芥菜胰蛋白酶抑制剂基因mti2转化拟南芥菜的研究
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摘要
虫害是影响农作物产量和品质的重要因素，因此培育出抗虫品种在生产上具有重要意义。但常规抗虫育种由于育种周期长，种质资源有限，加上抗虫机制不太明了等原因，其利用受到极大的限制。植物基因工程的快速发展为抗性育种提供了一条崭新而有效的途径。芥菜胰蛋白酶抑制剂2(MTI2)是从白芥的种子中分离出来的，它对鳞翅目幼虫中肠内的蛋白酶具有较强抑制作用。因此，芥菜胰蛋白酶抑制剂基冈mti2可作为一种很好的提高植物抗虫性的铒选基因。
本研究通过原化渗入转化法，将构建在质粒载体PKYLX-71上的mtj2基因编码的cDNA序列导入拟南芥菜，以进一步研究该基因在寄主植物体内的表达特点和抗虫性。主要实验结果如下：
1 利用原位渗入法将mti2基因转入拟南芥菜，最终的转化频率为0.2～2.8％。在转化的过程中用0.02％的表面活性剂Tween 20替代常用的silwet L-77。
2 对部分通过卡那霉素筛选的抗性植株进行了PCR和PCR-Southern检测，其中PCR检测的阳性率为98.9％，PCR-Southern检测的阳性率为100％。可以初步确定目的基因已经整合到寄主植物的基因组中。
3 对部分通过PCR检测的植株进行一步法RT-PCR检测，在80个被检测单株中，每个单株都获得了特异的扩增条带，所以初步确定目的基因在寄主植物体内得到表达。
The pests is a main factor that restrain the yield and quality of crops, so it is great importance for agriculture production that breeding new cullivars which can resistance to pest, but the common methods of pest-resistance breeding are restrained greatly because the breeding week is too long or resource is limited and the pest-resistance mechanism are not clear. But the quickly development of plant genetic engineering provides a new and powerful method for breeding pest-resistance cultivars. Mustar trypsin inhibitor has been isolated from white mustar seeds, it can significantly affected the activity of overall digestive protease in the gut of Lepidopteran pest larvals.Therefore, mti2 is thought to be a good candidate for enhancing pest resitancc in plant.
In this reaserch,the mti2 encoding cDNA sequence that recombinated in PKYLX-71 vector has been transfer into arabidopsis by in planta infiltration to study the character of expression and pest resistance in transgenic plants. The main results in this experiment were as follows:
1. The mti2 gene has been transferred into arabidopsis thaliana(Columbia ecotype)by in planta infiltration, the final transformation frequency is about 0.2%~2.8%. in the course of this transformation, the surfactants Tween 20 has been used as effective substitutes for Silwet L-77.the final Tween 20 concentration is 0.02%.
2. The kanamycin-resist plants had been test by PCR and PCR-Southern bloting, the positive frequency of PCR test is about 98.9% and the positive frequency of PCR-Southern bloting is 100%.it can be confirmed that the target gene has recombinated into the genomic of arabidopsis plants.
3. One-step RT-PCR test had been done in 80 plants that have passed the PCR test, a special bind had been acquired in every plant, it can be confirmed that the target gene which recombinated into the genomic of plant had expressed in plant.
本研究通过原化渗入转化法，将构建在质粒载体PKYLX-71上的mtj2基因编码的cDNA序列导入拟南芥菜，以进一步研究该基因在寄主植物体内的表达特点和抗虫性。主要实验结果如下：
1 利用原位渗入法将mti2基因转入拟南芥菜，最终的转化频率为0.2～2.8％。在转化的过程中用0.02％的表面活性剂Tween 20替代常用的silwet L-77。
2 对部分通过卡那霉素筛选的抗性植株进行了PCR和PCR-Southern检测，其中PCR检测的阳性率为98.9％，PCR-Southern检测的阳性率为100％。可以初步确定目的基因已经整合到寄主植物的基因组中。
3 对部分通过PCR检测的植株进行一步法RT-PCR检测，在80个被检测单株中，每个单株都获得了特异的扩增条带，所以初步确定目的基因在寄主植物体内得到表达。
The pests is a main factor that restrain the yield and quality of crops, so it is great importance for agriculture production that breeding new cullivars which can resistance to pest, but the common methods of pest-resistance breeding are restrained greatly because the breeding week is too long or resource is limited and the pest-resistance mechanism are not clear. But the quickly development of plant genetic engineering provides a new and powerful method for breeding pest-resistance cultivars. Mustar trypsin inhibitor has been isolated from white mustar seeds, it can significantly affected the activity of overall digestive protease in the gut of Lepidopteran pest larvals.Therefore, mti2 is thought to be a good candidate for enhancing pest resitancc in plant.
In this reaserch,the mti2 encoding cDNA sequence that recombinated in PKYLX-71 vector has been transfer into arabidopsis by in planta infiltration to study the character of expression and pest resistance in transgenic plants. The main results in this experiment were as follows:
1. The mti2 gene has been transferred into arabidopsis thaliana(Columbia ecotype)by in planta infiltration, the final transformation frequency is about 0.2%~2.8%. in the course of this transformation, the surfactants Tween 20 has been used as effective substitutes for Silwet L-77.the final Tween 20 concentration is 0.02%.
2. The kanamycin-resist plants had been test by PCR and PCR-Southern bloting, the positive frequency of PCR test is about 98.9% and the positive frequency of PCR-Southern bloting is 100%.it can be confirmed that the target gene has recombinated into the genomic of arabidopsis plants.
3. One-step RT-PCR test had been done in 80 plants that have passed the PCR test, a special bind had been acquired in every plant, it can be confirmed that the target gene which recombinated into the genomic of plant had expressed in plant.
引文
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2．王镜岩，朱圣庚，徐长法．生物化学．第三版．北京：高等教育出版社，2002
3．萨姆布鲁克等著，金冬雁，黎孟枫等译．分子克隆实验指南(第三版)，科学出版社，2003
4．曹鸣庆，刘凡，姚磊．通过农杆菌原位真空渗入法获得转基因小白菜．林忠平等编著：《走向21世纪的植物分子生物学》，科学出版社，2000
5．王文全，孟玲，许键．苦豆子胰蛋白酶抑制剂对几种害虫的抑制作用．中国生物防治，1997，13(4)：176～178
6．傅荣昭，孙勇如，贾士荣主编．《植物遗传转化技术手册》北京：中国科学技术出版社，1994
7．苏军．蛋白酶抑制剂基因的杀虫机理及其应用．福建果树，1998，100：19～20
8．秦新民等．小白菜苏云金芽抱杆菌CryIB基因的遗传转化．广西师范大学学报(自然科学版)，1999，17：77～82
9．杨广东，朱祯，李燕娥等．转修饰豇豆胰蛋白酶抑制剂基因(sck)抗虫甜椒植株的获得．应用与环境生物学报．2002，8(3)：239～244
10．张智奇，周音，钟维瑾等．慈姑蛋白酶抑制剂基因转化小白菜获抗虫转基因植株．上海农业学报，1999，15(4)：4～9
11．李慧芬，李旭刚，吴茜等．转修饰豇豆胰蛋白酶抑制剂基因(sck)玉米(Zea mays L．)植株的获得及其抗虫性分析．高技术通讯，2001，10：7～12
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