苑乂川 陳小雨 李明明 李 萍 賈亞濤 韓淵懷,2,3 邢國芳,2,3,*

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谷子苗期耐低磷種質篩選及其根系保護酶系統(tǒng)對低磷脅迫的響應

苑乂川1,**陳小雨1,**李明明1李 萍1賈亞濤1韓淵懷1,2,3邢國芳1,2,3,*

1山西農業(yè)大學農學院, 山西太谷 030801;2山西農業(yè)大學農業(yè)生物工程研究所, 山西太谷 030801;3雜糧種質資源發(fā)掘與遺傳改良山西省重點實驗室, 山西太谷 030801

本研究旨在探討不同基因型谷子苗期耐低磷特性, 建立其篩選的評價體系, 篩選出苗期耐低磷谷子種質材料。對160份核心谷子種質資源的苗期株高、根長、地上鮮重、地下鮮重、葉長、葉寬、莖粗、地上磷含量、地下磷含量、地上干重和地下干重11個指標的耐低磷系數(shù)進行變異分析、相關性分析、主成分分析和聚類分析, 采用隸屬函數(shù)法綜合評價不同谷子耐低磷相關指標和耐低磷特性; 針對篩選出的低磷敏感和耐低磷品種, 在生理水平上分析其根系超氧化物歧化酶(SOD)、過氧化物酶(POD)、過氧化氫酶(CAT)的活性。結果表明, 9份材料屬于低磷敏感品種, 66份材料屬于低磷較敏感品種, 70份材料屬于較耐低磷品種, 15份材料屬于耐低磷品種; 低磷脅迫促使谷子根系SOD、POD、CAT活性增加。總之, 以綜合指標對谷子耐低磷特性的鑒定更為客觀; 根系保護酶系統(tǒng)對谷子在低磷脅迫下的適應性具有重要作用。

谷子; 低磷脅迫; 種質資源; 綜合評價; 根系保護酶

磷(P)是作物必需的營養(yǎng)元素之一[1], 對植物生長發(fā)育起著至關重要的作用。但土壤中能被作物吸收利用的有效磷僅占土壤全磷的1%[2], 土壤有效磷不足已成為制約作物產量和品質提高的主要因素之一。另外, 磷是不可再生資源, 50~400年內將會被耗盡[3-4]。為了應對未來的磷缺陷對農業(yè)生產帶來的危機, 了解作物磷脅迫響應機制, 培育耐低磷品種顯得尤為重要[5]。

了解作物磷脅迫響應機制, 培育耐低磷品種, 篩選耐低磷種植資源, 鑒定耐低磷材料是關鍵。目前關于耐低磷種質資源的篩選與評價, 在水稻、玉米等作物中已經開展了大量的試驗。郭再華等[6]對來自不同國家的多個水稻品種開展苗期土培篩選, 設置多個磷濃度處理, 確定了水稻苗期耐低磷指標篩選的最適營養(yǎng)液配比和鑒定指標。湛靜等[7]在缺磷條件下對玉米苗期進行了性狀和生理機制的研究, 獲得了一套比較完善的玉米耐低磷指標篩選體系。劉淵等[8]以酸性磷酸酶的耐低磷系數(shù)作為評判基礎, 分析了不同磷水平下大豆株高、干重、磷含量等指標, 發(fā)現(xiàn)不同基因型品種耐低磷差異極大, 并篩選出耐低磷種質。深入研究同一作物不同基因型品種間所表現(xiàn)的磷吸收和利用的生理特點差異, 它的意義不僅在于篩選磷高效品種, 還致力于形成完善的耐低磷篩選體系。

谷子(L)是原產我國北方的小雜糧, 耐旱, 耐貧瘠, 具有養(yǎng)分高效利用的特質和潛力[9-10], 是中國北方地區(qū)最主要的種植作物之一[11]。谷子對磷缺乏有著相應的適應機制, 不同基因型谷子在磷吸收方面存在很大差異[12-14]。缺磷會對谷子的生長發(fā)育造成一定影響, 楊艷君等[15]發(fā)現(xiàn)磷缺乏導致收獲期谷穗重量降低, Ceasar等[16]在處理后前2周, 發(fā)現(xiàn)300 μmol L–1與10 μmol L–1供磷量對谷子生物量的影響無差異, 5周后, 10 μmol L–1供磷水平下的谷子出現(xiàn)葉片變紫等缺磷癥狀。據(jù)報道, 谷子中存在12個磷轉運載體PHR家族成員, 并且不同成員之間存在組織特異性表達[16]。然而, 目前關于谷子耐低磷基因型品種篩選的相關研究并未深入。

植物體內的保護性酶如超氧化物歧化酶(SOD)、過氧化物酶(POD)和過氧化氫酶(CAT), 是抵抗營養(yǎng)脅迫引起氧化應激的關鍵[17]。逆境脅迫不僅會影響植物的正常生理功能, 還會導致植物的新陳代謝失去平衡[18]。植物的抗逆能力與植物保護性酶系統(tǒng)的活性變化之間存在密切的關聯(lián)性。本研究從實驗室1500多份材料中挑選了160份核心種質資源, 綜合鑒定與評價了它們苗期的株高、根長、地上部鮮重、地下部鮮重、葉長、葉寬、莖粗、地上部磷含量、地下部磷含量、地上部干重和地下部干重, 篩選出耐低磷特性的指標和苗期具有耐低磷特性的谷子種質, 并進一步研究苗期根系保護酶系統(tǒng)在低磷脅迫下的響應, 為在遺傳育種研究中利用這些優(yōu)異種質資源提供理論依據(jù)。

1 材料與方法

1.1 供試材料

160份核心谷子種質資源, 由中國農業(yè)科學院作物科學研究所刁現(xiàn)民研究員提供。

1.2 試驗設計

在山西農業(yè)大學農學院溫室內進行隨機排列的盆栽試驗, 以石英沙培養(yǎng)和營養(yǎng)液澆灌, 篩選耐低磷品種。挑選整齊飽滿的種子, 播于育苗盆(長32.5 cm, 寬25.0 cm, 深7.0 cm), 盆內放置總體積4/5的石英沙, 播種行距4.5 cm, 株距3.5 cm, 雙粒點播。待幼苗生長至一葉一心期時, 向盆內加Hoagland營養(yǎng)液, 待幼苗至三葉期時間苗, 培育至30 d。另以水培試驗用于谷子根系酶活性鑒定, 先在石英沙中盆栽培育至一心一葉期, 然后移栽到Hoagland營養(yǎng)液中, 每3天更換1次營養(yǎng)液, 培育至30 d。

試驗條件均為溫度25℃, 光照175 μmol m–2s–1, 光周期16 h光照/8 h黑暗。設低磷水平(LP, 0.005 mmol L–1)和正常磷水平(NP, 0.25 mmol L–1) 2個處理。

1.3 指標測定

測量生長30 d谷子幼苗的株高、葉長、葉寬、莖粗和根長; 用 H2SO4-H2O2消解和釩鉬黃比色法測定地上和地下磷含量[19], 氮藍四唑(NBT)光化還原法測定根系SOD活性, 愈創(chuàng)木酚法測定根系POD活性, 紫外吸收法測定根系CAT活性[20]。

1.4 數(shù)據(jù)處理

采用Origin 8和Microsoft Excel 2003作圖表, 利用SPSS 19.0進行相關性分析、主成分分析、方差分析, 采用LSD進行顯著性檢驗, 利用DPS 7.05和R語言進行聚類分析, 采用隸屬函數(shù)法對160份核心谷子種質進行耐低磷綜合評價, 參考相關文獻[21]計算指標。

耐低磷系數(shù)= 低磷時的測量值/正常磷的測量值 (1)

(X)(X–min)/(max–min)=1, 2, 3, …,(2)

式中,(X)表示第個綜合指標的隸屬函數(shù)值,X表示第個綜合指標值;X表示第個綜合指標的最大值,X表示第個綜合指標的最小值。

(3)

式中,W表示第個綜合指標在所有綜合指標中的重要程度及權重;P為各品種第個綜合指標的貢獻率。

式中,表示在低磷脅迫條件下各品種耐低磷能力的綜合評價值。

2 結果與分析

2.1 不同品種單項指標耐低磷系數(shù)的分析

160份不同基因型的谷子在NP與LP環(huán)境下各指標均存在不同程度的變化。為避免作物的生物學固有差異, 采用了能準確反映品種耐低磷能力的耐低磷系數(shù)(相對值)作為判斷谷子耐低磷特性的依據(jù)[22]。株高、地上部鮮重、葉長、地上部磷含量、地下部磷含量和地上部干重耐低磷系數(shù)均小于1, 葉寬有9個品種耐低磷系數(shù)大于1, 莖粗僅有1個品種耐低磷系數(shù)大于1, 綜合說明低磷脅迫抑制地上部各性狀生長, 低磷下谷子磷含量低于正常環(huán)境下磷含量。大部分品種根長、地下部鮮重和地下部干重耐低磷系數(shù)大于1, 說明低磷影響植物的根系發(fā)育。11個指標的相對值變異系數(shù)均在10%以上, 可以看出, 不同基因型谷子的耐低磷特性存在較大差異, 160份種質在不同磷水平下存在廣泛的遺傳變異。鑒于上述各指標在不同磷環(huán)境中變異情況均較大, 說明這些指標都可能是鑒定谷子耐低磷能力較好的篩選指標(表1)。

表1 谷子部分性狀耐低磷系數(shù)(LP/NP)的變化范圍及變異系數(shù)

2.2 指標間耐低磷指數(shù)的相關性分析

地上部磷含量的耐低磷系數(shù)與株高、地上部鮮重、葉長、葉寬、地下部磷含量、地上部干重、地下部干重等指標的耐低磷系數(shù)呈極顯著正相關, 與地下部鮮重耐低磷系數(shù)呈極顯著負相關; 株高的耐低磷系數(shù)與地上部鮮重、葉長、葉寬、莖粗、地上部磷含量、地下部干重等指標的耐低磷系數(shù)呈極顯著正相關, 與地上部干重耐低磷系數(shù)呈顯著正相關, 與地下部鮮重耐低磷系數(shù)呈極顯著負相關; 根長的耐低磷系數(shù)與地下部鮮重耐低磷系數(shù)極顯著正相關, 與葉長耐低磷系數(shù)呈極顯著負相關; 株高、地上部鮮重、葉長和莖粗4個指標的耐低磷系數(shù)均存在兩兩之間極顯著正相關; 地上部磷含量、地上部干重和地下部干重3個指標之間的耐低磷系數(shù)均為極顯著正相關(表2)?？梢? 部分指標間存在一定相關關系, 不同指標間可能存在信息重疊, 單靠單項指標難以對表現(xiàn)復雜的耐低磷性綜合評價, 綜合各項指標能更有效地篩選低磷耐性種質資源。

表2 谷子各性狀耐低磷系數(shù)(LP/NP)的相關性分析

**代表顯著差異< 0.01;*代表顯著差異< 0.05。 PH: plant height; RL: root length; AFW: aboveground fresh weight; UFW: underground fresh weight; LL: leaf length; LW: leaf width; DMS: diameter of main stem; APC: aboveground phosphorus content; SPC: subsurface phosphorus content; ADW: aboveground dry weight; UDW: underground dry weight.*represents significant correlation at< 0.05;**represents significant correlation at< 0.01.

2.3 主成分分析

為達到簡化指標及濃縮數(shù)據(jù)的目的, 將上述單項指標轉化為有效的少量指標[21], 以160份核心谷子種質各單項指標的耐低磷系數(shù)為基礎進行主成分分析。從11個主要耐低磷特征指標在各個主成分上的權重系數(shù)即特征向量看, 可以發(fā)現(xiàn), 第1主成分貢獻率最大, 主要特征向量包括株高、地上部鮮重、葉長、葉寬、莖粗、地上部磷含量和地上部干重7個單項指標, 特征值為4.657, 反映了地上因子, 可說明原始指標信息量的42.334%; 第2主成分載荷較高的是根長和地下部鮮重, 反映了根系因子, 可說明原始指標信息量的20.793%; 第3主成分載荷較高的是地下部磷含量, 可反映總信息量的11.206%; 第4主成分載荷較高的是地下部干重, 可反映總信息量的8.719%。前4個主成分累積貢獻率達83.049%, 足以代表原指標的極大部分信息, 其余貢獻率可忽略不計(表3和表4)。因此, 利用4個相互獨立的綜合指標可以對不同谷子的耐低磷特性進行客觀的分析。

2.4 品種耐低磷能力的綜合分析

2.4.1 隸屬函數(shù)鑒定品種耐低磷能力 根據(jù)主成分分析結果, 利用公式(2)計算每個綜合指標的隸屬函數(shù)值(X)。從表5可以看出, 低磷脅迫下, 綜合指標中69號品種(1)最小, 為0, 表明其在中耐低磷能力最弱; V147號品種(1)最大, 為1.0000, 表明其在中耐低磷能力最強; 綜合指標和中()最小的品種分別為V151、V19、V81號,()最大的品種分別V72、V35、V69?？梢?個綜合指標鑒定的結果各不相同, 因此, 為對谷子耐低磷性進行全面充實的分析, 通過公式(3), 結合各綜合指標的貢獻率, 得出4個綜合指標的權重W分別為50.97%、25.04%、13.49%和10.05%, 進一步根據(jù)公式(4)計算4個綜合指標的綜合評價值, 并根據(jù)值大小對其耐低磷能力的強弱進行排序。結果見表5, 排名較前的品種值較小, 表明耐低磷能力弱, 稱之為磷敏感品種, 排名較后的品種值較大, 說明耐低磷力強, 稱之為磷耐性品種。

表3 各綜合指標特征值及貢獻率

表4 各因子載荷矩陣

PH: plant height; RL: root length; AFW: aboveground fresh weight; UFW: underground fresh weight; LL: leaf length; LW: leaf width; DMS: diameter of main stem; APC: aboveground phosphorus content; SPC: subsurface phosphorus content; ADW: aboveground dry weight; UDW: underground dry weight.

表5 各品種的綜合性狀指標值、權重、()及綜合評價值()

Table 5 Value of each comprehensive index (CI), index weight,(), and comprehensive evaluation value ()

品種VarietyZ1Z2Z3Z4μ(X1)μ(X2)μ(X3)μ(X4)綜合評價值 Comprehensive assessment value (D)排名 Order V1-1.63230.38301.7162-0.39550.22640.64790.77650.31230.415227 V2-1.4500-2.16821.3999-0.50850.25390.35130.72440.29080.34567 V3-1.2102-0.6284-0.20631.00630.29010.53030.45970.57930.403520 V41.63350.7376-2.40041.18950.71970.68910.09810.61420.6171134 V52.1822-0.2511-2.44190.47810.80260.57420.09130.47870.6154132 V60.03190.61620.2194-0.21650.47780.67500.52980.34640.520479 V7-0.0242-1.3712-0.19590.17460.46930.44400.46140.42090.456849 V80.18180.6668-0.6091-1.66410.50040.68090.39330.07070.486067 V93.0438-2.09661.90040.60230.93270.35960.80690.50240.7270158 V100.33141.3360-1.27080.12520.52300.75870.28420.41150.538195 V11-1.3979-0.04560.3979-0.11250.26180.59810.55930.36620.397117 V12-1.10211.03750.43910.09010.30650.72400.56600.40480.456447 V130.79831.0699-1.3880-0.95060.59350.72780.26490.20660.542297 V14-1.67372.0691-0.23221.20510.22010.84390.45540.61720.449842 V151.0917-0.5273-1.66330.75570.63780.54210.21960.53160.546399 V16-0.5638-0.14780.0901-1.28530.38780.58620.50850.14280.428030 V17-1.8131-0.7841-0.25501.87890.19910.51220.45170.74550.368912 V18-2.9829-0.81470.6199-0.86140.02240.50870.59580.22360.24261 V190.02092.1536-2.99561.18850.47610.85370.00000.61400.520981 V20-1.71780.1697-1.55860.59140.21350.62310.23680.50030.34939 V210.25870.17130.10041.09740.51200.62330.51020.59670.5485102 V22-0.3407-1.10731.5481-0.65800.42150.47470.74880.26230.462252 V23-2.75873.3932-0.15981.46130.05630.99790.46730.66600.411524 V242.37250.2468-1.48640.57680.83130.63210.24870.49750.6678149 V252.87940.2426-1.22861.45580.90790.63160.29120.66490.7300159 V261.94960.0191-2.30270.94410.76740.60560.11420.56750.6178135

(續(xù)表5)

品種VarietyZ1Z2Z3Z4μ(X1)μ(X2)μ(X3)μ(X4)綜合評價值 Comprehensive assessment value (D)排名 Order V27-0.07351.0066-0.26750.02850.46180.72040.44960.39310.517776 V28-1.4796-2.7691-1.54850.43260.24950.28150.23850.47000.27912 V29-0.3805-1.49940.0644-0.83020.41550.42910.50430.22950.411323 V30-2.30100.55680.8604-0.45320.12540.66810.63550.30130.34868 V31-0.3831-0.63840.3210-0.26120.41510.52920.54660.33790.453345 V32-0.3526-0.83410.24910.39580.41970.50640.53470.46300.461551 V33-1.25722.06350.1175-0.65590.28300.84330.51300.26270.452244 V34-2.0066-0.07411.2416-0.09450.16990.59480.69830.36960.368511 V35-2.96881.90813.07230.24690.02450.82521.00000.43470.399718 V36-1.11282.66231.45800.29400.30490.91290.73400.44360.529689 V37-1.03200.6961-0.01910.76110.31710.68430.49050.53260.455146 V382.3586-0.0702-0.7951-0.55120.82920.59520.36260.28270.6503146 V391.3125-0.14260.71840.83120.67120.58680.61210.54590.6289142 V401.7558-1.0203-1.92451.55020.73820.48480.17650.68290.5931123 V41-0.4456-0.30040.9464-0.01200.40560.56850.64960.38530.477263 V42-1.8962-2.17700.88290.17630.18650.35030.63920.42120.31324 V43-0.82740.2928-1.8849-0.77250.34800.63740.18300.24050.386914 V44-1.39860.4234-0.7386-1.07000.26170.65260.37200.18380.366310 V450.0631-0.24750.0927-0.79900.48250.57460.50900.23550.483264 V46-0.09551.4289-0.23990.31590.45850.76950.45410.44780.534791 V471.78381.5395-2.23101.25220.74240.78240.12600.62610.6570147 V480.38311.5463-1.2741-0.83370.53080.78310.28370.22880.529088 V49-0.54360.55571.08391.14370.39080.66800.67230.60550.520780 V50-1.38870.81531.05060.86400.26320.69820.66680.55220.456950 V512.1270-1.1928-0.1009-0.01650.79420.46470.47710.38450.6259140 V520.32420.7315-0.8191-0.00010.52190.68840.35870.38760.527587 V530.2900-0.6707-1.9185-1.24740.51680.52540.17750.15000.434732 V54-0.67560.2877-0.4629-0.10390.37090.63680.41740.36780.443437 V553.0862-0.84190.26820.25870.93910.50550.53790.43690.7237156 V56-0.17201.2193-1.3032-0.07380.44700.74510.27890.37360.491368 V57-0.67190.9067-1.0655-0.67820.37150.70880.31810.25850.436933 V582.88440.6624-0.28681.05810.90860.68040.44640.58920.7556160 V591.34632.29630.12290.28760.67630.87030.51390.44240.6784151 V60-1.57992.00500.9341-0.02460.23430.83650.64760.38290.456548 V61-1.27371.9840-0.61191.31800.28060.83400.39280.63870.471960 V62-1.99482.21610.15070.58010.17160.86100.51850.49810.425329 V630.72811.95430.00450.45040.58290.83060.49440.47340.6215137 V64-1.9503-0.9239-0.0778-0.53690.17840.49600.48090.28540.30993 V65-0.45701.55841.35890.46300.40390.78460.71760.47580.5491103 V66-0.1959-0.8819-0.12430.53030.44340.50090.47320.48860.466555 V67-1.4532-1.53551.54591.45330.25340.42490.74840.66440.406322 V68-0.09351.1747-1.09370.94990.45880.73990.31340.56860.521182 V69-3.13110.7949-1.46023.21510.00000.69580.25301.00000.31345 V70-0.8250-0.4004-0.0584-0.26870.34830.55680.48410.33650.417628

(續(xù)表5)

品種VarietyZ1Z2Z3Z4μ(X1)μ(X2)μ(X3)μ(X4)綜合評價值 Comprehensive assessment value (D)排名 Order V71-1.49842.04801.6374-0.57840.24660.84150.76350.27750.468557 V720.38223.41160.4863-0.64900.53071.00000.57380.26400.6260141 V73-1.30441.93191.5631-0.20740.27590.82800.75130.34810.485966 V740.35360.6448-0.2815-1.01170.52640.67830.44730.19490.518977 V75-0.44501.4046-0.1209-0.54430.40570.76670.47380.28400.492569 V76-1.49301.56810.4010-0.42360.24740.78570.55980.30700.430631 V770.2232-1.9715-0.29160.92740.50670.37420.44560.56430.471359 V78-0.90941.02810.16850.08510.33560.72290.52140.40380.464853 V79-0.00050.73171.1602-0.99470.47290.68840.68490.19820.526686 V800.82242.6255-0.6271-0.71170.59720.90860.39030.25210.6110130 V811.2214-2.9115-0.8400-2.03520.65740.26490.35520.00000.449340 V820.82090.5664-0.5639-0.74690.59690.66920.40070.24540.5517107 V83-0.4357-1.28310.80761.52090.40710.45420.62680.67730.476962 V84-0.5671-0.83290.4943-0.18600.38730.50660.57510.35220.438834 V851.24931.38140.0058-0.80980.66170.76400.49460.23340.6198136 V860.34291.1338-0.7069-1.01880.52470.73520.37720.19360.522883 V870.17750.9740-0.43910.45960.49980.71660.42130.47520.540996 V880.8198-0.3046-0.19720.44070.59680.56800.46120.47160.5581109 V89-1.26210.16560.4366-1.30000.28230.62260.56560.14000.390816 V900.3801-0.07291.1570-0.24610.53040.59490.68440.34080.5474100 V910.75661.38460.2228-0.27480.58720.76430.53040.33530.5975125 V920.98680.30510.5359-0.38560.62200.63890.58200.31420.5885119 V93-0.63070.04251.6762-1.11830.37770.60830.76990.17460.467056 V941.7287-0.0029-0.6032-0.97310.73410.60300.39430.20230.5996128 V950.74461.2538-0.3449-0.75490.58540.74910.43680.24390.5705115 V960.8910-0.6957-1.4774-0.79260.60750.52250.25020.23670.499171 V971.01611.5727-0.8139-0.45040.62640.78620.35950.30180.5964124 V98-2.40211.16521.60250.26950.11010.73880.75780.43900.389415 V990.85370.3506-0.34810.12390.60190.64410.43630.41120.5701114 V1000.22191.2902-1.88621.08820.50650.75340.18280.59490.533990 V1012.1516-0.82180.69161.48010.79790.50780.60770.66950.6861153 V1020.23590.39411.05081.91530.50860.64920.66690.75240.5907121 V1031.5819-1.5365-0.24661.05360.71190.42480.45300.58830.5921122 V104-0.9350-0.79841.62141.74020.33170.51060.76090.71910.475161 V105-0.9468-0.82730.4000-0.49060.32990.50720.55960.29420.401619 V1061.2936-0.49931.24250.85760.66830.54530.69840.55100.6293143 V1070.8802-3.09490.14131.00830.60590.24360.51700.57970.500472 V1081.72612.4421-0.6870-1.10930.73370.88730.38050.17640.6660148 V1090.0716-0.89171.1465-1.00740.48380.49970.68260.19580.484365 V1101.1676-0.64500.4010-1.49960.64930.52840.55980.10200.5495105 V1112.2048-2.31440.42980.59730.80600.33430.56450.50140.6233138 V1121.65660.4933-0.7047-1.37960.72320.66070.37750.12490.5981126 V1131.0447-1.7958-1.3696-1.34060.63070.39460.26800.13230.470358 V1142.5698-1.6432-0.9985-1.39060.86110.41230.32910.12280.5995127

(續(xù)表5)

品種VarietyZ1Z2Z3Z4μ(X1)μ(X2)μ(X3)μ(X4)綜合評價值 Comprehensive assessment value (D)排名 Order V1150.7084-0.2833-0.62631.38650.57990.57040.39050.65170.5595111 V1160.2016-0.9832-0.6574-1.41540.50340.48910.38530.11810.443438 V1170.33190.52341.0628-0.79340.52310.66420.66880.23650.5480101 V118-0.09041.9288-0.2509-1.70920.45930.82760.45230.06210.508974 V119-0.6536-0.46281.4120-1.38660.37420.54960.72640.12350.439335 V1200.23461.2992-0.3514-1.09190.50840.75440.43580.17970.525785 V1210.14000.3997-0.1264-0.88320.49410.64980.47280.21940.501473 V122-1.06251.07880.6917-0.70880.31250.72880.60770.25260.450343 V1230.28270.6421-0.1297-0.57240.51560.67800.47230.27860.525684 V1241.31920.35502.73951.64870.67220.64470.94520.70170.7052155 V1251.0251-1.48672.1165-0.56070.62780.43050.84250.28080.5709116 V126-0.7859-0.5590-0.53032.71690.35420.53840.40630.90510.465254 V1270.6901-0.7110-0.26950.56430.57720.52070.44930.49510.537293 V128-0.1924-2.14550.87920.07680.44390.35400.63860.40230.443336 V1292.0218-3.09020.85320.01160.77830.24410.63430.38980.5843118 V1302.30300.69750.1622-0.76510.82080.68450.52040.24190.6854152 V1311.11581.61940.8286-0.80070.64150.79160.63020.23510.6349144 V132-1.34670.5684-0.78760.93070.26950.66950.36390.56490.413425 V133-0.8406-0.8182-0.46820.19580.34600.50830.41650.42490.404421 V1342.0695-4.68580.40210.89450.78550.05860.55990.55800.5492104 V1350.66910.41280.1090-0.20920.57400.65140.51160.34780.5612112 V136-0.30191.50291.1814-0.10330.42730.77810.68840.36800.544298 V1371.05401.42761.22761.11560.63220.76930.69600.60010.6718150 V1381.73740.5377-0.0778-1.14120.73540.66590.48090.17030.6243139 V139-0.0621-2.09030.5436-1.55200.46360.36040.58330.09200.414926 V1401.6572-1.9967-0.2135-0.30410.72330.37130.45850.32970.5581110 V141-1.27720.95261.0633-0.14210.28000.71410.66890.36060.449641 V1420.8626-0.1704-0.99080.60970.60320.58360.33040.50380.5511106 V143-0.42071.6479-0.2193-0.63410.40940.79500.45750.26690.497570 V144-0.0266-0.02410.58381.41580.46890.60060.58990.65730.538094 V1450.01941.1904-0.91150.19980.47590.74180.34350.42570.519378 V1461.40470.5100-0.6107-0.96830.68510.66270.39300.20320.5895120 V1473.4893-3.9982-0.5485-0.21631.00000.13860.40330.34640.6352145 V1481.2168-0.67072.6366-0.82290.65670.52540.92820.23090.6158133 V1493.4272-1.4023-0.49870.61990.99060.44040.41150.50570.7238157 V1501.2416-0.0553-0.4547-1.47530.66050.59700.41870.10660.5538108 V1511.2981-5.1901-0.50880.56950.66900.00000.40980.49610.448439 V1520.6812-0.48610.0443-1.30830.57580.54690.50100.13840.512675 V1531.7589-0.4240-1.2320-1.32310.73860.55410.29060.13560.5687113 V1541.8504-0.34160.5364-1.58950.75240.56370.58210.08490.6121131 V155-3.06640.55772.78881.88800.00980.66820.95330.74720.379413 V156-2.4860-0.5374-0.12142.25820.09740.54090.47370.81770.33496 V1572.9685-1.3375-0.44480.84330.92130.44790.42040.54830.6960154 V1582.0892-2.21470.58430.08750.78850.34590.59000.40430.6106129 V1591.5189-2.88611.9919-0.23440.70240.26790.82190.34300.5720117 V1600.5692-0.66430.8973-0.35380.55890.52620.64160.32020.536892 Weights0.50970.25040.13490.1050

2.4.2 各品種耐低磷能力的聚類分析 為使本試驗結果更加客觀, 使用最大距離法對4個耐低磷綜合指標的綜合評價值進行聚類(圖1)。結果將實驗中谷子種質分為4類。第I類為低磷敏感型品種, 耐低磷值范圍為0.2426~0.3493, 耐低磷能力最弱, 共9份; 第II類為低磷較敏感型品種, 耐低磷能力中等偏弱, 耐低磷值范圍為0.3663~0.5126, 共66份; 第III類為較耐低磷品種, 耐低磷能力中等偏強, 耐低磷值范圍為0.5177~ 0.6352, 共70份; 第IV類為耐低磷品種, 耐低磷能力最強, 耐低磷值范圍為0.6503~0.7556, 共15份。

圖1 160個谷子品種的耐低磷性聚類圖

2.5 不同品種在磷脅迫下的根系保護酶活性差異

逆境下, 植物會產生破壞細胞膜結構的超氧自由基。SOD是植物體內天然存在的自由基清除因子, 與POD和CAT結合把毒害的H2O2轉換分解為完全無害的水, 保持植物正常的生長發(fā)育。這3種酶被認為是植物脅迫評估的重要生理指標。由于在LP環(huán)境下, 植物會優(yōu)先將光合產物分配到地下部分, 地上部分生長受到抑制的同時地下部分促進根系發(fā)育[23]。因此, 我們根據(jù)篩選結果選擇具有代表性的耐低磷品種(V47、V124、V130)與低磷敏感品種(V18、V69、V156)各3份進行水培, 測定其苗期根系SOD、POD、CAT活性。在NP環(huán)境下, SOD、POD和CAT根系酶活力平均值分別為282.83 U g–1FW、914.80 U g–1min–1FW和3.51 U g–1min–1FW; LP環(huán)境下, 3種酶活力平均值分別為297.83 U g–1, FW、998.46 U g–1min–1FW和3.81 U g–1min–1FW。低磷脅迫促使6個谷子根系酶活力平均增加了5.30%、9.14%和8.54%?？梢? 不同谷子根系在LP環(huán)境下相對于NP環(huán)境的SOD、POD、CAT活力均增大。與NP環(huán)境下相比, 低磷脅迫使V47、V124、V136、V18、V69和V156的SOD活力分別升高8.67%、6.20%、5.07%、4.42%、1.96%和5.74%。其中耐低磷品種V47增長幅度最大, 低磷敏感品種V69增長幅度最小, 6個谷子根系酶活力NP與LP處理間均達到顯著差異。V47、V124、V136、V18、V69和V156在LP條件下的POD活力與NP條件下相比, 分別升高7.23%、14.61%、19.74%、5.02%、3.96%和3.29%。其中耐低磷品種V124和V136根系酶活力顯著高于其余4個谷子品種。與NP環(huán)境下相比, 低磷脅迫使V47、V124、V136、V18、V69和V156的CAT活力分別升高5.41%、12.82%、7.91%、6.00%、1.05%和5.00%, 其中耐低磷品種V124增長幅度最大, 顯著高于其余5個谷子根系酶活力, 低磷敏感品種V156增長幅度最小(圖2)。

圖2 不同供磷強度下谷子SOD、POD、CAT活性的比較

圖柱上不同的字母表示5%水平下差異的顯著性。

Bars superscripted by different letters are significantly different at the 5% probability level.

3 討論

3.1 篩選耐低磷種質資源的方法

苗期是谷子需磷量的瓶頸期和篩選低磷耐性指標的關鍵時期[24]。苗期篩選不僅能快速、準確地鑒定和篩選品種, 還可以顯著減少田間篩選的工作量與成本[25]。而盆栽和水培是實施苗期試驗簡便有效的方法, 且精確度較高。因此本試驗采取盆栽方法對160份谷子苗期耐低磷性進行種質篩選, 進一步采用水培方法對耐低磷和低磷敏感品種進行根系酶活性分析。

3.2 苗期耐低磷綜合指標的確定

谷子的耐低磷表現(xiàn)是一個復雜的過程, 缺磷會抑制植株某些生理特性, LP環(huán)境下地上部指標株高、地上部鮮重、葉長、地上部磷含量和地上部干重均小于NP環(huán)境下的各對應指標, 同時缺磷也會促進根系生長, 引起地下部鮮重增大。本試驗通過主成分分析, 將所有單項指標劃分為具有代表性的綜合指標, 對160份核心谷子種質進行耐低磷能力的區(qū)分。結果表明, 缺磷對低磷敏感品種的影響要遠遠高于耐低磷品種, 由于部分耐低磷品種本身含有少量可用于谷物吸收的磷元素, 因而出現(xiàn)部分品種在LP環(huán)境下的莖粗、葉長等性狀沒有受到抑制反而促進生長的趨勢, 表明磷元素的缺乏對不同基因型谷子苗期的單項指標有不同的推動作用, 為避免利用單個指標描述耐低磷的局限性, 最終采用4個綜合指標分析不同品種的耐低磷特性。采用綜合指標對作物的抗逆能力進行評價已在苜蓿[22]、花生[26]、水稻[27]等中取得了一定的成果。

3.3 根系保護酶系統(tǒng)對低磷脅迫的響應

許多研究發(fā)現(xiàn)SOD、POD、CAT等酶類是植物抗逆的重要酶保護系統(tǒng), 這3種酶在低磷脅迫下可以反映植物的逆境脅迫效應[28]。萬美亮等[29]研究認為, 低磷脅迫誘導甘蔗體內保護酶系統(tǒng)活性升高, 是植物耐低磷的生理機制之一。本研究發(fā)現(xiàn), 低磷環(huán)境誘導SOD、POD和CAT 3種酶活性增加以清除谷子幼苗根系內的H2O2, 但不同谷子根系酶活性的變化幅度存在一定差異, 與NP環(huán)境相比, LP環(huán)境中3種酶活性增幅最大的均為耐低磷品種, 增幅最小的均為低磷敏感性品種。表明低磷脅迫對不同基因型谷子根系酶活性具有不同的調控作用, 耐低磷品種和低磷敏感性品種在逆境下的脅迫響應存在差異, 因而清除植物體內產生的自由基的能力也不同。本研究結果與李俊等[17,30-32]對油菜、大豆、玉米等研究結果類似。

4 結論

本研究對160份核心谷子種質的苗期耐低磷特性進行研究, 根據(jù)耐低磷能力強弱可分為低磷敏感型、低磷較敏感型、較耐低磷型和耐低磷型4種不同類型。低磷脅迫下, 本研究所測得的11個指標均可作為鑒定谷子苗期耐低磷能力和品種選育的參考指標。其中低磷耐性品種和低磷敏感品種根系保護酶系統(tǒng)對低磷脅迫響應能力的不同, 是谷子磷脅迫可能的重要逆境脅迫效應。

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Screening of germplasm tolerant to low phosphorus of seedling stage and response of root protective enzymes to low phosphorus in foxtail millet

YUAN Yi-Chuan1,**, CHEN Xiao-Yu1,**, LI Ming-Ming1, LI Ping1, JIA Ya-Tao1, HAN Yuan-Huai1,2,3, and XING Guo-Fang1,2,3,*

1College of Agronomy, Shanxi Agricultural University, Taigu 030801, Shanxi, China;2Institute of Agricultural Bioengineering, Shanxi Agricultural University, Taigu 030801, Shanxi, China;3Shanxi Key Laboratory of Genetic Resources and Genetic Improvement of Minor Crop, Taigu 030801, Shanxi, China

The purpose of this paper was to explore the characteristics of low-phosphate tolerance in different genotypes of foxtail millet and establish an evaluation system for screening and breeding new cultivars with low-phosphate tolerance. Correlation analysis, principal component analysis, membership function and cluster analysis were carried out on 11 indicators of 160 core foxtail millet varieties, including plant heights, root lengths, fresh weights of aerial part and underground part, leaf lengths and widths, stem thicknesses, phosphorus contents of aerial part and underground part, dry weights of aerial part and underground part, to comprehensively evaluate indicators and characters related to low-phosphorous tolerance. Furthermore, a hydroponic experiment was conducted to cultivate selected varieties with low-phosphorus sensitivity and low-phosphorus tolerance under low phosphorus (LP) and normal phosphorus (NP) conditions, and superoxide dismutase (SOD) activity, peroxidase (POD) activity, and catalase (CAT) activity in roots were analyzed. There were nine varieties with low-phosphorus sensitivity, 66 and 70 varieties with medium low-phosphate tolerance and 15 varieties with low-phosphorus tolerance. The activities of SOD, POD, and CAT were increased under low phosphorus condition. It was concluded that the comprehensive index is more objective to evaluate the low-phosphorus tolerance of 160 foxtail millet variation at the seedling stage, and the root protective enzymes are important for the adaptability of foxtail millet to low-phosphorus stress.

foxtail millet; low-phosphorus stress; germplasm resources; comprehensive evaluation; root protecting enzyme

2018-07-25;

2018-12-24;

2019-01-04.

10.3724/SP.J.1006.2019.82029

邢國芳, E-mail: sxauxgf@126.com

**同等貢獻(Contributed equally to this work)

E-mail: sxtyyyc@163.com

本研究由國家自然科學基金項目(31200914, 31501323), 山西省高等學?？萍紕?chuàng)新項目(2015145), 山西省重點研發(fā)計劃項目(201803D221008-4)和山西省主要農作物種質創(chuàng)新與分子育種重點科技創(chuàng)新平臺(201605D151002)資助。

This study was supported by the National Natural Science Foundation of China (31200914, 31501323), the Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (2015145), the Key R&D Program of Shanxi Province (201803D221008-4), and the Key Scientific and Technological Innovation and Molecular Breeding Platform of Major Crop Germplasm in Shanxi Province (201605D151002).

URL: http://kns.cnki.net/kcms/detail/11.1809.S.20190103.1710.009.html