相对含量。结果表明,NaCl处理后南瓜自根苗和黄瓜嫁接苗地下部Na+的相对含量比黄瓜高,地上部分Na+的相对含量比黄瓜低而K+和Ca2+的相对含量比黄瓜高。南瓜自根苗和黄瓜嫁接苗根系皮层的Na+相对含量高于黄瓜自根苗,但是中柱中Na+相对含量低于黄瓜自根苗,南瓜及嫁接苗通过其根系皮层较强的控制Na+向木质部的装载的能力,降低了地上部分Na+的含量。2.以津春2号黄瓜和超级拳王南瓜为材料,测定了NaCl处理后南瓜和黄瓜不同组织中Na+的浓度,结果显示南瓜地下部Na+浓度较高,而地上部Na+浓度较低。采用非损伤微测技术(NMT)研究了NaCl处理对黄瓜和南瓜茎横切面Na+、K+流速的影响,以及NaCl、Na+H+向转运蛋白抑制剂阿米洛利、质膜H+-ATPase的抑制剂钒酸钠处理对黄瓜和南瓜根系Na+、K+、H+流速的影响。结果表明,南瓜根系比黄瓜储存Na+的能力强,同时南瓜从地下部向地上部Na+运输速率比黄瓜小,而从地上部分向地下部分Na+流速比黄瓜大,而且南瓜根系有较强的排出Na+和吸收H+的能力,抑制剂处理显著抑制了Na+的排出和H+的吸收,说明Na+的排出是受质膜Na+/H+反转运系统调控的,此结果为解析黄瓜和南瓜Na+从地下部向地上部运输的差异提供了直接证据。3.以津春2号黄瓜为接穗,超级拳王南瓜为砧木,黄瓜自嫁植株为对照,对嫁接黄瓜在0和90mM NaCl处理下植株的生长、Na+浓度、质膜和液泡膜H+-ATPase、液泡膜H+-PPase活性及其编码基因的转录水平进行了研究。结果表明,NaCl胁迫抑制了黄瓜自嫁和嫁接植株的生长,提高了自嫁和嫁接植株地上部和地下部Na+浓度,但黄瓜嫁接植株受抑制程度较轻。NaCl处理提高了黄瓜自嫁和嫁接植株根系质膜和液泡膜H+-ATPase和液泡膜H+-PPase勺活性,影响了编码酶的相关基因的表达水平。但是耐盐砧木嫁接可维持其根系较高的质膜和液泡膜]H+-ATPase、液泡膜H+-PPase活性,编码质膜和液泡膜H+-ATPase基因的调节可能发生在转录后水平,而编码液泡膜H+-PPase的基因的调节可能发生在转录水平。4.以津春2号黄瓜为接穗,超级拳王南瓜为砧木,黄瓜自嫁植株为对照,进行以下处理：(1)CK(对照)：营养液；(2)Ca:10mM Ca(N03)2+营养液；(3)NaCl:90mM NaCl+营养液；(4)NaCl+Ca:90mM NaCl+10mM Ca(N03)2+营养液,研究了Ca2+对NaCl处理时黄瓜自嫁和嫁接植株的生长、离子积累以及质膜H+-ATPase活性和其编码基因表达的影响。结果表明,添加Ca(N03)2处理缓解了NaCl处理对植株的生长抑制,显著提高了地上部K+和Ca2+的浓度,降低了Na+的浓度,对NaCl处理时黄瓜嫁接植株的缓解作用更显著。黄瓜嫁接植株通过其较敏感的Ca2+响应途径维持了较高的质膜H+-ATPase活性和PMA以及SOS1的表达,以及较强的限制Na+和调节K+和Ca2+从地下部向地上部运输的能力,具有较强的维持离子平衡的能力,表现出较强的耐盐性。5.以津春2号黄瓜为接穗,超级拳王南瓜为砧木,进行以下处理：(1)CK(对照)：营养液；(2)K:10mM KN03+营养液；(3)NaCl:90mM NaCl+营养液；(4)NaCl+K:90mM NaCl+10mM KN03+营养液,研究了K+对NaCl处理下自嫁和砧木嫁接植株的生长、离子积累的影响。结果表明,添加KN03处理显著提高了地上部分的K+和Ca2+浓度,降低了Na+的浓度,提高了K+/Na+和Ca2+/Na+,嫁接植株具有较强的K+、Na+选择性,具有较强的维持离子平衡的能力,从而缓解了NaCl处理对植株生长的抑制作用,表现出较强的耐盐性。总之,本研究结果表明,与耐盐性弱的黄瓜相比,耐盐南瓜砧木根系具有较强的限制Na+向中柱运输、限制木质部Na+的装载、以及根系较强的Na+外排能力,是耐盐南瓜砧木嫁接黄瓜后能维持地上部分较低的Na+浓度,具有较强耐盐性的主要原因。嫁接和自嫁黄瓜Na+外排受质膜Na+/H+逆向反转运系统调控,较强的质膜质子泵活性、Na+/H+逆向反转运能力,是嫁接黄瓜Na+外排能力强的重要原因。更多还原

【Abstract】 In recent years, with the development of protected culture, soil salinization has been more and more severe and has become the biggest obstacle of limiting the development of protected culture. Thus, the vegetable production in greenhouses has become a severe problem. Cucumber (Cucumis sativus L.) is one of the most popular vegetables under the protected cultivation conditions, and is sensitive to salt stress, but pumpkin (Cucurbita moschata Duch.) is salt-tolerant to salt stress. Previous studies suggested that the key reason is that pumpkin roots had higher capacity to limit the transport of Na+to the shoot than cucumber; however, the mechanism is not completely known. Cucumber plants ’Jinchun No.2’(Cucumis sativus L.), pumpkin ’Chaojiquanwang’(Cucurbita moschata Duch),’Jinchun No.2’grafted onto itself (self-grafted) and onto ’Chaojiquanwang’(rootstock-grafted) were used, X-ray microanalysis, non-invasive micro-test technique and gene expression were used to determine the differences of Na+uptake and transport between the self-grafted and rootstock-grafted plants, and to supply theoretic basis for clarify grafting enhance salt tolerance of cucumber. The main results are as follows:1. Experiments were conducted to investigate the relative content of ions in roots, stems below and above cotyledon of cucumber (Cucumis sativus L.), pumpkin (Cucurbita moschata Duch.) and rootstock-grafted cucumber under salt stress using X-ray microanalysis. The findings show that, the relative Na+content of rootstock-grafted and pumpkin plants were higher than cucumber in the roots, but lower than cucumber in the shoots, and the relative K+and Ca2+content were higher than cucumber. The relative Na+content in the cortex of root of pumpkin and grafted plants were higher, but the relative Na+content in the stele of root of pumpkin and grafted plants was lower than those of cucumber. The cortex of root of pumpkin and grafted plants had higher ability to restrict excess Na+loading in the xylem, resulting in a less accumulation of Na+in the shoot.2. Cucumber and pumpkin were used to investigate the Na+concentration in different parts of plants. This study showed that more Na+was stored in pumpkin roots compared with cucumber roots under200mM NaCl stress, resulting in the less accumulation of Na+in the shoot. The non-invasive micro-test technique (NMT) showed that the stem below cotyledon of pumpkin exhibited a smaller efflux of Na+from roots to shoots but a higher efflux of Na+from shoot to root, and pumpkin roots showed a higher capacity to extrude Na+, and a correspondingly increased H+influx. However, the200mM NaCl induced Na+/H+exchange in the root was inhibited by amiloride (a Na+/H+antiporter inhibitor) or vanadate (a plasma membrane H+-ATPase inhibitor). This result indicated that the Na+exclusion in salt stressed pumpkin and cucumber roots was the result of an active Na+/H+antiporter across the plasma membrane. These results provide direct evidence of contrasting Na+transport ability from root to shoot in salt-sensitive cucumber and salt-tolerant pumpkin under NaCl stress.3. To assess the effects of salt-tolerant rootstock on plant growth, Na+concentration, the plasma membrane (PM) and vacuolar H+-ATPases and vacuolar H+-PPase systems and the expression of the encoding genes of these enzymes of grafted cucumber, cucumber were grafted onto itself and pumpkin rootstocks, respectively. Grafted plants were grown hydroponically and were exposed to0and90mM NaCl for5days. The results showed that NaCl induced significant decrease in growth parameters, increased Na+concentrations in roots and shoots of both types of plants, whereas smaller changes were observed in rootstock-grafted plants. NaCl distinctly stimulated the activities of plasma membrane H+-ATPase, vacuolar H+-ATPase and vacuolar H+-PPase, the expressions of plasma membrane H+-ATPase(PMA), vacuolar H+-ATPase (VHA) and vacuolar H+-PPase (VPP) encoding genes were also changed, but the rootstock-grafted plants maintained higher activity of these three enzymes in the roots. Results suggested that the higher salt tolerance of the rootstock-grafted cucumber seedlings could be partially attributed to the higher activities of PM H+-ATPase, vacuolar H+-ATPase and vacuolar H+-PPase under NaCl stress, and the regulation of the encoding PM H+-ATPase, vacuolar H+-ATPase genes may ocuured at post-transcriptional level, and the regulation of the encoding vacuolar H+-PPase may ocuured at transcriptional level.4. Self-grafted and pumpkin rootstock-grafted cucumber plants were subjected to the following four treatments:(1) CK (Control):nutrient solution alone;(2) Ca:10mM Ca(NO3)2+nutrient solution;(3) NaCl:90mM NaCl+nutrient solution; and (4) NaCl+Ca:90mM NaCl+10mM Ca(NO3)2+nutrient solution, to assess the effects of Ca2+on plant growth, ion concentration and the activity of plasma membrane H+-ATPases and the expression of the encoding genes. The results showed that, supplementary Ca(NO3)2ameliorated the negative effects of NaCl on plant dry mass, relative growth rate (RGR), as well as Ca+, K+, and Na+concentration, especially for pumpkin rootstock-grafted plants. The pumpkin rootstock-grafted plants had higher PM H+-ATPase activity as well as higher PMA and SOS1expression, higher ability to regulate the transport of Na+、K+, and Ca2+than the self-grafted plants under NaCl+Ca treatment through the highly sensitive Ca2+signal. Therefore, pumpkin rootstock grafting had higher ability to maintain ion balance, and showed higher salt tolerance.5. Self-grafted and pumpkin rootstock-grafted cucumber plants were subjected to the following four treatments:(1) CK (Control):nutrient solution alone;(2) K:10mM KNO3+nutrient solution;(3) NaCl:90mM NaCl+nutrient solution; and (4) NaCl+K:90mM NaCl+10mM KNO3+nutrient solution, to assess the effects of K+on plant growth, ion concentration. The results showed that, supplementary KNO3ameliorated the negative effects of NaCt on plant dry mass,as well as Ca2+, K+, and Na+concentration, increased K+/Na+and Ca2+/Na+, induced increased salt tolerance. Rootstock-grafted plants had higher K+/Na+selectivity and higher ability to maintain ion balance, showed higher salt tolerance than self-grafted plants.In conclusion, the present study suggests that, compared with the salt sensitive cucumber, pumpkin roots showed higher capacity to restrict the transport of Na+to steler and loading of Na+to xylem, the roots of the salt-tolerant rootstock also had higher ability to exclude Na+, which resulted in the lower Na+of the shoot and the higher salt tolerance of grafted plants. Na+efflux was regulated by the Na+/H+antiport of the plasma membrane, higher activity of proton pump and Na+/H+antiport on the plasma membrane could be attributed to the higher salt tolerance of grafted plants.更多还原