2个转基因抗虫杂交棉Bt蛋白含量的时空表达特性研究

余恩,蔡芸菲,赵茹冰,陈进红,祝水金(浙江大学农业与生物技术学院农学系/浙江省作物种质资源重点实验室,杭州310058)

2个转基因抗虫杂交棉Bt蛋白含量的时空表达特性研究

余恩,蔡芸菲,赵茹冰,陈进红,祝水金*
(浙江大学农业与生物技术学院农学系/浙江省作物种质资源重点实验室,杭州310058)

摘要采用双抗夹心酶联免疫(enzyme linked immuno-sorbent assay,ELISA)的方法,分析了2个转苏云金杆菌(Bacillus thuringiensis,Bt)基因杂交棉(浙大13-1和浙大13-2)以及转Bt基因纯系亲本不同生育期和不同器官的Bt蛋白表达规律.结果表明,Bt基因在所检测的转基因抗虫杂交棉品种和转基因纯系亲本各器官中均有表达,但不同器官Bt蛋白含量明显不同,品种之间各器官的Bt蛋白含量也有明显差异.2个转基因抗虫杂交棉品种苗期以三叶期的子叶最高,苗期叶片次之;苗期根系和茎秆中Bt蛋白表达呈抛物线形,分别在第33天和第40天达到峰值,其中根系表达量高于茎秆.叶片中的Bt蛋白含量随着棉花生长发育进程而降低,盛花期Bt蛋白含量最低,之后又有所增强,整个生育期呈“V”字形规律.转基因抗虫杂交棉品种盛花期棉株各器官中,花药的Bt蛋白含量显著高于同期其他器官.2个杂交棉品种之间各器官的Bt蛋白含量的趋势基本相同,但浙大13-1器官中的Bt蛋白含量大多高于浙大13-2.亲本各器官中的Bt蛋白含量高于其杂交棉品种,但植株生长后期的叶片和花药中的Bt蛋白低于其杂交棉品种,二者有明显差异.

关键词棉花;抗虫杂交棉;Bt蛋白;时空表达;酶联免疫

浙江大学学报(农业与生命科学版) 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

第一作者联系方式: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)

棉花是一种重要的经济作物,棉纤维是重要的纺织和工业原料.在棉花生长过程中受棉铃虫等病虫害的为害,造成棉花生产大幅度减产.转基因抗虫棉的问世对于棉花生产中虫害控制具有重要意义.1987年世界上首例苏云金杆菌(Bacillus thuringiensis,Bt)杀虫蛋白基因转入棉花,随后进行大规模商业应用[1].1992年,我国的国产抗虫棉投入生产应用,之后又培育出双价基因抗虫棉并进行商业化应用.2014年我国的转基因抗虫棉种植面积占总棉花面积的93%,并取得了巨大的经济、社会效益和生态效益.转基因抗虫棉植株器官中的Bt蛋白含量的高低直接影响其抗虫性.为此,国内外学者采用多种方法研究了转基因抗虫棉植株Bt基因表达和蛋白含量与抗虫性之间的关系,发现转Bt基因抗虫棉前期抗虫性强,后期抗虫性弱等特点[2-5].转基因抗虫杂交棉大多由转基因抗虫纯系与普通棉亲本之间的品种间杂交F1,因协调了抗虫性与产量、品质和抗病性等之间的关系,深受棉农的欢迎,在我国长江流域棉区占90%以上[6].然而,有关转Bt基因抗虫杂交棉不同生育期以及不同器官之间的Bt蛋白表达特性的研究相对较少.本文采用双抗夹心酶联免疫(enzyme linked immuno-sorbent assay,ELISA)的方法,系统的测定了2个转Bt基因杂交棉(浙大13-1和浙大13-2)及其亲本全生育期不同器官的Bt蛋白含量,旨在为转基因抗虫杂交棉的生产应用,特别是病虫害防治提供理论依据.

1　材料与方法

1.1试验材料

供试的2个转Bt基因抗虫棉杂交品种为浙大13-1和13-2,均由浙江大学农业与生物技术学院选育而成.其中浙大13-1为高产型转基因抗虫杂交棉品种,浙大13-2为大铃优质型抗虫杂交棉品种,两者的抗虫亲本均为同一转Bt基因纯系品种.试验于2014年在浙江大学紫金港实验农场进行.试验采用3行区、3次重复,随机区组排列,小区种植面积30 m2.4月30日播种,田间管理如同大田生产.此外,将各材料种于控温控湿的智能温室,重复3次.控制温度为白天/黑夜:28℃/18℃,湿度70%.

1.2取样时间

每个小区在棉花生长的苗期、蕾期、初花期、盛花期、铃期和吐絮期分别取功能叶(主茎平展倒三叶)、老叶(花铃期以后主茎平展倒7～9叶)、幼蕾(直径5～7 mm)、铃(直径2～3 cm)、根,以及开花当日的花瓣、花药、雌蕊和子房.每个样品3次重复,样品立即放入冰盒,20 min内存放于-80℃冰箱保存备用.温室种植的材料自真叶平展后,每7 d分别取根茎叶存放于-80℃冰箱保存备用.

1.3Bt蛋白含量ELISA检测

使用Enviro Logix公司Cry1Ab/Cry1Ac的抗体夹心ELISA定量检测试剂盒(AP003,检测下限0.3 ng/g)进行Bt蛋白含量测定.所有样品的吸光度值均由酶标仪(Biotek Synergy H1)读取,波长设定为450 nm.每次检测均用进口Cry1A蛋白标样制作标准曲线,其相关系数为0.999 0.根据标准曲线求Bt蛋白含量.

1.4数据处理

用SAS 9.1软件进行方差分析,多重比较采用LSD法.

2　结果与分析

2.1不同时期功能叶Bt蛋白含量动态变化

棉花播种后第35、60、77、95、112、129和144 天,即棉花生长的三叶期、七叶期、蕾期、初花期、盛花期、铃期和吐絮期分别取主茎平展倒三叶测定其Bt蛋白含量.结果表明,转基因抗虫杂交棉浙大13-1和浙大13-2功能叶的Bt蛋白含量均随着生长发育的推进逐渐降低,盛花期功能叶的Bt蛋白含量最低,之后又逐渐升高,整个生育期呈“V”字形表达(图1).2个杂交棉品种材料均以苗期叶片Bt蛋白的表达量最高,盛花期最低.显著性测验表明,从苗期到盛花期浙大13-2品种每个阶段Bt蛋白的表达量都显著性减少,盛花期后又呈显著性增加的趋势.浙大13-1品种与之相似,只是前期Bt蛋白减少量个别阶段没有达到显著水平.而其亲本杂优2012(转Bt基因纯系品种)功能叶片中的Bt蛋白含量均随着棉花生长发育而下降,不呈“V”形曲线,且每一时期的差异基本上达到显著水平.

从图1可见,浙大13-1三叶期的Bt蛋白含量为468.85 ng/g,亲本杂优2012为687.04 ng/g,说明亲本Bt纯合基因的表达量较浙大13-1 Bt杂合基因的表达量高.浙大13-2三叶期Bt蛋白表达量为610.04 ng/g,显著地高于浙大13-1,因2个杂交棉之间的抗虫基因来源相同,他们之间的差异只能说明遗传背景或另一亲本的基因对Bt基因的表达存在互作.因此,在杂交棉亲本选配时不仅要考虑亲本的抗虫性,同时还要考虑到非抗虫亲本对其的影响.

2.2植株不同器官的Bt蛋白含量变化

苗期和盛花期对2个转基因抗虫杂交棉及其亲本不同叶片和花期器官的Bt蛋白进行测定.结果(表1)表明,棉株不同叶片中Bt蛋白的含量差异较大,其中以苗期(2片真叶展开时)子叶中Bt蛋白含量最高,盛花期不同叶片Bt蛋白含量总体呈主茎下部叶＞果枝叶＞上部功能叶＞苞叶的规律;花器官Bt蛋白含量从高到低依次为花药＞雌蕊＞子房＞花瓣,其中两抗虫杂交棉品种花药Bt蛋白含量显著高于其他器官中Bt蛋白含量.在所有检测的器官中,棉籽仁中Bt蛋白含量最高,高达2 000 ng/g,棉籽壳含量最低仅为50 ng/g左右.

从表1中还可以看出,浙大13-1主茎下部叶、果枝叶、花苞Bt蛋白的表达量高于浙大13-2,且主茎下部叶达到了显著水平,而上部功能叶浙大13-2 较13-1高;浙大13-2其花器官、蕾、铃壳器官的Bt蛋白表达量要高于浙大13-1.这可能是因为浙大13-2为大铃优质型抗虫杂交棉品种,生殖器官的代谢强度较浙大13-1高.浙大13-1各器官Bt蛋白含量与亲本杂优2012相比,总体呈偏低的趋势,但花药的Bt蛋白表达量高于亲本,且达到了极显著水平.浙大13-2与亲本相比也表现出与浙大13-1类似的规律.在浙大13-1所有检测的器官中,Bt蛋白含量从高到低依次为种仁＞子叶＞花药＞根系＞茎秆＞主茎下部叶＞雌蕊＞果枝叶＞上部功能叶＞苞叶＞铃壳＞蕾＞子房＞花瓣＞种壳.

2.3苗期根茎叶Bt蛋白含量的动态变化

为了进一步探究转基因抗虫杂交棉及其亲本之间根茎叶生长发育过程中的Bt含量动态变化规律,将各材料种于温室内,每隔7 d取根茎叶测定其Bt蛋白含量,结果见图2.

由图2可以看出,2个转基因杂交棉根(图2A)、茎(图2B)中Bt蛋白的动态变化均呈“抛物线形”的规律,根系和茎秆中Bt蛋白的表达量分别在播种后的第33天和第40天达到峰值,之后又逐渐降低;而亲本材料杂优2012苗期根、茎中Bt蛋白的表达规律性不强.两转基因杂交棉及其亲本苗期叶片(图2C)中Bt蛋白含量的变化规律基本一致,均呈现逐渐减少的趋势.苗期棉株各器官中,子叶Bt蛋白含量最高,根部Bt蛋白含量总体高于茎秆.2个杂交棉品种之间,浙大13-1根、茎、叶中Bt蛋白的含量均高于浙大13-2,这可能与2个杂交棉品种的特点有一定的关系.

结合图1结果,温室与大田生产种植的材料相同,但棉株各器官Bt蛋白含量差异性较大,说明环境条件对Bt蛋白表达有较大的影响.

3　讨论

本试验通过温室培养和大田栽培研究发现,转基因抗虫杂交棉浙大13-1和浙大13-2主茎功能叶整个生育期Bt蛋白的表达量前期较多,盛花期最少,而后期又有所增多,总体呈“V”字形分布,与崔金杰等[7]、张永军等[8]、WANG等[9]研究结果基本一致,与沈平等[10]部分一致.邢朝柱等[11]和李汝忠等[12]的研究结果是在转Bt基因棉整个生育期上部叶片Bt蛋白含量呈倒“V”字形,在花期或蕾期Bt蛋白含量最高.可见,参试品种、生长环境、栽培管理措施等对于Bt蛋白含量有显著的影响.此外,本试验中亲本(杂优2012)整个生育期叶片Bt蛋白含量逐渐降低,没有呈现“V”字形表达规律.由于转Bt基因棉使用的是Ca MV35S启动子,只要有生长就有外源基因表达,而杂交棉具有杂种优势,后期开花后仍有较强的生长势和营养生长.所以,后期Bt蛋白的表达量增加,而杂优2012为纯系品种,后期长势较转基因杂交棉弱,整个生育期叶片中Bt蛋白呈逐渐降低的规律.因此,在转基因杂交棉种植生产实践中,可结合Bt蛋白的表达规律,在棉花盛花期更应注意棉铃虫的发生情况,及时喷施农药进行防控.本试验结果表明,转基因抗虫杂交棉品种植株的不同器官组织中Bt蛋白含量测定结果是:种仁＞花药＞子叶＞根系＞茎秆＞主茎下部叶＞雌蕊＞果枝叶＞上部功能叶＞苞叶＞铃壳＞蕾＞子房＞花瓣＞种壳,这与张桂芬等[13]研究结果基本相似;纯系亲本为:种仁＞子叶＞根系＞茎秆＞果枝叶＞主茎下部叶＞上部功能叶＞花药＞雌蕊＞铃壳＞苞叶＞蕾＞子房＞花瓣＞种壳.杂交棉品种与其纯系亲本之间各器官Bt蛋白含量的差异可能与杂交棉的杂种优势特点有关.花铃期是棉花生殖生长旺盛时期,此时大量营养物质运输到花、铃等器官,而转基因杂交棉与纯系品种相比此时期果枝数多,花芽分化速度快[14],花器官代谢强度高于亲本材料可能是导致其花药中Bt蛋白显著高于纯系亲本的原因之一.花器中花瓣中Bt蛋白含量最低,但花瓣中含有较多的棉酚,可增强Bt蛋白的杀虫活性[15].花铃期是棉花遭受棉铃虫危害最严重的时期,而棉铃虫幼虫的取食特性偏好于花器官、幼叶.转基因抗虫杂交棉(浙大13-1和浙大13-2)此时花药和雌蕊Bt蛋白的大量表达对于棉铃虫的控制更为有利.

相关研究发现,室内外不同气候条件(温度、湿度、降雨量)下生长的Bt棉叶片的抗虫性有显著差异[16].本试验结果也表明大田与温室棉株苗期Bt蛋白表达量存在较大的差异,其中转基因抗虫杂交棉的差异较纯系亲本大,说明杂交的Bt基因表达易受环境的影响.

参考文献(References):

[1]VAECK M,REYNAERTS A,HOFTE H,et al.Transgenic plants protected from insect attack.Nature,1987,328(6125):33-37.

[2]陈松,吴敬音,周宝良,等.转Bt基因棉Bt毒蛋白表达量的时空变化.棉花学报,2000,12(4):189-193.CHEN S,WU J Y,ZHOU B L,et al.On the temporal and spatial expressions of Bt toxin protein in Bt transgenic cotton.Cotton Science,2000,12(4):189-193.(in Chinese with English abstract)

[3]ROCHESTER I J.Effect of genotype,edaphic,environmental conditions,and agronomic practices on Cry1Ac protein expression in transgenic cotton.Journal of Cotton Science,2006,10:252-262.

[4]王保民,李召虎,李斌,等.转Bt抗虫棉各器官毒蛋白的含量及表达.农业生物技术学报,2002,10(3):215-219.WANG B M,LI Z H,LI B,et al.Content and expression of Bacillus thuringiensis insecticidal protein in transgenic cotton.Journal of Agricultural Biotechnology,2002,10 (3):215-219.(in Chinese with English abstract)

[5]王冬梅,李海强,丁瑞丰,等.新疆北部地区转Bt基因棉外源杀虫蛋白表达量时空动态研究.棉花学报,2012,24(1):18-26.WANG D M,LI H Q,DING R F,et al.Spatio-temporal expression of foreign Bt insecticidal protein in transgenic Bt cotton varieties in northern Xinjiang Province,China.Cotton Science,2012,24(1):18-26.(in Chinese with English abstract)

[6]秦志勇.发展杂交棉三系不育系的必要性和可能性.中国种业,2010(8):45-46.QIN Z Y.The necessity and possibility of developing hybrid GMS cotton,Chiana Seed Industry,2010(8):45-46.(in Chinese)

[7]崔金杰,夏敬源,马丽华,等.转双价基因抗虫棉对棉铃虫的抗虫性及时空动态.棉花学报,2002,14(6):323-329.CUI J J,XIA J Y,MA L H,et al.Studies on the efficacy dynamic of the transgenic Cry IAc plus Cp TI cotton (ZGK9712)to cotton bollworm.Cotton Science,2002,14 (6):323-329.(in Chinese with English abstract)

[8]张永军,吴孔明,郭予元.转Bt基因棉花杀虫蛋白含量的时空表达及对棉铃虫的毒杀效果.植物保护学报,2001,28(1):1-6.ZHANG Y J,WU K M,GUO Y Y.On the spatio-temporal expression of the contents of Bt insecticidal protein and the resistance of Bt transgenic cotton to cotton bollworm.Acta Phytophylacica Sinica,2001,28(1):1-6.(in Chinese with English abstract)

[9]WAN P,ZHANG Y J,WU K M,et al.Seasonal expression profiles of insecticidal protein and control efficacy against Helicoverpa armigera for Bt cotton in the Yangtze River valley of China.Journal of Economic Entomology,2005,98 (1):195-201.

[10]沈平,林克剑,张永军,等.转Bt基因棉不同品种杀虫蛋白季节性表达及其对棉铃虫的控制作用.棉花学报,2010,22(5):393-397.SHEN P,LIN K J,ZHANG Y J,et al.Seasonal expression of Bacillus thuringiensis insecticidal protein and the control to cotton bollworm in different varieties of transgenic cotton.Cotton Science,2010,22(5):393-397.(in Chinese with English abstract)

[11]邢朝柱,靖深蓉,崔学芬,等.转Bt基因棉杀虫蛋白含量时空分布及对棉铃虫产生抗性的影响.棉花学报,2001,13(1):11-15.XING C Z,JING S R,CUI X F,et al.The spatio-temporal distribution of Bt(Bacillus thuringiensis)insecticidal protein and the effect of transgenic Bt cotton on bollworm resistance.Cotton Science,2001,13(1):11-15.(in Chinese with English abstract)

[12]李汝忠,沈法富,王宗文,等.转Bt基因抗虫棉Bt基因表达的时空动态.山东农业科学,2002(2):7-9.LI R Z,SHEN F F,WANG Z W,et al.Temporal and spatial dynamics of Bt gene expression in Bt transgenic cotton.Shandong Agricultural Sciences,2002(2):7-9.(in Chinese with English abstract)

[13]张桂芬,万方浩,郭建英,等.Bt毒蛋白在转Bt基因棉中的表达及其在害虫—天敌间的转移.昆虫学报,2004,47(3):334-341.ZHANG G F,WAN F H,GUO J Y,et al.Expression of Bt toxin in transgenic Bt cotton and its transmission through pests Helicoverpa armigera and Aphis gossypii to natural enemy Propylaea japonica in cotton plots.Acta Entomologica Sinica,2004,47(3):334-341.(in Chinese with English abstract)

[14]陈德华,吴云康,段海,等.转基因抗虫杂交棉杂交种生长规律及其生理特性的研究.中国棉花,1997,24(6):15-17.CHEN D H,WU Y K,DUAN H,et al.Research on growth regulation and physiological features of transgenic hybrid cotton.China Cotton,1997,24(6):15-17.(in Chinese)

[15]OLSEN K M,DALY J C,TANNER G J.The effect of cotton condensed tannin on the efficacy of the Cry IAcδendotoxin of Bacillus thuringiensis//Australian Cotton Growers Research Association.Proceedings of the 9th Australian Cotton Conference,Wee Waa,NSW,Australia,1998:337-342.

[16]孟凤霞,沈晋良,褚姝频.Bt棉叶对棉铃虫抗虫性的时空变化及气象因素的影响.昆虫学报,2003,46(3):299-304.MENG F X,SHEN J L,CHU S P.Temporal-spatial variation in efficacy of Bt cotton leaves against Helicoverpa armigera(Hǜbner)and effect of weather conditions.Acta Entomologica Sinica,2003,46(3):299-304.(in Chinese with English abstract)

收稿日期(Received):2015-07-15;接受日期(Accepted):2015-09-05;网络出版日期(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

基金项目:转基因生物新品种培育重大专项(2014ZX08005-005).

DOI:10.3785/j.issn.1008-9209.2015.07.151

中图分类号Q 946.1;S 562

文献标志码A