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Transportation of the Foreign Bt-Cry1Ac Protein and Insect Resistance Detection in Transgenic Poplar (Populus spp.) with Different Anvil Grafting |
CHEN Xing-Hao1, ZHANG Jun1, *, LI Zheng1, YU Xiao-Yue1, ZHANG De-Jian2, YANG Min-Sheng1 |
1 Hebei Key Laboratory for Tree Genetic Resources and Forest Protection / College of Forestry Science, Hebei Agricultural University, Baoding 071000, China; 2 College of Life Science, Inner Mongolia University, Hohhot 010021, China |
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Abstract With the extensive planting of poplar (Populus spp.) in China, the pest problem of poplar comes along.Nowadays, the application of chemical insecticides can not only fail to effectively reduce the harm of pests to poplar trees, but also seriously damage the ecological environment.Therefore, it is feasible to improve the insect resistance of poplar through grafting and propagation of transgenic poplar and non-transgenic poplar.In this study, in order to understand the ecological safety of grafted transgenic poplar, hybrid Populus tomentosa and Hb1 transgenic Populus × euramericana cv.'Neva' with Bt-Cry1Ac gene were used as scions and rootstocks to study whether the mRNA and protein of Cry1Ac gene were transported between scions and rootstocks, and whether the leaves of grafted seedlings were resistant to the insect pest.The results of reverse transcription-PCR (RT-PCR) and third-generation digital PCR showed that no Cry1Ac gene mRNA was detected in the shoots and leaves of hybrid P.tomentosa as scions or rootstocks, and the copy number of Cry1Ac gene was much lower than that of positive control, indicating that the exogenous Cry1Ac gene mRNA could not be transported between rootstocks and scions.Cry1Ac protein was detected in leaves, phloem and xylem of scions and rootstocks in 2 grafting combination by enzyme-linked immuno sorbent assay (ELISA), which proved that Cry1Ac protein could be transported between rootstocks and scions by grafting.The non-transgenic poplar grafted on the transgenic poplar could inhibit the growth and development of the larvae of the Hyphantria cunea, and slow down the development of the larvae, showing a certain degree of insect resistance.This study clarified the expression, transmission and insect resistance of Cry1Ac gene expression products between transgenic and non-transgenic tissues, and could provide theoretical basis and scientific guidance for the rational use of grafted poplar in insect resistance practice.
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Received: 20 September 2018
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Corresponding Authors:
zhangjunem@126.com
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[1] 陈发毅.2017.利用转录组分析研究柑橘砧穗间mRNA的移动[D].硕士学位论文, 华中农业大学, 导师: 胡春根, pp.3-9. (Chen F Y.2017.Using transcriptome analysis to investigate the movement of mRNA between citrus rootstock and scion[D].Thesis for M.S., Huazhong Agriculture University, Supervisor: Hu C G, pp.3-9.) [2] 陈盼飞, 任亚超, 张军, 等.2016.8年生嫁接转基因杨树 Bt 毒蛋白的表达与运输[J].林业科学, 52(7): 46-52. (Chen P F, Ren Y C, Zhang J, et al.2016.Expression and Transportation of Bt toxic protein in 8-year-old grafted transgenic poplar[J].Scientla Silvae Sinicae, 52(7): 46-52.) [3] 侯军铭, 韩会智, 王树洪, 等.2018.不同注药时间对杨树自流式打孔注药药液吸收速率的影响[J].东北林业大学学报, 46(7): 99-101. (Hou J M, Han H Z, Wang S H, et al.2018.Effect of injection time on absorption rate of poplar to pesticide with auto flowing trunk injection[J].Journal of Northeast Forestry University, 46(7): 99-101.) [4] 胡建军, 刘庆一, 王克胜, 等.1999.欧洲黑杨转Bt毒蛋白基因植株大田抗虫性测定[J].林业科学研究, 12(2): 202-205. (Hu J J, Liu Q Y, Wang K S, et al.1999.Field test on insect-resistance of transgenic plants (Populus nigra) transformed with Bt toxin gene[J].Forest Research, 12(2): 202-205.) [5] 金涛, 林玉英, 马光昌, 等.2018.新入侵害虫海枣异胸潜甲幼虫的龄数划分及在银海枣树上的垂直分布[J].环境昆虫学报, 40(03): 695-701. (Jin T, Lin Y Y, Ma G C, et al.2018.Division of larval instars and its vertical distribution on the Phoenix sylvestris trees of Javeta pallida baly 1858, a new invasive pest[J].Journal of Environmental Entomology, 40(03): 695-701.) [6] 牛春阳, 王峰, 李丹蕾, 等.2016.C14族R2R3-MYB基因调控杨树抗锈菌过敏性反应[J].北京林业大学学报, 38(07): 25-32. (Niu C Y, Wang F, Li D L, et al.2016.Impact of clade 14 R2R3-MYB genes on hypersensitive response of the poplar infected with Melampsora larici-populina[J].Journal of Beijing Forestry University, 38(07): 25-32.) [7] 任亚超, 董研, 张文林, 等.2017.转Bt基因烟草中Cry1Ac基因的叠加效应[J].农业生物技术学报, 25(12):1903-1917. (Ren Y C, Dong Y, Zhang W L, et al.2017.Superposition effect of Cry1Ac gene in transgenic Bt tobacco(Nicotiana tabacum)[J].Journal of Agricultural Biotechnology, 25(12): 1903-1917.) [8] 芮玉奎, 朱本忠, 罗云波.2005.转Bt基因抗虫棉(Gossyposium)伤流中Bt毒蛋白的运输[J].植物学报, 22(3): 320-324. (Rui Y K, Zhu B Z, Luo Y B.2005.Long-distance transportation of Bt-toxin through xylem sap in Bt-cotton (Gossyposium)[J].Chinese Bulletin of Botany, 22(3): 320-324.) [9] 孙燕红, 马龙彪, 兴旺, 等.2018.甜菜DAMD引物的筛选及不同凝胶系统对扩增产物多态性的影响[J].中国农学通报, 34(16): 42-45. (Sun Y H, Ma L B, Xing W, et al.2018.Screening of beet DAMD primers and the effects of different gel systems on polymorphism of amplified products[J].Chinese Agricultural Science Bulletin, 34(16): 42-45.) [10] 王建武, 冯远娇, 骆世明.2003.Bt玉米抗虫蛋白表达的时空动态及其土壤降解研究[J].中国农业科学, 36(11):1279-1286. (Wang J W, Feng Y J, Luo S M.2003.Studies on spatial-temporal dynamics of insecticidal protein expression of Bt corn and its degradation in soil[J].Scientia Agricultura Sinica, 36(11): 1279-1286.) [11] 王连荣, 杨敏生.2010.转基因杨树中外源Bt基因mRNA及其蛋白运输[J].林业科学, 46(7): 49-54. (Wang L R, Yang M S.2010.Transportation of the mRNA and protein of the foreign Bt gene in transgenic poplar[J].Scientla Silvae Sinicae, 46(7): 49-54.) [12] 翁绿水, 王作林, 肖国樱.2018.转基因水稻B2A68中抗虫蛋白Cry2Aa的表达特征和抗性分析[J].农业生物技术学报, 26(5):756-763. (Weng L S, Wang Z L, Xiao G Y.2018.Expression profile of insecticidal protein Cry2Aa and lepidopteran resistance in transgenic rice (Oryza sativa) B2A68[J].Journal of Agricultural Biotechnology, 26(5): 756-763.) [13] 吴斌, 肇慧君, 王玫, 等.2011.贝类及其产品中甲型肝炎病毒RT-PCR检测方法的研究[J].中国国境卫生检疫杂志, (1): 43-47. (Wu B, Zhao H J, Wang M, et al.2011.Studies on detection hepatitis A virus in shellfish and shellfish products with RT-PCR method[J].Chinese Frontier Health Quarantine, (1): 43-47.) [14] 杨敏生, 李志兰, 王颖, 等.2006.双抗虫基因对三倍体毛白杨的转化和抗虫性表达[J].林业科学, 42(9): 61-68. (Yang M S, Li Z L, Wang Y, et al.2006.Transformation and expression of two insect-resistant genes to hybrid triploid of Chinese white poplar[J].Scientla Silvae Sinicae, 42(9): 61-68.) [15] 张益文, 任亚超, 刘娇娇, 等.2015.转双抗虫基因欧美杨107杨中外源基因的表达[J].林业科学, 51(12): 45-52. (Zhang Y W, Ren Y C, Liu J J, et al.2015.Exogenous gene expression on transgenic Populus×euramericana cv.'74 /76' carrying bivalent insect-resistant genes[J].Scientla Silvae Sinicae, 51(12): 45-52.) [16] 周立东, 郑莲香, 南垚, 等.2007.蜂胶与杨树叶和杨树芽中叶绿素含量的比较研究[J].中国蜂业, 58(6): 5-7. (Zhou L D, Zheng L X, Nan Y, et al.2007.Compare of chlorophyll content of propolis with poplar leaves and buds[J].Apiculture of China, 58(6): 5-7.) [17] 朱路青, 曹越平.2005.转Bt基因大豆植株中Bt毒蛋白的表达[J].上海交通大学学报(农业科学版), 23(3): 234-238. (Zhu L Q, Cao Y P.Expression of insecticidal protein of Bacillus thuringiensis in Bt transgenic soybean[J].Journal of Shanghai Jiaotong University (Agricultural Science), 23(3): 234-238.) [18] Deeken R, Engelman J C, Efetova M, et al.2006.An integrated view of gene expression and solute profiles or Arabidopsis tumors: A genome-wide approach[J].Plant Cell, 18(12): 3617-3634. [19] Kehr J, Buhtz A.2008.Long distance transport and movement of RNA through the phloem[J].Journal of Experimental Botany, 59(1): 85-92. [20] Mlotshwa S, Pruss G J, Vance V.2008.Small RNAs in viral infection and host defense[J].Trends in Plant Science, 13(7): 375-382. [21] Mohammadi K, Movahedi A, Maleki S S, et al.2018.Functional analysis of overexpressed PtDRS1, involved in abiotic stresses enhances growth in transgenic poplar[J].Plant Physiology & Biochemistry, 126: 22-31. [22] Moldován N, Szucs A, Tombácz D, et al.2018.Multi-platform next-generation sequencing identifies novel RNA molecules and transcript isoforms of the endogenous retrovirus isolated from cultured cells[J].Fems Microbiology Letters, 365(5): fny013. [23] Roberto R M, Beatriz X C,William J L.2001.The phloem as a conduit for inter-organ communication.[J].Current Opinion in Plant Biology, 4(3): 202-209. [24] Sivasupramaniam S, Moar W J, Ruschke L G, et al.2008.Toxicity and characterization of cotton expressing Bacillus thuringiensis Cry1Ac and Cry2Ab2 proteins for control of lepidopteran pests[J].Journal of Economic Entomology, 101(2): 546-554. [25] Yoo B C, Kragler F, Varkonyi-Gasic E, et al.2004.A systemic small RNA signaling system in plants[J].Plant Cell,(16)8: 1979-2000. |
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