伯氏嗜线虫致病杆菌蛋白酶抑制剂在烟草中的表达及抗蚜功能研究
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摘要
蛋白酶抑制剂能够抑制昆虫肠道中蛋白酶活性影响昆虫的生长和发育，因而可以应用于农业害虫防治。研究发现来自伯氏嗜线虫致病杆菌（Xenorhabdus bovienii strain BJFS526）的蛋白酶抑制剂（Xbpi-1）对蚜虫有良好的防治效果。为探讨Xbpi-1在植物中的抗蚜功能，本研究将Xbpi-1基因导入烟草，检测了该基因在烟草中的整合、转录和表达，检验了转基因烟草对烟蚜的抗虫效果并分析了转基因烟草的农艺性状与生理指标。
本研究获得的主要结果如下：
（1）构建了伯氏嗜线虫致病杆菌蛋白酶抑制剂基因（Xbpi-1）与绿色荧光蛋白（Greenfluorescent protein, gfp）基因的植物融合表达载体pCAMBIA2300-35S-Xbpigfp-Ocs和对照载体pCAMBIA2300-35S-gfp-Ocs。对载体进行了酶切和测序检验，将检验正确的载体通过冻融法转化到根癌农杆菌EHA105中。通过根癌农杆菌介导转化与抗生素筛选获得了烟草转化子，并将其繁育至T2代。
（2）通过PCR检测初步确认了阳性转化株，Southern blot分析表明了Xbpigfp基因在烟草基因组中的整合模式，RT-PCR验证了目的基因在烟草中的转录，ELISA，Western blot和荧光蛋白检测证实了目的基因在烟草中的表达。
（3）转基因和野生型烟草的农艺性状与生理指标研究表明，转基因烟草的PPO活性和5184株系的叶片重显著增加，其他农艺性状没有显著变化。
（4）鉴定了转基因烟草对烟蚜的抗性，与取食表达gfp烟草的烟蚜相比，取食表达Xbpigfp烟草的烟蚜存活率没有显著变化，但成虫体重降低了33.7%，繁殖量降低了36.2%；通过分析取食表达Xbpigfp基因烟草后烟蚜体内蛋白酶活性，发现与取食表达gfp基因烟草的烟蚜相比，取食表达Xbpigfp基因烟草的烟蚜体内总蛋白酶活性和氨肽酶活性都显著下降。
Protease inhibitor can reduce the damage of pest by inhibiting the activty of insect protease.Study demonstrates the Xbpi-1(protease inhibitor from Xenorhabdus bovienii) had adverse effects onaphids, such as green peach aphid (Myzus persicae). To study the resistantance of Xbpi-1againstaphids, the gene of Xbpi-1was transferred into tobacco. The transgene’s integration, teanscription andexpression were identified. Insect-resistance and agronomic traits of transgenic plants were alsostudied.
Key results:
(1) Plant expression vectors, pCAMBIA2300-35S-Xbpigfp-Ocs with Xbpigfp gene andpCAMBIA2300-35S-gfp-Ocs with gfp gene, were constructed, which are harboring CaMV35Spromoter and OCS terminator. The vectors were verified by restriction analysis and sequencing,and introduced into Agrobacterium tumefaciens strain EHA105by freeze-thaw method.
(2) The Xbpigfp gene integration was confirmed by PCR and Southern blot. The transcription wasverified by RT-PCR. The expression of Xbpigfp in tobacco was detected by ELISA and greenfluorescence protein (GFP) signal.
(3) The PPO activity of transgenic tobacco and the leaf weights in transgenic line5184weredramatically increased, however other changes in agronomic traits of transgenic tobacco have notbeen observed.
(4) The resistance of transgenic tobacco against aphids (M. persicae), and effects on the proteaseactivity in aphids were studied. The results showed that the mean adult weights and biomassproduced were reduced about33.7%and36.2%, respectively, when aphids fed on line PI3transgenic plants compared to those fed on control plants (wild type and plants expressed GFPonly) after two weeks and the protease activity of aphids were significantly reduced.
本研究获得的主要结果如下：
（1）构建了伯氏嗜线虫致病杆菌蛋白酶抑制剂基因（Xbpi-1）与绿色荧光蛋白（Greenfluorescent protein, gfp）基因的植物融合表达载体pCAMBIA2300-35S-Xbpigfp-Ocs和对照载体pCAMBIA2300-35S-gfp-Ocs。对载体进行了酶切和测序检验，将检验正确的载体通过冻融法转化到根癌农杆菌EHA105中。通过根癌农杆菌介导转化与抗生素筛选获得了烟草转化子，并将其繁育至T2代。
（2）通过PCR检测初步确认了阳性转化株，Southern blot分析表明了Xbpigfp基因在烟草基因组中的整合模式，RT-PCR验证了目的基因在烟草中的转录，ELISA，Western blot和荧光蛋白检测证实了目的基因在烟草中的表达。
（3）转基因和野生型烟草的农艺性状与生理指标研究表明，转基因烟草的PPO活性和5184株系的叶片重显著增加，其他农艺性状没有显著变化。
（4）鉴定了转基因烟草对烟蚜的抗性，与取食表达gfp烟草的烟蚜相比，取食表达Xbpigfp烟草的烟蚜存活率没有显著变化，但成虫体重降低了33.7%，繁殖量降低了36.2%；通过分析取食表达Xbpigfp基因烟草后烟蚜体内蛋白酶活性，发现与取食表达gfp基因烟草的烟蚜相比，取食表达Xbpigfp基因烟草的烟蚜体内总蛋白酶活性和氨肽酶活性都显著下降。
Protease inhibitor can reduce the damage of pest by inhibiting the activty of insect protease.Study demonstrates the Xbpi-1(protease inhibitor from Xenorhabdus bovienii) had adverse effects onaphids, such as green peach aphid (Myzus persicae). To study the resistantance of Xbpi-1againstaphids, the gene of Xbpi-1was transferred into tobacco. The transgene’s integration, teanscription andexpression were identified. Insect-resistance and agronomic traits of transgenic plants were alsostudied.
Key results:
(1) Plant expression vectors, pCAMBIA2300-35S-Xbpigfp-Ocs with Xbpigfp gene andpCAMBIA2300-35S-gfp-Ocs with gfp gene, were constructed, which are harboring CaMV35Spromoter and OCS terminator. The vectors were verified by restriction analysis and sequencing,and introduced into Agrobacterium tumefaciens strain EHA105by freeze-thaw method.
(2) The Xbpigfp gene integration was confirmed by PCR and Southern blot. The transcription wasverified by RT-PCR. The expression of Xbpigfp in tobacco was detected by ELISA and greenfluorescence protein (GFP) signal.
(3) The PPO activity of transgenic tobacco and the leaf weights in transgenic line5184weredramatically increased, however other changes in agronomic traits of transgenic tobacco have notbeen observed.
(4) The resistance of transgenic tobacco against aphids (M. persicae), and effects on the proteaseactivity in aphids were studied. The results showed that the mean adult weights and biomassproduced were reduced about33.7%and36.2%, respectively, when aphids fed on line PI3transgenic plants compared to those fed on control plants (wild type and plants expressed GFPonly) after two weeks and the protease activity of aphids were significantly reduced.
引文
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