倪 郁，宋 超，李加納

西南大學(xué)農(nóng)學(xué)與生物科技學(xué)院，南方山地農(nóng)業(yè)教育部工程技術(shù)中心，重慶 400716

UV-B輻射增強對擬南芥表皮蠟質(zhì)的影響

倪 郁，宋 超，李加納*

西南大學(xué)農(nóng)學(xué)與生物科技學(xué)院，南方山地農(nóng)業(yè)教育部工程技術(shù)中心，重慶 400716

以野生型擬南芥、蠟質(zhì)不同程度缺失突變體CER1、CER3、CER4、CER6、CER10、CER20及KCS1為試驗材料，通過施加50 μW/cm2、長達(dá)10 d的UV-B輻射，研究了擬南芥表皮蠟質(zhì)晶體結(jié)構(gòu)、組分及蠟質(zhì)基因?qū)V-B輻射的響應(yīng)機制。結(jié)果表明：UV-B輻射增強改變了擬南芥表皮蠟質(zhì)晶體結(jié)構(gòu)，表皮蠟質(zhì)松針狀(CER1)、柱狀、桿狀(CER3、CER10與KCS1)晶體結(jié)構(gòu)顯著減少，球狀蠟質(zhì)晶體類型出現(xiàn)在CER6表面，無規(guī)則片狀、膜狀結(jié)構(gòu)覆蓋在KCS1與CER10莖表面。野生型擬南芥蠟質(zhì)晶體結(jié)構(gòu)類型無明顯變化，但在部分區(qū)域積累了大量水平桿狀、管狀結(jié)構(gòu)，增加了蠟質(zhì)層厚度。UV-B輻射增強也改變了擬南芥表皮蠟質(zhì)組分的分泌量。野生型在UV-B處理后一級醇、酸、醛含量顯著上升，烷、次級醇及酮含量顯著下降，蠟質(zhì)總量增加不顯著。一級醇含量的增加及酮和次級醇含量的減少在擬南芥各材料響應(yīng)UV-B輻射中具有普遍性。UV-B輻射增強誘導(dǎo)了野生型CER3、CER4、KCS1基因表達(dá)的上調(diào)，其中CER4大量表達(dá)，促進(jìn)了蠟質(zhì)組分中一級醇、酸和醛含量的積累；CER1在UV-B處理后表達(dá)量下調(diào)，可能導(dǎo)致烷合成下游分支途徑相關(guān)產(chǎn)物(烷類、次級醇及酮類)的減少。WIN1表達(dá)量的下調(diào)對蠟質(zhì)總量沒有顯著影響。UV-B輻射增強使蠟質(zhì)前體從烷合成分支途徑更多地轉(zhuǎn)向一級醇分支途徑。

擬南芥；表皮蠟質(zhì)；基因；UV-B輻射；蠟質(zhì)突變體

大氣臭氧層減薄導(dǎo)致的紫外線B(UV-B，280—320 nm)輻射增強是當(dāng)今人類面臨的全球性重大環(huán)境問題之一[1- 4]。研究UV-B 輻射增強對植物的傷害作用以及植物響應(yīng)UV-B輻射的防御與修復(fù)機制，對于促進(jìn)UV-B輻射環(huán)境下植物的正常生長發(fā)育以及作物的高產(chǎn)、穩(wěn)產(chǎn)均具有重要的理論意義。植物葉片形態(tài)特征的變化是植物對環(huán)境脅迫的防御反應(yīng)之一。前人研究表明，植物葉表皮毛的結(jié)構(gòu)與密度、蠟質(zhì)層及表皮細(xì)胞層的厚度均影響紫外線輻射能否穿透到葉肉細(xì)胞[5]；蠟質(zhì)含量高的植物葉片比含量低的葉片能吸收更多的紫外線[6]。表皮蠟質(zhì)主要由可溶性的超長鏈脂肪酸、烷烴、一級醇、二級醇、脂肪醛、酮類和酯類組成。前人對UV-B輻射與植物表皮蠟質(zhì)互作研究主要集中在對表皮蠟質(zhì)組分及含量的化學(xué)分析上[7- 11]，而對表皮蠟質(zhì)響應(yīng)UV-B輻射變化的分子機制了解甚少。擬南芥作為研究遺傳學(xué)和分子生物學(xué)的一種模式植物，具有典型的角質(zhì)層結(jié)構(gòu)及組份。因此，擬南芥在植物角質(zhì)層的生態(tài)學(xué)特性研究上也應(yīng)該是個模式系統(tǒng)。在包括擬南芥在內(nèi)的大多數(shù)植物中，角質(zhì)層蠟生物合成主要依靠蠟質(zhì)前體超長鏈脂肪酸(very long chain fatty acids, VLCFAs)通過脫羰基途徑(烷合成途徑)與?；€原途徑(一級醇合成途徑)進(jìn)行[12]。在擬南芥蠟質(zhì)合成途徑中，一些蠟質(zhì)相關(guān)基因陸續(xù)被克隆，如蠟質(zhì)前體VLCFAs合成基因KCS1[13]、催化生成一級醇的脂肪酰-CoA還原酶基因CER4[14]、醛類合成相關(guān)基因CER3[15]、烷類合成基因CER1[16,17]及表皮蠟質(zhì)過量表達(dá)轉(zhuǎn)錄因子WIN1[18]等。這些蠟質(zhì)基因的分離鑒定為揭示蠟質(zhì)組分在植物中的功能奠定了分子基礎(chǔ)。本試驗以野生型擬南芥以及蠟質(zhì)不同程度缺失突變體為試驗材料，研究UV-B輻射增強條件下表皮蠟質(zhì)含量、組分及蠟質(zhì)晶體結(jié)構(gòu)的變化規(guī)律；分析UV-B輻射誘導(dǎo)表達(dá)的主要蠟質(zhì)基因、特異性蠟質(zhì)組分，為深入揭示植物表皮蠟質(zhì)與UV-B輻射互作機制積累基礎(chǔ)資料。

1 材料與方法

1.1 試驗材料

哥倫比亞野生型(Columbia- 0 ecotype) 擬南芥種子來源于西南大學(xué)植物生理生化研究室，其余表皮蠟質(zhì)突變體CER1、CER3、CER4、CER6、CER10、CER20及KCS1種子均購于擬南芥資源中心(ABRC，USA)。突變體特征如表1所示。蛭石、珍珠巖和有機土按1∶1∶1的比例混勻，裝入花盆(7 cm×8 cm)，將低溫春化后的種子播在花盆中，每盆4 株，然后用塑料薄膜覆蓋5 d以利于出苗。試驗材料放置在人工氣候培養(yǎng)箱中培養(yǎng)，溫度為21 ℃/23 ℃(黑夜/白天)，光照每天16 h，空氣濕度為75%。待植株生長至5—6 周時進(jìn)行UV-B脅迫處理。

表1 擬南芥表皮蠟質(zhì)突變體特征Table 1 Characteristics of the Arabidopsis thaliana cuticular wax mutants

1.2 UV-B輻射處理

在植物培養(yǎng)室內(nèi)使用日光燈管提供正常光照，使用Philips 公司的UV-B燈管(TL30W/03，波峰308 nm)進(jìn)行UV-B輻射處理，UV-B燈管用0.13 mm厚度的醋酸纖維素膜濾除少量UV-C，每隔3 d更換1 次。UV-B 處理的擬南芥植株每天10:00—12:00 增施UV-B輻射，輻射強度50 μW/cm2。通過調(diào)節(jié)紫外燈管與材料間的高度控制紫外輻射強度。紫外輻射劑量用UV- 340A紫外光照度計(Lutron, 臺灣) 測定。連續(xù)處理10 d后，采集莖稈用于表皮蠟質(zhì)提取、電鏡掃描分析及基因表達(dá)檢測。每種材料不同處理各設(shè)3 個重復(fù)。

1.3 表皮蠟質(zhì)形態(tài)結(jié)構(gòu)觀察

采用掃描電子顯微鏡(S3000-N，Hitachi)觀察蠟質(zhì)晶體結(jié)構(gòu)。將植株莖稈分成1 cm長小段，間隔貼于電鏡載物臺上，進(jìn)行離子濺射鍍金膜，鍍金后材料進(jìn)行電鏡掃描觀察、拍照。

1.4 表皮蠟質(zhì)含量和組分分析

利用氣相色譜-質(zhì)譜聯(lián)用儀(GCMS-QP2010 plus)測定分析蠟質(zhì)組成與含量。采集植株莖桿放入含有10 mL氯仿的試管中浸提30 s，以提取表面蠟質(zhì)，樣品中加入C16烷作為內(nèi)部標(biāo)準(zhǔn)。氮吹儀吹干浸提液，在100 ℃下用80 μL BSTFA衍生20 min，再經(jīng)氮氣吹干后，溶于200 μL正己烷中，進(jìn)行色譜-質(zhì)譜分析。程序升溫方式：初溫80 ℃，保持2 min；每分鐘15 ℃升溫至260 ℃，保持10 min。然后每分鐘5 ℃升溫至320 ℃，保持5 min。基于FID峰值量化蠟質(zhì)，根據(jù)內(nèi)部標(biāo)準(zhǔn)物(C16烷)的濃度計算各蠟質(zhì)組分含量。蠟質(zhì)含量用單位面積上的微克數(shù)(μg/cm2)進(jìn)行表示。莖稈面積測定采用數(shù)字化掃描儀(EPSON V750)和WinFOLIA專業(yè)葉片圖像分析系統(tǒng)(Regent Instrument Inc, Canada)進(jìn)行分析并記錄。

1.5 qRT-PCR分析

1.5.1 樣品RNA的提取與cDNA合成

采用Trizol試劑盒(invitrogen)分別提取對照與UV-B處理野生型擬南芥莖稈總RNA，DNase I(寶生物工程有限公司)消化去除基因組DNA。樣品mRNA的反轉(zhuǎn)錄依照北京全式金生物技術(shù)有限公司的TransScript First-Strand cDNA Synthesis SuperMix反轉(zhuǎn)錄試劑盒進(jìn)行。

1.5.2 qPCR 分析

qPCR反應(yīng)在Mx3000P熒光定量PCR儀(StrataGene, USA)上進(jìn)行。反應(yīng)體系按照iTaq Universal SYBR Green Supermix試劑盒(BIO-RAD)說明配制，設(shè)3 次重復(fù)。待分析的蠟質(zhì)基因有：參與烷類物質(zhì)合成的CER1、參與醛類物質(zhì)合成的CER3、催化VLCFAs還原生成一級醇的脂肪酰-CoA 還原酶基因CER4、催化VLCFAs合成的第一步縮合反應(yīng)的β-酮脂酰-COA合酶KCS1、控制表皮蠟質(zhì)積累的轉(zhuǎn)錄因子WIN1，Actin7為內(nèi)參基因。本實驗所使用引物序列如表2所示。數(shù)據(jù)收集和處理在MXPro v4.10軟件(StrataGene, USA)中進(jìn)行。UV-B脅迫下蠟質(zhì)基因表達(dá)水平的變化以相對表達(dá)量表示，即UV-B脅迫下蠟質(zhì)基因的表達(dá)量與對照植株蠟質(zhì)基因表達(dá)量的比值。

表2 qPCR相關(guān)引物Table 2 Sequences of the primers used in qPCR

1.6 數(shù)據(jù)分析

采用SPSS13.0軟件分析UV-B處理對擬南芥莖表皮蠟質(zhì)含量、蠟質(zhì)基因表達(dá)量的影響。顯著水平為P<0.05(LSD檢驗)。

2 結(jié)果與分析

2.1 UV-B輻射增強對擬南芥蠟質(zhì)晶體結(jié)構(gòu)的影響

野生型擬南芥莖蠟質(zhì)結(jié)構(gòu)主要由垂直柱狀結(jié)構(gòu)與水平片狀結(jié)構(gòu)組成，此外還存在少量桿狀、管狀、和傘狀結(jié)構(gòu)。與野生型相比較，各蠟質(zhì)突變體總體上表現(xiàn)出蠟質(zhì)晶體分布密度減少，晶體結(jié)構(gòu)的形狀與大小發(fā)生改變。UV-B輻射處理后，擬南芥各材料表現(xiàn)出不同程度的莖干與蓮座葉枯黃現(xiàn)象，其中突變體材料受UV-B輻射影響普遍較野生型嚴(yán)重。掃描電鏡顯示(圖1,圖2)，CER1在受到UV-B輻射處理后，松針狀晶體結(jié)構(gòu)減少并顯著變短。CER3、CER10與KCS1在UV-B輻射處理后柱狀、桿狀晶體結(jié)構(gòu)顯著減少；CER10局部出現(xiàn)邊界不清的“無定型”膜狀結(jié)構(gòu)覆蓋莖表面；而KCS1則出現(xiàn)許多水平無規(guī)則片層結(jié)構(gòu)。CER20蠟質(zhì)晶體體積減小，但數(shù)量增加。UV-B輻射處理對一級醇減少突變體CER4結(jié)構(gòu)無顯著影響，處理前后均以垂直片狀結(jié)構(gòu)為主。CER6在UV-B輻射處理后蠟質(zhì)晶體形狀發(fā)生變化，一些小型球狀蠟質(zhì)晶體出現(xiàn)。UV-B輻射處理后，野生型擬南芥局部區(qū)域積累了大量水平桿狀、管狀結(jié)構(gòu)。

圖1 增強UV-B輻射下擬南芥野生型(WT)和蠟質(zhì)突變體(CER1、CER3、CER4)莖表皮蠟質(zhì)晶體結(jié)構(gòu)的變化(10 μm)Fig.1 Effects of enhanced UV-B radiation on crystalloid structures of epicuticular wax on stem in Arabidopsis thaliana wild type (WT) and mutants (CER1, CER3, CER4)

圖2 增強UV-B輻射下擬南芥突變體(CER6、CER10、CER20、KCS1)莖表皮蠟質(zhì)晶體結(jié)構(gòu)的變化(10 μm)Fig.2 Effects of enhanced UV-B radiation on crystalloid structures of cuticular wax on stem in Arabidopsis thaliana mutants(CER6, CER10, CER20, KCS1)

圖3 增強的UV-B輻射對擬南芥野生型和蠟質(zhì)突變體莖表皮蠟質(zhì)含量的影響Fig.3 Effects of enhanced UV-B radiation on contents of wax constituents and total wax in stems of Arabidopsis wild type and mutants

2.2 UV-B輻射增強對擬南芥表皮蠟質(zhì)組分含量的影響

擬南芥各材料經(jīng)UV-B處理(圖3)，一級醇含量顯著上升(CER6、CER10增加不顯著)，醛類與酸類含量增加或無顯著變化(KCS1酸類含量顯著減少)，次級醇、酮含量下降或無顯著變化，烷及蠟總量在不同材料中變化不一致。野生型在UV-B處理后一級醇、酸、醛含量顯著上升，烷、次級醇及酮含量顯著下降，蠟質(zhì)總量無顯著變化(圖3)。

2.3 蠟質(zhì)合成相關(guān)基因表達(dá)對UV-B輻射的響應(yīng)

本試驗對蠟質(zhì)前體VLCFAs合成基因KCS1、催化生成一級醇的脂肪酰-CoA還原酶基因CER4、醛類合成相關(guān)基因CER3、烷類合成基因CER1、及表皮蠟質(zhì)過量表達(dá)轉(zhuǎn)錄因子WIN1對UV-B輻射的響應(yīng)進(jìn)行了調(diào)查。結(jié)果表明，這5個蠟質(zhì)基因在野生型擬南芥中均有表達(dá)。在增強的UV-B輻射下，蠟質(zhì)相關(guān)基因CER3、CER4、KCS1表達(dá)量上調(diào)，其中CER4表達(dá)量是對照植株的13.64倍。而UV-B處理植株的CER1與WIN1表達(dá)量下調(diào)，分別是對照植株的0.09倍和0.01倍(圖4)。

圖4 增強的UV-B輻射對野生型擬南芥相關(guān)蠟質(zhì)基因表達(dá)的影響Fig.4 Effects of enhanced UV-B radiation on the transcript of wax-related gene in Arabidopsis wild type

3 討論

強紫外線輻射下，植物采取一系列防御措施以適應(yīng)環(huán)境條件的改變，如減少葉面積、節(jié)間長度及生物量[19]；葉片表面玻璃化[20]；合成大量類黃酮類物質(zhì)以提高對紫外線的吸收能力等[21- 22]。植物表皮蠟質(zhì)在減少紫外線輻射傷害方面也發(fā)揮著積極作用，如玉米蠟質(zhì)缺失突變體出現(xiàn)了比野生型更多的卷葉及DNA損傷現(xiàn)象[6,23]。各擬南芥材料經(jīng)UV-B處理后表現(xiàn)出不同程度的莖干與蓮座葉枯黃現(xiàn)象，其中蠟質(zhì)突變體材料枯黃、萎蔫程度大于野生型擬南芥。

前人研究報道，葉表皮蠟質(zhì)晶體結(jié)構(gòu)不同，其葉片對光的反射率也不盡相同，如水平片狀蠟質(zhì)結(jié)構(gòu)比垂直片狀結(jié)構(gòu)及光滑表面有更強的反射率[24]。本實驗中UV-B輻射處理除了對CER4與CER20蠟質(zhì)晶體結(jié)構(gòu)無顯著影響外，其余突變體材料蠟質(zhì)晶體結(jié)構(gòu)均發(fā)生了一定的變化。例如，UV-B輻射處理后，表皮蠟質(zhì)松針狀(CER1)、柱狀、桿狀(CER3、CER10與KCS1)晶體結(jié)構(gòu)顯著減少；CER10與KCS1不同程度出現(xiàn)無規(guī)則膜狀、片狀結(jié)構(gòu)覆蓋在莖表面等。野生型擬南芥蠟質(zhì)結(jié)構(gòu)類型雖然無明顯變化，但在部分區(qū)域積累了大量水平桿狀、管狀結(jié)構(gòu)，增加了蠟質(zhì)層厚度。紫外線輻射下，植物蠟質(zhì)層厚度是影響紫外線輻射到達(dá)葉肉細(xì)胞的因素之一[5]。UV-B輻射誘導(dǎo)的擬南芥蠟質(zhì)晶體結(jié)構(gòu)變化反映了植物的一種主動調(diào)節(jié)適應(yīng)過程。

蠟質(zhì)總量對UV-B輻射的響應(yīng)在不同植物中是不一樣的。UV-B處理條件下大麥(Hordeumvulgare)、黃瓜(Cucumissativus)等蠟質(zhì)含量增加[25]，云杉(Piceaasperata)蠟質(zhì)含量變化不顯著[10]，煙草(Nicotianatabacum)蠟質(zhì)含量降低[26]。UV-B處理后的不同材料間蠟質(zhì)總量總體上呈現(xiàn)出不變或增加趨勢(CER10蠟質(zhì)總量在UV-B處理后下降)。蠟質(zhì)組分中一級醇含量在UV-B處理后普遍上升，酮和次級醇含量普遍下降，暗示了擬南芥植株可通過改變特定蠟質(zhì)組分的分泌量來響應(yīng)UV-B脅迫。在增強的UV-B輻射下，挪威云杉葉表蠟質(zhì)中二十九烷含量顯著增加，烷基酯含量顯著下降[10]；黃瓜子葉的主要蠟質(zhì)組分——烷和一級醇的種類和含量發(fā)生改變[11]。

擬南芥角質(zhì)層蠟生物合成主要依靠蠟質(zhì)前體VLCFAs通過一級醇分支途徑與烷合成分支途徑進(jìn)行[12]。野生型擬南芥qRT-PCR結(jié)果表明，CER3、CER4、KCS1在UV-B輻射處理后表達(dá)量上調(diào)，增強的UV-B輻射誘導(dǎo)了CER4基因的大量表達(dá)。這3個基因的表達(dá)上調(diào)解釋了蠟質(zhì)組分中一級醇、酸和醛含量在受到UV-B脅迫后的顯著增加。另外一方面，CER1與WIN1在UV-B輻射處理后表達(dá)量下調(diào)。CER1催化長鏈醛到烷的轉(zhuǎn)化，其表達(dá)量的降低可能會導(dǎo)致烷合成下游分支途徑相關(guān)產(chǎn)物的減少及醛的積累。野生型擬南芥在受到UV-B脅迫后烷類、次級醇及酮類的顯著下降證明了這一點。WIN1作為表皮蠟質(zhì)過量表達(dá)時的轉(zhuǎn)錄因子，其表達(dá)量的降低將影響蠟質(zhì)總量的積累。但在本實驗中，野生型擬南芥蠟質(zhì)總量無顯著變化，說明蠟質(zhì)數(shù)量的積累受蠟質(zhì)合成途徑中上、下游相關(guān)蠟質(zhì)基因的共同作用。綜合分析蠟質(zhì)組分與基因表達(dá)結(jié)果表明，增強UV-B輻射使蠟質(zhì)前體從烷合成分支途徑更多地轉(zhuǎn)向了一級醇分支途徑。

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Effect of enhanced ultraviolet-B radiation on epicuticular wax inArabidopsisthaliana

NI Yu, SONG Chao, LI Jiana*

CollegeofAgronomyandBiotechnology,SouthwestUniversity,EngineeringResearchCenterofSouthUplandAgriculture,MinistryofEducation,Chongqing400716,China

Due to the depletion of stratospheric ozone (O3), the quantity of UV-B radiation reached the earth′s surface has increased significantly. Enhanced UV-B radiation influenced plant growth, development and productivity by damaging DNA, RNA, and proteins. Plants are thought to employ a variety of UV-B protective mechanisms, including accumulation of a range of secondary metabolites and adaptive changes in plant morphological features. As the outer surface of aerial plant tissues, the epicuticular wax is believed to play an important role in protecting plants against ultraviolet-B radiation. Epicuticular wax was composed primarily of saturated free fatty acids, aldehydes, alkanes, primary alcohols, secondary alcohols, ketones, and wax esters.Arabidopsis, as a model system to study the genetics and the molecular biology, has the typical structure and composition of cuticle, and it should be a model system to study the ecology of epicuticular waxes. However, little is known as to the responsees of different wax constituents to enhanced UV-B radiation, and the role of epicuticular waxes in providing protection forArabidopsisfrom UV-B has not been examined either. Therefore, in the current study, seven wax mutants (CER1,CER3,CER4,CER6,CER10,CER20 andKCS1) and wild type ofA.thalianawere selected to analyze the responses of epicuticular wax in crystalloid structure, content and constituents, and wax related gene to UV-B radiation (50 μW/cm2) . The plants accepted UV-B radiation 2 h every day for consecutive 10 days. The results showed that enhanced UV-B radiation altered the crystalloid structure of epicuticular wax onArabidopsisstems. Under the enhanced UV-B radiation, amounts of pine needle-shaped crystalloids (CER1), columnar-shaped crystalloids and rods (CER3、CER10 andKCS1) decreased significantly. Some small globular-shaped crystalloids appeared on the surface ofCER6 mutant. Some irregular plate- and membrane-shaped structures covered the surface ofKCS1 andCER10. No significant change of crystalloid types was observed on the stem of theArabidopsiswild type; however, many horizontal rod and tube crystalloids accumulated in specific zones, which increased the thickness of cuticle. Enhanced UV-B radiation also altered the secretion quantities of wax constituents. Under UV-B radiation, the contents of primary alcohols, fatty acids and aldhydes inArabidopsiswild type increased significantly, the contents of alkanes, secondary alcohols and ketones decreased significantly, while the contents of total wax changed insignificantly. Under UV-B radiation, the increase of primary alcohols and the decrease of secondary alcohols and ketones were universal inArabidopsismutants. Enhanced UV-B radiation upregulated the expression ofCER3,CER4 andKCS1, which promoting the accumulation of primary alcohols, fatty acids and aldehydes. The decreased expression ofCER1 under UV-B radiation might lead to the reduction of products from alkane-synthesizing branch of the pathway, including alkanes, secondary alcohols and ketones. The decreased expression ofWIN1 under UV-B radiation had no effect on the content of total wax, implying that the accumulation of total wax was a result of comprehensive effects of multi genes involved in wax synthesis pathway. Enhanced UV-B radiation shunted wax precursors away from the alkane-synthesizing branch to primary alcohol branch of the pathway.

Arabidopsisthaliana; epicuticular wax; gene; UV-B radiation; wax mutants

國家自然科學(xué)基金(31000122, 31270450); 重慶市自然科學(xué)基金(cstc2012jjA80022); 教育部作物資源利用創(chuàng)新引智基地(B12006)

2013- 05- 07;

日期:2014- 04- 17

10.5846/stxb201305070972

*通訊作者Corresponding author.E-mail: ljn1950@swu.edu.cn

倪郁，宋超，李加納.UV-B輻射增強對擬南芥表皮蠟質(zhì)的影響.生態(tài)學(xué)報,2015,35(5):1505- 1512.

Ni Y, Song C, Li J N.Effect of enhanced ultraviolet-B radiation on epicuticular wax inArabidopsisthaliana.ActaEcologicaSinica,2015,35(5):1505- 1512.