【作者】 王连荣；

【导师】 杨敏生；

【作者基本信息】 河北农业大学 ， 林木遗传育种， 2010， 博士

【摘要】 本试验以741杨、毛白杨、Pb29(转Btcry1Ac基因741杨株系)和CC71(转Btcry3A基因741杨株系)为材料,通过嫁接手段,采用RT-PCR、ELISA等试验技术,进行外源Bt基因转录产物mRNA运输规律,外源Bt毒蛋白运输机制和规律,以及转基因嫁接杨树抗虫性的研究；以741杨、毛白杨、Pb29、转基因棉花国审GK45(转Btcry1Ac基因)和非转基因棉花新陆早36号为材料,建立4种杨棉复合系统,对外源Bt毒蛋白在系统中的富集、分布和降解规律进行研究。1.以741杨和Pb29互为接穗和砧木进行嫁接,利用RT-PCR技术,对Btcry1Ac基因的mRNA是否在砧木与接穗间运输进行了研究。RT-PCR检测结果表明：以741杨为接穗或砧木的嫩枝和嫩叶中均未检测到Btcry1Ac基因的mRNA,说明Btcry1Ac基因的mRNA没有在砧木与接穗间进行运输。2.以741杨和CC71互为接穗和砧木,以Pb29为接穗CC71为砧木进行嫁接,利用RT-PCR技术,对Btcry3A基因的mRNA是否在砧木与接穗间运输进行了研究。RT-PCR检测结果表明：以741杨为接穗或砧木的嫩枝和嫩叶,以及以Pb29为接穗的嫩枝和嫩叶中均未检测到Btcry3A基因的mRNA,说明Btcry3A基因的mRNA没有在砧木与接穗间进行运输。3.以741杨和Pb29互为接穗和砧木,以毛白杨和Pb29互为接穗和砧木,以CC71和Pb29互为接穗和砧木,以Pb29为中间砧741杨为接穗和砧木进行嫁接,利用ELSA技术对BtCry1Ac毒蛋白运输机制及规律进行了研究。ELISA检测结果表明：以741杨、毛白杨为砧木或接穗的叶片、韧皮部、木质部和髓中均检测出BtCry1Ac毒蛋白的存在；以CC71为砧木或接穗的叶片和韧皮部中均检测出BtCry1Ac毒蛋白,木质部和髓中没有检测到BtCry1Ac毒蛋白存在。各组织均以韧皮部中BtCry1Ac毒蛋白含量较高。这说明BtCry1Ac毒蛋白可以通过嫁接的方式在砧木和接穗间进行运输,且以韧皮部运输为主,但是2种外源蛋白(BtCry1Ac和BtCry3A)在运输的过程中可能发生了拮抗。4.以741杨和CC71互为接穗和砧木,以毛白杨和CC71互为接穗和砧木,以Pb29和CC71互为接穗和砧木,以CC71为中间砧741杨为接穗和砧木进行嫁接,利用ELISA技术对BtCry3A毒蛋白运输机制及规律进行了研究。ELISA检测结果表明：以741杨、毛白杨、Pb29为砧木或接穗的叶片、韧皮部、木质部和髓中均检测出BtCry3A毒蛋白的存在。各组织均以木质部中BtCry1Ac毒蛋白含量较高,但是以Pb29为砧木或接穗的木质部中BtCry3A毒蛋白含量相对较低。这说明BtCry3A毒蛋白可以通过嫁接的方式在砧木和接穗间进行运输,且以木质部运输为主,但是2种外源蛋白(BtCry1Ac和BtCry3A)在运输的过程中可能发生了拮抗。5.用转Btcry1Ac基因为砧木或接穗的嫁接杨树接穗叶片在室内喂饲杨扇舟蛾和美国白蛾的幼虫,研究嫁接处理对杨扇舟蛾和美国白蛾幼虫的影响。结果表明：转Btcry1Ac基因嫁接杨树对低龄杨扇舟蛾和美国白蛾幼虫具有一定的毒杀作用,而对存活的高龄幼虫则表现为对其生长发育的抑制作用,各嫁接处理均有效延长幼虫的发育历期,降低幼虫体长和体重增加速率,减少幼虫排粪量和食叶面积,导致幼虫生长发育不良,具有一定的抗虫性。6.用转Btcry3A基因为砧木或接穗的嫁接杨树接穗叶片在室内喂饲柳蓝叶甲幼虫,研究嫁接处理对柳蓝叶甲幼虫的影响。结果表明：转Btcry3A基因嫁接杨树对低龄柳蓝叶甲幼虫具有一定的毒杀作用,而对存活的高龄幼虫具有延长幼虫发育历期,降低幼虫体长增加速率,减少幼虫蛹重的作用,具有一定的抗虫性。7.利用ELISA技术,对杨棉复合系统中转基因杨树Pb29和转基因棉国审GK45外源BtCry1Ac毒蛋白表达进行研究,结果表明：转基因杨树Pb29长枝叶、短枝叶和根系等组织中,BtCry1Ac毒蛋白含量在整个生长发育期均呈先升后降的趋势。短枝叶BtCry1Ac毒蛋白含量一直高于长枝叶,根系中BtCry1Ac毒蛋白含量6月20低于短枝叶和长枝叶,以后高于短枝叶和长枝叶。转基因棉国审GK45根、茎、叶等组织中,BtCry1Ac毒蛋白含量在整个生长发育期呈动态下降趋势。茎中BtCry1Ac毒蛋白含量下降较缓慢,一直维持较高的水平,而根和叶中BtCry1Ac毒蛋白含量下降较快,到植株生长发育后期降到最低。茎和叶中BtCry1Ac毒蛋白含量高于根系。8.利用ELISA技术,对杨棉复合系统土壤中是否存在BtCry1Ac毒蛋白进行检测。结果表明：转基因杨树Pb29和转基因棉花国审GK45根际土,非转基因杨树与转基因棉花复合系统(B,D)、转基因杨树与转基因棉花复合系统(E,G)、转基因杨树与非转基因棉花复合系统(F,H)的表土,有少数的采样点检测到BtCry1Ac毒蛋白的存在,但是含量都很低。更多还原

【Abstract】 In this paper, poplar 741, Populus tomentosa, Pb29 (transgenic poplar 741 with BtcrylAc gene) and CC71 (transgenic poplar 741 with Btcry3A gene) were used as the materials, mRNA transport law of exogenous Bt gene, transport mechanisms and rules of exogenous Bt toxin protein and insect-resistance of transgenic poplar 741 grafted were studied by using the grafting procedure; to poplar 741, Populus tomentosa, Pb29 (transgenic poplar 741 with BtcrylAc gene), transgenic cotton Guoshen GK45 (transgenic BtcrylAc gene) and non-transgenic cotton Handan109 for the material, the establishment of four kinds of Poplar-cotton composite system, the law of concentration, distribution and degradation of exogenous Bt toxin protein was studied.1 In order to discover whether mRNA of BtcrylAc gene was transported between the stock and scion, a study was made by means of RT-PCR technology while poplar741 and Pb29 were grafted as scion and stock mutually. The result of RT-PCR test was that mRNA of BtcrylAc gene was not detected in the branch and leaf of poplar741 as scion or stock; therefore it meant that mRNA of BtcrylAc gene was not transported between the stock and scion.2 In order to discover whether mRNA of Btcry3’A gene was transported between the stock and scion, a study was made by means of RT-PCR technology while poplar741 and CC71 were grafted as scion and stock mutually, and Pb29 was grafted onto CC71 rootstock. The result of RT-PCR test was that mRNA of Btcry3Agene was not detected in the branch and leaf of poplar741 and Pb29 as scion or stock; therefore, it meant that mRNA of Btcry3A gene was not transported between the stock and scion.3 While poplar741 and Pb29 were grafted as scion and stock mutually, Populus tomentosa and Pb29 were grafted as scion and stock mutually, CC71 and Pb29 were grafted as scion and stock mutually and poplar741 were grafted Pb29 as intermediate stock, in order to study on transport mechanisms and rules of exogenous BtCryl Ac toxin protein by means of ELS A technology. The results of ELISA test were as follows:BtCryl Ac toxin protein was all detected in the leaf, phloem, xylem and pith of poplar741 and Populus tomentosa as scion or stock; BtCry1 Ac toxin protein was only detected in the leaf, phloem of CC71 as scion or stock, but BtCry1 Ac toxin protein of xylem and pith were not detected. Therefore, it demonstrated that BtCry1 Ac toxin protein was transported between the stock and scion by means of grafting and phloem was mainly transport. but two kinds of exogenous proteins (BtCry1Ac and BtCry3A) antagonized during transport.4 While poplar741 and CC71 were grafted as scion and stock mutually, Populus tomentosa and CC71 were grafted as scion and stock mutually, Pb29 and CC71were grafted as scion and stock mutually and poplar741 were grafted CC71 as intermediate stock, in order to study on transport mechanisms and rules of exogenous BtCry3A toxin protein by means of ELISA technology. The results of ELISA test were as follows BtCry3A toxin protein was all detected in the leaf, phloem, xylem and pith of poplar741, Populus tomentosa and Pb29 as scion or stock, but BtCry3A toxin protein content of xylem and pith were lower. therefore, it demonstrated that BtCry3A toxin protein was transported between the stock and scion by means of grafting and xylem were mainly transport. But two kinds of exogenous proteins (BtCry1Ac and BtCry3A) antagonized during transport.5 With the aim of finding out the influence of grafting poplar741 with transgenic BtcrylAc gene on Clostera anachoreta Fabricius and Hyphantria cunea (Drury), the larvae of C. anachoreta and H. cunea were fed indoors on the leaves of scion which had been grafted. The results showed that grafting poplar741 with transgenic BtcrylAc gene had some toxin effects on the younger larvae of C. anachoreta and H. cunea and inhibited of growth and development of the older ones, could extend the larval developmental period of C. anachoreta and H. cunea, lower rates of larval body length and weight, reduced frass quantity and eating leaf area of the larvae, resulted in poor larval growth and development, and even express certain resistance to insects.6 With the aim of finding out the influence of grafting poplar741 with transgenic Btcry3A gene on Plagiodera versicolora (Laicharting), the larvae of P. versicolora were fed indoors on the leaves of scion which had been grafted. The results showed that grafting poplar741 with transgenic Btcry3A gene had some toxin effects on the younger larvae of P. versicolora and inhibited of growth and development. It could extend the larval developmental period of P. versicolora, lower rates of larval body length, reduced pupa weight of the older ones, and even express certain resistance to insects.7 It was studied that the expression of exogenous BtCry1Ac gene of transgenic poplar Pb29 and transgenic cotton Guoshen GK45 in poplar-cotton composite system by means of ELSA technology. The results showed that the contents of exogenous BtCry1Ac toxin protein branch leaves, short branch leaves and root firstly increased and then decreased in the growth and development of transgenic poplar Pb29. The contents of exogenous BtCry1Ac toxin protein branch leaves were higher than that of long branch leaves. On 20 June the contents of exogenous BtCrylAc toxin protein were less that of long branch leaves and short branch leaves and higher than that of long branch leaves and short branch leaves later. The contents of exogenous BtCrylAc toxin protein, stems and leaves decreased along with the growth of transgenic Guoshen GK45. The contents of exogenous BtCrylAc toxin protein of stems decreased more slowly and always maintained a high level, while that of roots and leaves decreased rapidly and reached a minimum level to the late plant growth and development, were less than that of stems.8 It was tested whether soil were the presence of BtCrylAc toxin protein in poplar-cotton composite system or not by means of ELSA technology. The results showed that a few sampling points had the presence of BtCrylAc toxin protein, but the concentrations were very low in the rhizosphere soil of transgenic poplar Pb29 and transgenic cotton Guoshen GK45, topsoil of non-transgenic poplar and transgenic cotton composite system (B, D), topsoil of transgenic poplar and transgenic cotton composite system (E, G), topsoil of transgenic poplar and non-transgenic cotton composite system (F, H).更多还原