余恩,蔡蕓菲,趙茹冰,陳進紅,祝水金(浙江大學農(nóng)業(yè)與生物技術(shù)學院農(nóng)學系/浙江省作物種質(zhì)資源重點實驗室,杭州310058)

2個轉(zhuǎn)基因抗蟲雜交棉Bt蛋白含量的時空表達特性研究

余恩,蔡蕓菲,趙茹冰,陳進紅,祝水金*
(浙江大學農(nóng)業(yè)與生物技術(shù)學院農(nóng)學系/浙江省作物種質(zhì)資源重點實驗室,杭州310058)

摘要采用雙抗夾心酶聯(lián)免疫(enzyme linked immuno-sorbent assay,ELISA)的方法,分析了2個轉(zhuǎn)蘇云金桿菌(Bacillus thuringiensis,Bt)基因雜交棉(浙大13-1和浙大13-2)以及轉(zhuǎn)Bt基因純系親本不同生育期和不同器官的Bt蛋白表達規(guī)律.結(jié)果表明,Bt基因在所檢測的轉(zhuǎn)基因抗蟲雜交棉品種和轉(zhuǎn)基因純系親本各器官中均有表達,但不同器官Bt蛋白含量明顯不同,品種之間各器官的Bt蛋白含量也有明顯差異.2個轉(zhuǎn)基因抗蟲雜交棉品種苗期以三葉期的子葉最高,苗期葉片次之;苗期根系和莖稈中Bt蛋白表達呈拋物線形,分別在第33天和第40天達到峰值,其中根系表達量高于莖稈.葉片中的Bt蛋白含量隨著棉花生長發(fā)育進程而降低,盛花期Bt蛋白含量最低,之后又有所增強,整個生育期呈“V”字形規(guī)律.轉(zhuǎn)基因抗蟲雜交棉品種盛花期棉株各器官中,花藥的Bt蛋白含量顯著高于同期其他器官.2個雜交棉品種之間各器官的Bt蛋白含量的趨勢基本相同,但浙大13-1器官中的Bt蛋白含量大多高于浙大13-2.親本各器官中的Bt蛋白含量高于其雜交棉品種,但植株生長后期的葉片和花藥中的Bt蛋白低于其雜交棉品種,二者有明顯差異.

關(guān)鍵詞棉花;抗蟲雜交棉;Bt蛋白;時空表達;酶聯(lián)免疫

浙江大學學報(農(nóng)業(yè)與生命科學版) 42(1):17～22,2016

Journal of Zhejiang University(Agric.&Life Sci.)

http://www.journals.zju.edu.cn/agr

E-mail:zdxbnsb@zju.edu.cn

第一作者聯(lián)系方式:余恩(http://orcid.org/0000-0002-3827-8951),E-mail:yuen@zju.edu.cn

URL:http://www.cnki.net/kcms/detail/33.1247.S.20151221.1725.002.html

Studies on temporal-spatial expression characters of Bt protein in two transgenic hybrid cotton.Journal of Zhejiang University(Agric.&Life Sci.),2016,42(1):17-22

YU En,CAI Yunfei,ZHAO Rubing,CHEN Jinhong,ZHU Shuijin*(Department of Agronomy,College of Agriculture and Biotechnology,Zhejiang University/Zhejiang Key Laboratory of Crop Germplasm,Hangzhou 310058,China)

Summary Cotton bollworm,Helicoberpa armigera,was one of the most harmful pests in cotton-production areas all over the world.The transgenic insect resistant cotton with Bt gene from Bacillus thuringiensis was developed to control this pest in 1987.It was increased quickly in recent years,accounting for more than 93%of all cotton planted in China,and huge economic,social,and ecological benefits were obtained.

The levels of Bt protein in cotton organs can directly affect the insect resistance of cotton plants.Many reports showed that the Bt cotton expressed the Bt protein at a high level in early season which provided good control of the second generation of cotton bollworm,but it was decreased in middle and late stages which lead to the decrease of the controlling efficacy to bollworm.However,few reports had been found about the levels of Bt protein in differentorgans and tissues during the whole growth stages of the transgenic hybrid cotton.Whole season levels of Bt protein in two transgenic hybrid cotton,Zheda 13-1 and Zheda 13-2,and their pure line parent,Zayou 2012,were investigated in 2014 in Hangzhou City,Zhejiang Province,located in southeast coastal region of China.Enzyme linked immuno-sorbent assay(ELISA)method was used to quantify Bt protein.

The results showed that the Bt protein could be detected in all organs and tissues in the two hybrid cultivars and their parent,however,its levels changed significantly among the different organs from time to time.During the seedling stage,cotyledon was the highest organ,following by the first true leaf.The Bt protein level in root and stem was increased at the beginning,reached the peak value on 33 and 40 days after sowing,respectively for two cultivars,then decreased steadily afterward.In general,the Bt protein in root was much higher than that in stem.In the leaves of the two hybrid cottons,the upper third leaf,Bt protein was high in the early stage,declined in midseason but rebounded in late-season.While for their parent,it was high in early season,then declined steadily in middle and latter season.The difference might relate to the growth habit of the hybrid cotton which kept growing in late-season for relatively long time.So it is suggested that the pesticide control might be necessary for the hybrid cotton in the mid-season of cotton growth.In addition,the study showed that Bt protein contents in the anther and stamens were much higher than those in leaves,squares,bolls,ovules,and petals.While for their parent,it was much lower in the anther and stamens than that in other organs and tissues.It should be studied further whether or not this phenomenon was resulted from the heterosis of the hybrid cotton,the high concentration of Bt protein in the anther and stamens was beneficial to the cotton bollworm control of the hybrid cotton as the cotton bollworms were more interested in the cotton reproductive organs.

Key words cotton;hybrid cotton cultivars;Bt protein;temporal-spatial expression;enzyme linked immunosorbent assay(ELISA)

棉花是一種重要的經(jīng)濟作物,棉纖維是重要的紡織和工業(yè)原料.在棉花生長過程中受棉鈴蟲等病蟲害的為害,造成棉花生產(chǎn)大幅度減產(chǎn).轉(zhuǎn)基因抗蟲棉的問世對于棉花生產(chǎn)中蟲害控制具有重要意義.1987年世界上首例蘇云金桿菌(Bacillus thuringiensis,Bt)殺蟲蛋白基因轉(zhuǎn)入棉花,隨后進行大規(guī)模商業(yè)應(yīng)用[1].1992年,我國的國產(chǎn)抗蟲棉投入生產(chǎn)應(yīng)用,之后又培育出雙價基因抗蟲棉并進行商業(yè)化應(yīng)用.2014年我國的轉(zhuǎn)基因抗蟲棉種植面積占總棉花面積的93%,并取得了巨大的經(jīng)濟、社會效益和生態(tài)效益.轉(zhuǎn)基因抗蟲棉植株器官中的Bt蛋白含量的高低直接影響其抗蟲性.為此,國內(nèi)外學者采用多種方法研究了轉(zhuǎn)基因抗蟲棉植株Bt基因表達和蛋白含量與抗蟲性之間的關(guān)系,發(fā)現(xiàn)轉(zhuǎn)Bt基因抗蟲棉前期抗蟲性強,后期抗蟲性弱等特點[2-5].轉(zhuǎn)基因抗蟲雜交棉大多由轉(zhuǎn)基因抗蟲純系與普通棉親本之間的品種間雜交F1,因協(xié)調(diào)了抗蟲性與產(chǎn)量、品質(zhì)和抗病性等之間的關(guān)系,深受棉農(nóng)的歡迎,在我國長江流域棉區(qū)占90%以上[6].然而,有關(guān)轉(zhuǎn)Bt基因抗蟲雜交棉不同生育期以及不同器官之間的Bt蛋白表達特性的研究相對較少.本文采用雙抗夾心酶聯(lián)免疫(enzyme linked immuno-sorbent assay,ELISA)的方法,系統(tǒng)的測定了2個轉(zhuǎn)Bt基因雜交棉(浙大13-1和浙大13-2)及其親本全生育期不同器官的Bt蛋白含量,旨在為轉(zhuǎn)基因抗蟲雜交棉的生產(chǎn)應(yīng)用,特別是病蟲害防治提供理論依據(jù).

1　材料與方法

1.1試驗材料

供試的2個轉(zhuǎn)Bt基因抗蟲棉雜交品種為浙大13-1和13-2,均由浙江大學農(nóng)業(yè)與生物技術(shù)學院選育而成.其中浙大13-1為高產(chǎn)型轉(zhuǎn)基因抗蟲雜交棉品種,浙大13-2為大鈴優(yōu)質(zhì)型抗蟲雜交棉品種,兩者的抗蟲親本均為同一轉(zhuǎn)Bt基因純系品種.試驗于2014年在浙江大學紫金港實驗農(nóng)場進行.試驗采用3行區(qū)、3次重復(fù),隨機區(qū)組排列,小區(qū)種植面積30 m2.4月30日播種,田間管理如同大田生產(chǎn).此外,將各材料種于控溫控濕的智能溫室,重復(fù)3次.控制溫度為白天/黑夜:28℃/18℃,濕度70%.

1.2取樣時間

每個小區(qū)在棉花生長的苗期、蕾期、初花期、盛花期、鈴期和吐絮期分別取功能葉(主莖平展倒三葉)、老葉(花鈴期以后主莖平展倒7～9葉)、幼蕾(直徑5～7 mm)、鈴(直徑2～3 cm)、根,以及開花當日的花瓣、花藥、雌蕊和子房.每個樣品3次重復(fù),樣品立即放入冰盒,20 min內(nèi)存放于-80℃冰箱保存?zhèn)溆?溫室種植的材料自真葉平展后,每7 d分別取根莖葉存放于-80℃冰箱保存?zhèn)溆?

1.3Bt蛋白含量ELISA檢測

使用Enviro Logix公司Cry1Ab/Cry1Ac的抗體夾心ELISA定量檢測試劑盒(AP003,檢測下限0.3 ng/g)進行Bt蛋白含量測定.所有樣品的吸光度值均由酶標儀(Biotek Synergy H1)讀取,波長設(shè)定為450 nm.每次檢測均用進口Cry1A蛋白標樣制作標準曲線,其相關(guān)系數(shù)為0.999 0.根據(jù)標準曲線求Bt蛋白含量.

1.4數(shù)據(jù)處理

用SAS 9.1軟件進行方差分析,多重比較采用LSD法.

2　結(jié)果與分析

2.1不同時期功能葉Bt蛋白含量動態(tài)變化

棉花播種后第35、60、77、95、112、129和144 天,即棉花生長的三葉期、七葉期、蕾期、初花期、盛花期、鈴期和吐絮期分別取主莖平展倒三葉測定其Bt蛋白含量.結(jié)果表明,轉(zhuǎn)基因抗蟲雜交棉浙大13-1和浙大13-2功能葉的Bt蛋白含量均隨著生長發(fā)育的推進逐漸降低,盛花期功能葉的Bt蛋白含量最低,之后又逐漸升高,整個生育期呈“V”字形表達(圖1).2個雜交棉品種材料均以苗期葉片Bt蛋白的表達量最高,盛花期最低.顯著性測驗表明,從苗期到盛花期浙大13-2品種每個階段Bt蛋白的表達量都顯著性減少,盛花期后又呈顯著性增加的趨勢.浙大13-1品種與之相似,只是前期Bt蛋白減少量個別階段沒有達到顯著水平.而其親本雜優(yōu)2012(轉(zhuǎn)Bt基因純系品種)功能葉片中的Bt蛋白含量均隨著棉花生長發(fā)育而下降,不呈“V”形曲線,且每一時期的差異基本上達到顯著水平.

從圖1可見,浙大13-1三葉期的Bt蛋白含量為468.85 ng/g,親本雜優(yōu)2012為687.04 ng/g,說明親本Bt純合基因的表達量較浙大13-1 Bt雜合基因的表達量高.浙大13-2三葉期Bt蛋白表達量為610.04 ng/g,顯著地高于浙大13-1,因2個雜交棉之間的抗蟲基因來源相同,他們之間的差異只能說明遺傳背景或另一親本的基因?qū)t基因的表達存在互作.因此,在雜交棉親本選配時不僅要考慮親本的抗蟲性,同時還要考慮到非抗蟲親本對其的影響.

2.2植株不同器官的Bt蛋白含量變化

苗期和盛花期對2個轉(zhuǎn)基因抗蟲雜交棉及其親本不同葉片和花期器官的Bt蛋白進行測定.結(jié)果(表1)表明,棉株不同葉片中Bt蛋白的含量差異較大,其中以苗期(2片真葉展開時)子葉中Bt蛋白含量最高,盛花期不同葉片Bt蛋白含量總體呈主莖下部葉＞果枝葉＞上部功能葉＞苞葉的規(guī)律;花器官Bt蛋白含量從高到低依次為花藥＞雌蕊＞子房＞花瓣,其中兩抗蟲雜交棉品種花藥Bt蛋白含量顯著高于其他器官中Bt蛋白含量.在所有檢測的器官中,棉籽仁中Bt蛋白含量最高,高達2 000 ng/g,棉籽殼含量最低僅為50 ng/g左右.

從表1中還可以看出,浙大13-1主莖下部葉、果枝葉、花苞Bt蛋白的表達量高于浙大13-2,且主莖下部葉達到了顯著水平,而上部功能葉浙大13-2 較13-1高;浙大13-2其花器官、蕾、鈴殼器官的Bt蛋白表達量要高于浙大13-1.這可能是因為浙大13-2為大鈴優(yōu)質(zhì)型抗蟲雜交棉品種,生殖器官的代謝強度較浙大13-1高.浙大13-1各器官Bt蛋白含量與親本雜優(yōu)2012相比,總體呈偏低的趨勢,但花藥的Bt蛋白表達量高于親本,且達到了極顯著水平.浙大13-2與親本相比也表現(xiàn)出與浙大13-1類似的規(guī)律.在浙大13-1所有檢測的器官中,Bt蛋白含量從高到低依次為種仁＞子葉＞花藥＞根系＞莖稈＞主莖下部葉＞雌蕊＞果枝葉＞上部功能葉＞苞葉＞鈴殼＞蕾＞子房＞花瓣＞種殼.

2.3苗期根莖葉Bt蛋白含量的動態(tài)變化

為了進一步探究轉(zhuǎn)基因抗蟲雜交棉及其親本之間根莖葉生長發(fā)育過程中的Bt含量動態(tài)變化規(guī)律,將各材料種于溫室內(nèi),每隔7 d取根莖葉測定其Bt蛋白含量,結(jié)果見圖2.

由圖2可以看出,2個轉(zhuǎn)基因雜交棉根(圖2A)、莖(圖2B)中Bt蛋白的動態(tài)變化均呈“拋物線形”的規(guī)律,根系和莖稈中Bt蛋白的表達量分別在播種后的第33天和第40天達到峰值,之后又逐漸降低;而親本材料雜優(yōu)2012苗期根、莖中Bt蛋白的表達規(guī)律性不強.兩轉(zhuǎn)基因雜交棉及其親本苗期葉片(圖2C)中Bt蛋白含量的變化規(guī)律基本一致,均呈現(xiàn)逐漸減少的趨勢.苗期棉株各器官中,子葉Bt蛋白含量最高,根部Bt蛋白含量總體高于莖稈.2個雜交棉品種之間,浙大13-1根、莖、葉中Bt蛋白的含量均高于浙大13-2,這可能與2個雜交棉品種的特點有一定的關(guān)系.

結(jié)合圖1結(jié)果,溫室與大田生產(chǎn)種植的材料相同,但棉株各器官Bt蛋白含量差異性較大,說明環(huán)境條件對Bt蛋白表達有較大的影響.

3　討論

本試驗通過溫室培養(yǎng)和大田栽培研究發(fā)現(xiàn),轉(zhuǎn)基因抗蟲雜交棉浙大13-1和浙大13-2主莖功能葉整個生育期Bt蛋白的表達量前期較多,盛花期最少,而后期又有所增多,總體呈“V”字形分布,與崔金杰等[7]、張永軍等[8]、WANG等[9]研究結(jié)果基本一致,與沈平等[10]部分一致.邢朝柱等[11]和李汝忠等[12]的研究結(jié)果是在轉(zhuǎn)Bt基因棉整個生育期上部葉片Bt蛋白含量呈倒“V”字形,在花期或蕾期Bt蛋白含量最高.可見,參試品種、生長環(huán)境、栽培管理措施等對于Bt蛋白含量有顯著的影響.此外,本試驗中親本(雜優(yōu)2012)整個生育期葉片Bt蛋白含量逐漸降低,沒有呈現(xiàn)“V”字形表達規(guī)律.由于轉(zhuǎn)Bt基因棉使用的是Ca MV35S啟動子,只要有生長就有外源基因表達,而雜交棉具有雜種優(yōu)勢,后期開花后仍有較強的生長勢和營養(yǎng)生長.所以,后期Bt蛋白的表達量增加,而雜優(yōu)2012為純系品種,后期長勢較轉(zhuǎn)基因雜交棉弱,整個生育期葉片中Bt蛋白呈逐漸降低的規(guī)律.因此,在轉(zhuǎn)基因雜交棉種植生產(chǎn)實踐中,可結(jié)合Bt蛋白的表達規(guī)律,在棉花盛花期更應(yīng)注意棉鈴蟲的發(fā)生情況,及時噴施農(nóng)藥進行防控.本試驗結(jié)果表明,轉(zhuǎn)基因抗蟲雜交棉品種植株的不同器官組織中Bt蛋白含量測定結(jié)果是:種仁＞花藥＞子葉＞根系＞莖稈＞主莖下部葉＞雌蕊＞果枝葉＞上部功能葉＞苞葉＞鈴殼＞蕾＞子房＞花瓣＞種殼,這與張桂芬等[13]研究結(jié)果基本相似;純系親本為:種仁＞子葉＞根系＞莖稈＞果枝葉＞主莖下部葉＞上部功能葉＞花藥＞雌蕊＞鈴殼＞苞葉＞蕾＞子房＞花瓣＞種殼.雜交棉品種與其純系親本之間各器官Bt蛋白含量的差異可能與雜交棉的雜種優(yōu)勢特點有關(guān).花鈴期是棉花生殖生長旺盛時期,此時大量營養(yǎng)物質(zhì)運輸?shù)交ā⑩彽绕鞴?而轉(zhuǎn)基因雜交棉與純系品種相比此時期果枝數(shù)多,花芽分化速度快[14],花器官代謝強度高于親本材料可能是導(dǎo)致其花藥中Bt蛋白顯著高于純系親本的原因之一.花器中花瓣中Bt蛋白含量最低,但花瓣中含有較多的棉酚,可增強Bt蛋白的殺蟲活性[15].花鈴期是棉花遭受棉鈴蟲危害最嚴重的時期,而棉鈴蟲幼蟲的取食特性偏好于花器官、幼葉.轉(zhuǎn)基因抗蟲雜交棉(浙大13-1和浙大13-2)此時花藥和雌蕊Bt蛋白的大量表達對于棉鈴蟲的控制更為有利.

相關(guān)研究發(fā)現(xiàn),室內(nèi)外不同氣候條件(溫度、濕度、降雨量)下生長的Bt棉葉片的抗蟲性有顯著差異[16].本試驗結(jié)果也表明大田與溫室棉株苗期Bt蛋白表達量存在較大的差異,其中轉(zhuǎn)基因抗蟲雜交棉的差異較純系親本大,說明雜交的Bt基因表達易受環(huán)境的影響.

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收稿日期(Received):2015-07-15;接受日期(Accepted):2015-09-05;網(wǎng)絡(luò)出版日期(Published online):2015-12-21

*通信作者(

Corresponding author):祝水金(http://orcid.org/0000-0001-6208-9630),E-mail:shjzhu@zju.edu.cn

基金項目:轉(zhuǎn)基因生物新品種培育重大專項(2014ZX08005-005).

DOI:10.3785/j.issn.1008-9209.2015.07.151

中圖分類號Q 946.1;S 562

文獻標志碼A