转双抗虫基因欧美杨107杨中外源基因的表达

Exogenous Gene Expression on Transgenic Populus×euramericana cv. '74/76' Carrying Bivalent Insect-Resistant Genes

【目的】鉴定并分析转双抗虫基因(BtCryl Ac和API基因)欧美杨107杨(简称:转基因107杨)中外源基因的表达情况,并且筛选出对鳞翅目害虫美国白蛾和杨扇舟蛾具有高抗虫性的转基因107杨株系【方法】以10个转基因107杨株系2年生田间苗为试验材料,未转基因107杨为对照(CK),进行外源基因表达测定,包括PC R检测、绝对荧光定量PC R检测和毒蛋白检测以及鳞翅目害虫抗虫性对比试验【结果】PCR检测结果表明,10个转基因株系均检测到外源基因BtCryl Ac和API而对照未检测到,证明BtCryl Ac和API基因已稳定插入转基因植株基因组中。荧光定量PCR检测表明,8月份PB1,PB2,PB6,PB9和PB10这5个株系BtCryl Ac基因的转录丰度较高为1.72×10~8~7.91×10~9,PB1最高;PB3,PB7和PB8次之为1.03×10~7~2.35×10~7;PB4和PB5与对照一样未检测到BtCrylAc基因的转录表达。ELISA毒蛋白检测表明,CrylAc毒蛋白的表达量与转录丰度大小变化趋势一致,PBl最高,为665.11 ng·g^(-1),PB4和PB5与对照一样未检测到毒蛋白。室内饲虫试验证明转基因107杨对美国白蛾幼虫的杀虫效果高于杨扇舟蛾幼虫。8个表达毒蛋白的转基因株系对1-6龄美国白蛾幼虫的致死率均为100%,但致死时间存在一定差异,PB1和PB2对各龄幼虫的致死时间均较短;PB1,PB2,PB6,PB9和PB10对1~4龄杨扇舟蛾幼虫的致死率均为100%,其中PB1和PB2的致死时间较短,PB3,PB7和PB8对1~2龄杨扇舟蛾幼虫的致死率为100%,对3龄和4龄的致死率最高为22.22%。综合抗虫对比试验结果,可以将8个转基因株系划分为2个抗性水平:PB1,PB2,PB6,YB9和PB10为高抗株系对美国白蛾和杨扇舟蛾均表现高抗性;PB3,PB7和PB8为中抗株系,对杨扇舟蛾表现出中等抗性,但对美国白蛾表现出高抗性。【结论】通过对转基因107杨进行一系列分子检测及室内抗虫对比试验,证明外源基因已成功导入杨树基因组并稳定存在,并且有8个株系高效表达;抗虫试验证明转基因107杨对美国白蛾和杨扇舟蛾具有明显的抗性,筛选出的5个株系对2种害虫都具有高抗性,另外3个株系PB3,PB7和PB8对美国白蛾具有很高的抗性,而对杨扇舟虫我则表现出中低抗性。

[ Objective] In order to identify and analyze the expression of exogenous genes in transgenic Populus x euramericana cv. ＇ 74/76 ＇ carrying bivalent insect-resistant genes BtCrylAc and API （ Abbreviation： Transgenic 107 poplar） , and to select transgenic 107 poplar lines with high resistance to Lepidoptera pests Hlyphantria cunea and Clostera anachoreta. [ Method] Two-year-old field seedlings of ten transgenic 107 poplar lines were selected as materials and non- transgenic 107 poplar as c6ntrol. Detections of exogenous gene expression were conducted, including PCR detection, absolute fluorescence quantitative PCR, toxin detection and comparative studies on insect resistance of Lepidoptera pests. [ Result] PCR detection showed that exogenous genes BtCrylAc and API existed in the ten transgenic lines, but not in the CK, indicating that BtCrylAc and API were steadily inserted into transgenic plant genome. The fluorescence quantitative PCR showed transcription abundance of BtCrylAc in five lines PB1, PB2, PB6, PB9, and PB10 were higher, rangingfrom 1.72 × 10^8 to 7.91 × 10^9 , PB1 was the highest; and followed by PB3, PB7, and PB8, ranging from 1.03 × 10^7 to 2.35 × 10^7 ; PB4, PB5, and CK detected no transcriptional expression of BtCrylAc. ELISA toxin detection showed the content of CrylAc toxin expression was in accordance with variation tendency of transcription abundance. PB1 was the highest, with 665. 11 ng. g-l, PB4, PB5, and CK detected no toxin. The indoor insect feeding test showed that the insecticidal effect of transgenic 107 poplar on H. cunea larvae was higher than that of C. anachoreta. Eight transgenic lines expressed toxin had high resistance to L~ -L6 （L1 -L6 represent 1 to 6 instars）H, cunea larvae with a mortality of 100% , but there were differences on lethal time among these lines, the lethal time of PB1 and PB2 on every instars were shorter. PB1 , PB2, PB6, PB9 and PB10 had a high resistance to L1 - L4 C. anachoreta larvae with a mortality of 100% , the lethal time of PB1 and PB2 were shorter. The mortality of PB3, PB7, and PB8 on L~ - L2 C. anachoreta larvae was 100% , but the mortality of L3 - L4 C. anachoreta larvae was as high as 22.22%. The 8 transgenie lines were divided into two resistance levels： PBI , PB2, PB6, PB9 and PB10 were high resistant lines, which showed high insect resistance to H. cunea and C. anachoreta ; PB3, PB7, and PB8 were moderate resistant lines, which showed moderate insect resistance to C. anachoreta, but high resistance to H. cunea. [ Conclusion] A series of molecular detections and indoor insect feeding tests were conducted on transgenie 107 poplar in this study. The results indicated that exogenous genes were steadily inserted into the poplar genome, and the 8 lines were of high-efficiency expression. Insect resistance test showed that transgenie 107 poplar had high resistance to H. cunea and C. anachoreta. Five lines were selected and showed high resistance to two pests, the other three lines PB3, PB7, and PB8 showed high resistance to H. cunea, but low or moderate resistance to C. anachoreta.