拟南芥液泡膜H~+-焦磷酸酶基因AVP1改良紫花苜蓿（Medicago Sativa L.）抗逆性的研究
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摘要
干旱和盐碱化是人类面临的世界性问题，也是制约作物和牧草产量的2个主要的环境因子。特别在我国西北干旱半干旱地区，随着水资源的日益短缺，干旱和土壤盐碱化已成为该地区的农牧业发展和生态环境的严重威胁。紫花苜蓿(Medicago sativa L．)是一种优良的豆科牧草，在我国西北干旱半干旱地区农牧业生产和生态建设中发挥着极为重要的作用。然而，很多紫花苜蓿品种的耐盐、抗旱能力普遍不高，在盐渍或灌溉不足条件下种植难获高产。培育具有较强耐盐性和抗旱性的紫花苜蓿新品种，是提高西北干旱半干旱地区紫花苜蓿人工草地产量、节约灌溉以及利用大面积盐荒地的根本途径之一。传统的紫花苜蓿抗逆育种周期长，且容易受到种内抗逆基因资源的限制，基因工程技术的迅速发展为紫花苜蓿抗逆育种提供了一个新的手段。通过转基因技术培育耐盐、抗旱品种将是紫花苜蓿抗逆育种的一个重要的发展方向。液泡膜H~+-pyrophosphatase(H~+-PPase)已经被证明在植物离子区域化和生长素运输中有着重要的作用，其编码基因在一些模式植物和重要作物中的超表达显著提高了转基因植物的耐盐、抗旱性或耐瘠薄性，并促进了其生长发育，表明此类基因在改良紫花苜蓿的抗逆性方面具有潜在的应用价值。
在前期的研究中，我们已初步建立了农杆菌介导的紫花苜蓿栽培品种遗传转化体系，本研究对其做了进一步优化调整。为获得抗逆性强的转基因紫花苜蓿品系，本研究首先利用相关的农艺学和生理学指标，通过隶属函数法对西北地区广泛栽培的5个优良紫花苜蓿品种的耐盐性和抗旱性分别进行综合评价，筛选出具有较强耐盐性和抗旱性的材料新疆大叶苜蓿，将拟南芥液泡膜H~+-PPase编码基因AVP1导入该品种，并对转基因紫花苜蓿的抗逆性进行鉴定，主要研究结果如下：
1．与无NaCl胁迫的对照相比，在200 mmol·~(-1)NaCl胁迫下，5个紫花苜蓿品种的地上部干重和K~+／Na~+比均显著降低，叶片MDA含量和相对质膜透性均有所增加，但各指标的变化幅度因品种而异；利用上述4个指标对5个紫花苜蓿品种进行综合评价，发现它们的耐盐性由强到弱依次为：润布勒＞新疆大叶＞三得利＞甘农3号＞陇东。同样，与正常浇水的对照相比，在干旱胁迫下，紫花苜蓿各品种的地上部干重、叶片渗透势和相对含水量均显著下降，叶片相对质膜透性均显著增加，但各指标的变化幅度因品种的不同而不同；利用以上4个指标对5个紫花苜蓿品种进行综合评价，抗旱性由强到弱依次为：陇东＞新疆大叶＞甘农3号＞三得利＞润布勒。通过分析以上综合评价结果，发现新疆大叶苜蓿既有良好的耐盐性，又有较强的抗旱性，因此本研究将其选为遗传转化的种质材料，进一步改良其抗逆性。
2．在已有研究的基础上，进一步优化了农杆菌侵染方式和卡那霉素(Kan)选择方案，分别为：下胚轴在常压下用农杆菌侵染12 min；用75和40 mg·L~(-1)Kan分别于体胚和生根诱导的第2周开始选择抗性体胚和抗性植株。调整后的转化体系的转化效率(PCR阳性植株占侵染外植体的百分率)约为2．1％，转化周期约为19周。
3．首次用拟南芥液泡膜H~+-PPase基因AVP1对新疆大叶苜蓿进行遗传转化，获得了转基因植株；PCR分析表明外源基因已经整合到紫花苜蓿基因组中；对随机抽取的8个转基因系列进行RT-PCR分析，发现AVP1基因在这些转基因系列中都能稳定表达，但其表达量在不同系列间存在差异，其中系列1最高，系列8最低。
4．对转基因紫花苜蓿的耐盐、抗旱性分析表明，AVP1的超表达增强了转基因紫花苜蓿的耐盐性和抗旱性。200 mmol·L~(-1)NaCl处理10 d后，野生型植株生长严重受抑，出现萎蔫、甚至死亡，而转基因植株仍然能够正常生长。自然干旱处理6 d后，野生型植株开始萎蔫，而转基因植株依然正常生长，直到第8 d才发生萎蔫；复水后，转基因植株解除萎蔫并恢复正常的生长发育，而野生型植株却发生永久萎蔫，最后死亡。生理测定结果表明：不论在正常生长条件，还是在盐或干旱胁迫下，转基因紫花苜蓿比野生型植株在叶和根中积累了更多的Na~+、K~+和Ca2~+；大量的阳离子积累造成转基因植株的叶片渗透势显著低于野生型植株，使其在干旱胁迫下维持较高的叶片相对含水量。此外，在盐或水分胁迫下，转基因紫花苜蓿的叶片MDA含量和相对质膜透性均显著低于野生型植株，净光合速率和根系活力均显著高于野生植株。这些结果说明AVP1的超表达促进了转基因紫花苜蓿细胞中Na~+向液泡中的区域化，并增强了细胞对其它阳离子的吸收能力。一方面可以避免盐胁迫下细胞质中过量Na~+对细胞膜和各类细胞器的伤害，并稳定细胞内的离子平衡，另一方面提高细胞的渗透调节能力，使转基因紫花苜蓿能在盐或干旱胁迫下保持较多水分并维持其膨压。
5．盆栽实验表明，AVP1的超表达促进了转基因紫花苜蓿的根系增生和地上部发育。无论在正常生长条件下，还是长期处于盐或干旱环境中，转基因紫花苜蓿的生长状况总是好于野生型植株。在正常浇水条件下生长60 d(第20 d刈割1次)后，2个转基因系列的株高和地上部生物量分别比野生型植株显著高出15．5％-21．6％和27．8％-35．2％；经过较长时间盐或干旱处理后，转基因植株的以上指标同样高于野生型植株。这是因为无论在何种条件下，与野生型植株相比，AVP1转基因植株的根系更为粗壮发达、根干重和根冠比更高。发达的根系有助于转基因紫花苜蓿在胁迫条件下吸收更多的水分和养分，因而对其耐盐性和抗旱性也有着重要的贡献。
6．AVP1的超表达提高了转基因紫花苜蓿的耐瘠薄性。在缺磷(10μmol·L~(-1)Pi)条件下生长21 d后，2个转基因系列植株的生长受抑程度比野生型植株小，其根干重、地上部干重以及根冠比分别比野生型植株显著高出54．6％-96．5％、20．7％-39．7％和23．5％-41．2％。不论有无养分胁迫，转基因植株的单株含磷量(μmol·plant~(-1))显著高于野生型植株，分别是野生植株的1．3-1．4倍(1 mmol·L~(-1)Pi)和1．3-1．8倍(10μmol·L~(-1)Pi)；其单位干物质中的含磷量(μmol·g~(-1)DW)也高于野生型植株。说明转基因紫花苜蓿吸收了更多的Pi，从而促进其生长。
Salinity and drought are two major abiotic factors limiting crop production and forageyield.In particular,with ingravescent fresh water scarcity,drought and soil salinity havebeen threatening the development of agriculture and animal husbandry,and even thesecurity of whole ecosystem in the arid and semi-arid areas of northwest China.Alfalfa(Medicago sativa L.) is one of the most important legume forages and acts a veryimportant role in the agriculture and animal husbandry,and the ecological construction inthe arid and semi-arid areas of northwest China.However,most alfalfa cultivars aredifficult to maintain high forage yield in saline and arid soils since they are sensitive to saltand drought.Breeding enhanced salt and drought resistant alfalfa cultivars is one ofnecessary ways for increasing forage yield of alfalfa pastures,decreasing irrigation,andreusing salinity land,in the arid and semi-arid areas of northwest China.Salt and droughttolerance of alfalfa can be improved by conventional breeding method,but it often takes along time and is readily limited by narrow genes resources within species.Recentdevelopments in transgenetic technology have provided an efficient tool for improvingalfalfa,and will trigger an important direction of alfalfa stress tolerance breeding.It hasbeen confirmed that vacuolar H~+-pyrophosphatase (H~+-PPase) plays important roles invacuolar Na~+ sequestration and auxin fluxes.The overexpressions of H~+-PPase confersome model plants and crops enhanced growth and tolerance to salinity,water-deficit andlow-Pi stress conditions,suggesting that the H~+-PPase genes have potential benefits inimproving stress tolerance of alfalfa.
We have previously established an Agrobacterium-mediated transformation systemfor alfalfa cultivars.In this study,to generate transgenic alfalfa lines with enhanced stresstolerance,we comprehensively assessed the tolerance of five cultivars which are widelysowed in northwest China through subordinate function values analysis using relevantagricultural and physiological indicators together,and selected the alfalfa cultivar withwell salt and drought tolerance.Then an H~+-PPase gene AVP1 from Arabidopsis wasintroduced into this cultivar based upon the transformation system,which was furthermodified and evaluated.Finally,the stress tolerance of transgenic alfalfa was assayed.Themain results of this study are as following:
1.Compared with 0 mM NaCl,200 mM NaCl reduced significantly the shoot dryweight and shoot K~+/Na~+ ratio,and enhanced the leaf MDA content and relative membranepermeability in all five alfalfa cultivars,but the degree of either reduction or enhancementdiffered between these cultivars.Through comprehensively combining above fourindicators,the salt tolerance of the five cultivars decreased in the order:Rambler,XinjiangDaye,Sandili,Gannong No.3,and Longdong.Similarly,compared with well watered condition,after drought stress treatment,the shoot dry weight,leaf osmotic potential andrelative water content were significantly dropped,and the leaf relative membranepermeability was significantly increased in all five alfalfa cultivars,however the changerange of these indicators are different between five cultivars.Through comprehensiveassessment combining above four indicators,the drought tolerance of the five cultivarsdecreased in the order:Longdong,Xinjiang Daye,Gannong No.3,Sandili,and Rambler.According to analysis of above two comprehensive assessment results,we found that thecultivar Xinjiang Daye possessed well both salt and drought tolerance,and it thus wasselected as a potential germplasm material to further improve its stress tolerance in thisstudy.
2.Based upon our previous transformation system,we modified the Agrobacteriuminfection and kanamycin (Kan) selection procedures:the Agrobacterium infection wasperformed under normal pressure for 12 min;kanamycin resistant somatic embryos andplants were selected by 75 and 40 mg·L~(-1) Kan after 2 weeks when somatic embryos androots were induced,respectively.This adjusted transformation system took about 19 weeksto get a transformation efficiency (number of PCR-positive plants produced per onehundred infected hypocotyls.) of about 2.1%.
3.This study firstly transformed an H~+-PPase gene A VPI from Arabidopsis intoalfalfa cultivar Xinjiang Daye and obtained transgenic plants.PCR amplification indicatedthat A VPI was integrated into the chromosomal DNA of alfalfa.RT-PCR analysis inrandom eight transgenic lines showed that A VPI expressed in all tested lines,but itstranscript levels are different between eight lines.Compared with other lines,line 1 andline 8 showed the highest and lowest expression level ofA VPI,respectively.
4.The overexpression of AVPI enhanced salt and drought tolerance in transgenicalfalfa.After 10 days in the presence of 200 mM NaCl,wild type plants exhibited growthreduction,chlorosis,even die,whereas transgenic plants continue to grow well.Moreover,After 6 days of drought stress,wild-type plants showed chlorosis,whereas transgenicalfalfa still maintained normal growth until day 8 after subjected to water-deficit stress.After rewatered,wild-type plants showed irreversible chlorosis and die,but plants fromtransgenic lines survived and resumed normal growth.The examinations of physiologyshowed that transgenic alfalfa accumulate more Na~+,K~+ and Ca~(2+) in leaves and roots thanwild type,which resulted in lower leaf osmotic potential in transgenic plants;thus theirleaves retained more water during drought stress than wild-type plants.Furthermore,undersalt or water-deficit stress,transgenic alfalfa displayed significantly lower leaf MDAcontent and relative membrane permeability,significantly higher net photosynthetic rateand root activity compared with wild-type plants.These results suggested that theoverexpression ofA VPI increased sequestration of Na~(-1) into vacuole and uptake capacity of other cations in the transgenic alfalfa.These mechanisms might on the one hand reduce thedeleterious effects of excess Na~+ in the cytosol under salt stress and steady intracellular ionhomeostasis,on the other hand,enhance the osmoregulation capacity of cells,thus increasethe water retention and maintain the turgor of transgenic alfalfa under salt or droughtstress.
5.Pot experiments showed that the overexpression of AVPI resulted in increased rootproliferation and shoot development in transgenic alfalfa.Under normal condition,orlong-term salt/drought treatments,transgenic alfalfa always displayed enhanced growththan wild-type.The plants of two transgenic lines grown in well water soil for 60 days(cutting at 20th day) exhibited 15.5%-21.6% higher shoot height and 27.8%-35.2% highershoot dry weight than wild-type plants.Similar results were observed in long-term salt ordrought treatments.These phenotypes should be ascribed to that transgenic alfalfadeveloped more robust root systems,and thus got higher root biomasses and root/shootratios compared with wild-type under either normal condition or salt/drought stress.Therobust root systems allowed transgenic alfalfa to take up greater amounts of water andnutrient during the imposed salt or water deficit stress,thus also contributed to enhance saltand drought tolerance of transgenic plants.
6.The overexpression of AVPI increased the barren tolerance in transgenic alfalfa.Transgenic plants outperformed wild-type plants when challenged with inphosphorus-deficient condition (10μM Pi) for 21 days:the root biomass,shoot biomassand root/shoot ratio of transgenic plants from two transgenic lines were 54.6%-96.5%,20.7%-39.7%,and 23.5%-41.2% higher,respectively,than that of wild-type plants.Undereither normal or restrictive Pi condition,the toltal Pi content per plant (μmol·plant~(-1)) in twotransgenic lines were 1.3-1.4 fold (under 1 mM Pi) and 1.3-1.8 fold (under 10μM Pi) incomparison that of wild-type;the normalized Pi content (μmol·g~(-1) DW) was also higher intransgenic alfalfa than in wild-type plants,suggesting that transgenic plants absorbed morePi and thus grown accordingly.
在前期的研究中，我们已初步建立了农杆菌介导的紫花苜蓿栽培品种遗传转化体系，本研究对其做了进一步优化调整。为获得抗逆性强的转基因紫花苜蓿品系，本研究首先利用相关的农艺学和生理学指标，通过隶属函数法对西北地区广泛栽培的5个优良紫花苜蓿品种的耐盐性和抗旱性分别进行综合评价，筛选出具有较强耐盐性和抗旱性的材料新疆大叶苜蓿，将拟南芥液泡膜H~+-PPase编码基因AVP1导入该品种，并对转基因紫花苜蓿的抗逆性进行鉴定，主要研究结果如下：
1．与无NaCl胁迫的对照相比，在200 mmol·~(-1)NaCl胁迫下，5个紫花苜蓿品种的地上部干重和K~+／Na~+比均显著降低，叶片MDA含量和相对质膜透性均有所增加，但各指标的变化幅度因品种而异；利用上述4个指标对5个紫花苜蓿品种进行综合评价，发现它们的耐盐性由强到弱依次为：润布勒＞新疆大叶＞三得利＞甘农3号＞陇东。同样，与正常浇水的对照相比，在干旱胁迫下，紫花苜蓿各品种的地上部干重、叶片渗透势和相对含水量均显著下降，叶片相对质膜透性均显著增加，但各指标的变化幅度因品种的不同而不同；利用以上4个指标对5个紫花苜蓿品种进行综合评价，抗旱性由强到弱依次为：陇东＞新疆大叶＞甘农3号＞三得利＞润布勒。通过分析以上综合评价结果，发现新疆大叶苜蓿既有良好的耐盐性，又有较强的抗旱性，因此本研究将其选为遗传转化的种质材料，进一步改良其抗逆性。
2．在已有研究的基础上，进一步优化了农杆菌侵染方式和卡那霉素(Kan)选择方案，分别为：下胚轴在常压下用农杆菌侵染12 min；用75和40 mg·L~(-1)Kan分别于体胚和生根诱导的第2周开始选择抗性体胚和抗性植株。调整后的转化体系的转化效率(PCR阳性植株占侵染外植体的百分率)约为2．1％，转化周期约为19周。
3．首次用拟南芥液泡膜H~+-PPase基因AVP1对新疆大叶苜蓿进行遗传转化，获得了转基因植株；PCR分析表明外源基因已经整合到紫花苜蓿基因组中；对随机抽取的8个转基因系列进行RT-PCR分析，发现AVP1基因在这些转基因系列中都能稳定表达，但其表达量在不同系列间存在差异，其中系列1最高，系列8最低。
4．对转基因紫花苜蓿的耐盐、抗旱性分析表明，AVP1的超表达增强了转基因紫花苜蓿的耐盐性和抗旱性。200 mmol·L~(-1)NaCl处理10 d后，野生型植株生长严重受抑，出现萎蔫、甚至死亡，而转基因植株仍然能够正常生长。自然干旱处理6 d后，野生型植株开始萎蔫，而转基因植株依然正常生长，直到第8 d才发生萎蔫；复水后，转基因植株解除萎蔫并恢复正常的生长发育，而野生型植株却发生永久萎蔫，最后死亡。生理测定结果表明：不论在正常生长条件，还是在盐或干旱胁迫下，转基因紫花苜蓿比野生型植株在叶和根中积累了更多的Na~+、K~+和Ca2~+；大量的阳离子积累造成转基因植株的叶片渗透势显著低于野生型植株，使其在干旱胁迫下维持较高的叶片相对含水量。此外，在盐或水分胁迫下，转基因紫花苜蓿的叶片MDA含量和相对质膜透性均显著低于野生型植株，净光合速率和根系活力均显著高于野生植株。这些结果说明AVP1的超表达促进了转基因紫花苜蓿细胞中Na~+向液泡中的区域化，并增强了细胞对其它阳离子的吸收能力。一方面可以避免盐胁迫下细胞质中过量Na~+对细胞膜和各类细胞器的伤害，并稳定细胞内的离子平衡，另一方面提高细胞的渗透调节能力，使转基因紫花苜蓿能在盐或干旱胁迫下保持较多水分并维持其膨压。
5．盆栽实验表明，AVP1的超表达促进了转基因紫花苜蓿的根系增生和地上部发育。无论在正常生长条件下，还是长期处于盐或干旱环境中，转基因紫花苜蓿的生长状况总是好于野生型植株。在正常浇水条件下生长60 d(第20 d刈割1次)后，2个转基因系列的株高和地上部生物量分别比野生型植株显著高出15．5％-21．6％和27．8％-35．2％；经过较长时间盐或干旱处理后，转基因植株的以上指标同样高于野生型植株。这是因为无论在何种条件下，与野生型植株相比，AVP1转基因植株的根系更为粗壮发达、根干重和根冠比更高。发达的根系有助于转基因紫花苜蓿在胁迫条件下吸收更多的水分和养分，因而对其耐盐性和抗旱性也有着重要的贡献。
6．AVP1的超表达提高了转基因紫花苜蓿的耐瘠薄性。在缺磷(10μmol·L~(-1)Pi)条件下生长21 d后，2个转基因系列植株的生长受抑程度比野生型植株小，其根干重、地上部干重以及根冠比分别比野生型植株显著高出54．6％-96．5％、20．7％-39．7％和23．5％-41．2％。不论有无养分胁迫，转基因植株的单株含磷量(μmol·plant~(-1))显著高于野生型植株，分别是野生植株的1．3-1．4倍(1 mmol·L~(-1)Pi)和1．3-1．8倍(10μmol·L~(-1)Pi)；其单位干物质中的含磷量(μmol·g~(-1)DW)也高于野生型植株。说明转基因紫花苜蓿吸收了更多的Pi，从而促进其生长。
Salinity and drought are two major abiotic factors limiting crop production and forageyield.In particular,with ingravescent fresh water scarcity,drought and soil salinity havebeen threatening the development of agriculture and animal husbandry,and even thesecurity of whole ecosystem in the arid and semi-arid areas of northwest China.Alfalfa(Medicago sativa L.) is one of the most important legume forages and acts a veryimportant role in the agriculture and animal husbandry,and the ecological construction inthe arid and semi-arid areas of northwest China.However,most alfalfa cultivars aredifficult to maintain high forage yield in saline and arid soils since they are sensitive to saltand drought.Breeding enhanced salt and drought resistant alfalfa cultivars is one ofnecessary ways for increasing forage yield of alfalfa pastures,decreasing irrigation,andreusing salinity land,in the arid and semi-arid areas of northwest China.Salt and droughttolerance of alfalfa can be improved by conventional breeding method,but it often takes along time and is readily limited by narrow genes resources within species.Recentdevelopments in transgenetic technology have provided an efficient tool for improvingalfalfa,and will trigger an important direction of alfalfa stress tolerance breeding.It hasbeen confirmed that vacuolar H~+-pyrophosphatase (H~+-PPase) plays important roles invacuolar Na~+ sequestration and auxin fluxes.The overexpressions of H~+-PPase confersome model plants and crops enhanced growth and tolerance to salinity,water-deficit andlow-Pi stress conditions,suggesting that the H~+-PPase genes have potential benefits inimproving stress tolerance of alfalfa.
We have previously established an Agrobacterium-mediated transformation systemfor alfalfa cultivars.In this study,to generate transgenic alfalfa lines with enhanced stresstolerance,we comprehensively assessed the tolerance of five cultivars which are widelysowed in northwest China through subordinate function values analysis using relevantagricultural and physiological indicators together,and selected the alfalfa cultivar withwell salt and drought tolerance.Then an H~+-PPase gene AVP1 from Arabidopsis wasintroduced into this cultivar based upon the transformation system,which was furthermodified and evaluated.Finally,the stress tolerance of transgenic alfalfa was assayed.Themain results of this study are as following:
1.Compared with 0 mM NaCl,200 mM NaCl reduced significantly the shoot dryweight and shoot K~+/Na~+ ratio,and enhanced the leaf MDA content and relative membranepermeability in all five alfalfa cultivars,but the degree of either reduction or enhancementdiffered between these cultivars.Through comprehensively combining above fourindicators,the salt tolerance of the five cultivars decreased in the order:Rambler,XinjiangDaye,Sandili,Gannong No.3,and Longdong.Similarly,compared with well watered condition,after drought stress treatment,the shoot dry weight,leaf osmotic potential andrelative water content were significantly dropped,and the leaf relative membranepermeability was significantly increased in all five alfalfa cultivars,however the changerange of these indicators are different between five cultivars.Through comprehensiveassessment combining above four indicators,the drought tolerance of the five cultivarsdecreased in the order:Longdong,Xinjiang Daye,Gannong No.3,Sandili,and Rambler.According to analysis of above two comprehensive assessment results,we found that thecultivar Xinjiang Daye possessed well both salt and drought tolerance,and it thus wasselected as a potential germplasm material to further improve its stress tolerance in thisstudy.
2.Based upon our previous transformation system,we modified the Agrobacteriuminfection and kanamycin (Kan) selection procedures:the Agrobacterium infection wasperformed under normal pressure for 12 min;kanamycin resistant somatic embryos andplants were selected by 75 and 40 mg·L~(-1) Kan after 2 weeks when somatic embryos androots were induced,respectively.This adjusted transformation system took about 19 weeksto get a transformation efficiency (number of PCR-positive plants produced per onehundred infected hypocotyls.) of about 2.1%.
3.This study firstly transformed an H~+-PPase gene A VPI from Arabidopsis intoalfalfa cultivar Xinjiang Daye and obtained transgenic plants.PCR amplification indicatedthat A VPI was integrated into the chromosomal DNA of alfalfa.RT-PCR analysis inrandom eight transgenic lines showed that A VPI expressed in all tested lines,but itstranscript levels are different between eight lines.Compared with other lines,line 1 andline 8 showed the highest and lowest expression level ofA VPI,respectively.
4.The overexpression of AVPI enhanced salt and drought tolerance in transgenicalfalfa.After 10 days in the presence of 200 mM NaCl,wild type plants exhibited growthreduction,chlorosis,even die,whereas transgenic plants continue to grow well.Moreover,After 6 days of drought stress,wild-type plants showed chlorosis,whereas transgenicalfalfa still maintained normal growth until day 8 after subjected to water-deficit stress.After rewatered,wild-type plants showed irreversible chlorosis and die,but plants fromtransgenic lines survived and resumed normal growth.The examinations of physiologyshowed that transgenic alfalfa accumulate more Na~+,K~+ and Ca~(2+) in leaves and roots thanwild type,which resulted in lower leaf osmotic potential in transgenic plants;thus theirleaves retained more water during drought stress than wild-type plants.Furthermore,undersalt or water-deficit stress,transgenic alfalfa displayed significantly lower leaf MDAcontent and relative membrane permeability,significantly higher net photosynthetic rateand root activity compared with wild-type plants.These results suggested that theoverexpression ofA VPI increased sequestration of Na~(-1) into vacuole and uptake capacity of other cations in the transgenic alfalfa.These mechanisms might on the one hand reduce thedeleterious effects of excess Na~+ in the cytosol under salt stress and steady intracellular ionhomeostasis,on the other hand,enhance the osmoregulation capacity of cells,thus increasethe water retention and maintain the turgor of transgenic alfalfa under salt or droughtstress.
5.Pot experiments showed that the overexpression of AVPI resulted in increased rootproliferation and shoot development in transgenic alfalfa.Under normal condition,orlong-term salt/drought treatments,transgenic alfalfa always displayed enhanced growththan wild-type.The plants of two transgenic lines grown in well water soil for 60 days(cutting at 20th day) exhibited 15.5%-21.6% higher shoot height and 27.8%-35.2% highershoot dry weight than wild-type plants.Similar results were observed in long-term salt ordrought treatments.These phenotypes should be ascribed to that transgenic alfalfadeveloped more robust root systems,and thus got higher root biomasses and root/shootratios compared with wild-type under either normal condition or salt/drought stress.Therobust root systems allowed transgenic alfalfa to take up greater amounts of water andnutrient during the imposed salt or water deficit stress,thus also contributed to enhance saltand drought tolerance of transgenic plants.
6.The overexpression of AVPI increased the barren tolerance in transgenic alfalfa.Transgenic plants outperformed wild-type plants when challenged with inphosphorus-deficient condition (10μM Pi) for 21 days:the root biomass,shoot biomassand root/shoot ratio of transgenic plants from two transgenic lines were 54.6%-96.5%,20.7%-39.7%,and 23.5%-41.2% higher,respectively,than that of wild-type plants.Undereither normal or restrictive Pi condition,the toltal Pi content per plant (μmol·plant~(-1)) in twotransgenic lines were 1.3-1.4 fold (under 1 mM Pi) and 1.3-1.8 fold (under 10μM Pi) incomparison that of wild-type;the normalized Pi content (μmol·g~(-1) DW) was also higher intransgenic alfalfa than in wild-type plants,suggesting that transgenic plants absorbed morePi and thus grown accordingly.
引文
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