农杆菌介导甜高粱转Bt cry1Ah的研究

doi:10.3864/j.issn.0578-1752.2011.10.003

摘要/Abstract

摘要： 【目的】建立一套高效、稳定的甜高粱农杆菌遗传转化体系，并在甜高粱中验证来源于苏云金芽胞杆菌新型杀虫基因cry1Ah表达产物的杀虫活性。【方法】以甜高粱幼穗愈伤组织为转化受体，通过农杆菌介导法将密码子优化过的Bt cry1Ah导入2个甜高粱品种“BABUSH”和“MN-3025”中。对获得的再生植株进行PCR检测、RT-PCR分析及除草剂抗性、目的蛋白表达水平和抗虫性鉴定分析。【结果】对336块幼穗愈伤组织块进行了农杆菌侵染，经双丙氨膦（Bialaphos）梯度筛选后共获得66株再生植株，PCR鉴定在8个转化事件中有22株为阳性植株，平均转化率为2.38%。RT-PCR检测结果表明，cry1Ah在T0甜高粱转基因植株中能够正常转录。Western blotting分析和ELISA定量测定显示，有5株可检测到Bt蛋白，但Bt蛋白含量差异较大，最高可达165.69 ng•g-1叶片鲜重（FW），最低的仅为1.93 ng•g-1叶片鲜重（FW），平均为87.50 ng•g-1 FW。饲虫试验表明，有2株转基因甜高粱植株对亚洲玉米螟（Ostrinia furnacalis）表现为抗性。【结论】利用建立的以甜高粱幼穗为外植体的农杆菌遗传转化体系，可获得具有玉米螟抗性的转基因甜高粱植株，其后代遗传稳定性还有待于进一步的研究。

关键词: 甜高粱, cry1Ah, 农杆菌转化, 抗虫性

Abstract: 【Objective】 The aim of the study is to establish a high frequency and steady Agrobacterium tumefaciens- mediated transformation system of sweet sorghum, and validate the insecticidal function of a novel Bt cry1Ah gene in the transgenic plants.【Method】Using the callus induced from immature inflorescence as transformation recipients, the codon optimized Bt cry1Ah was transferred into sweet sorghum varieties “BABUSH” and “MN-3025” via Agrobacterium-mediated transformation. The obtained regenerative plants were identified by PCR, RT-PCR analysis, and their herbicide resistance, the expression of aim protein and insect-resistant identification were also analyzed.【Result】After gradient selection with Biolaphos, a total of 66 regenerated plants were produced from 336 agroinfected calli in these two sweet sorghum varieties. Among these plants, 22 PCR positive transformed plants of 8 independent transformation events were obtained, and the average transformation efficiency was 2.38%. The transcription of cry1Ah gene in the T0 transgenic sweet sorghum plants was further confirmed by RT-PCR. The Bt proteins could be detected by western blotting and ELISA assay in five transgenic plants, which showed different expression levels in a range of 1.93 ng•g-1 FW to 165.69 ng•g-1 FW, with an average of 87.50 ng•g-1 FW. Additionally, the results of bioassay indicated that two of the five transgenic plants displayed high insect-resistance to Ostrinia furnacalis.【Conclusion】An Agrobacterium-mediated genetic transformation system of sweet sorghum was established by using the callus derived from immature inflorescence as the recipients. The resulted T0 generation transgenic sweet sorghum plants with cry1Ah showed high insect-resistance to Ostrinia furnacalis. Further investigations on the genetic stability of cry1Ah in different transgenic lines and generations are undergoing.

Key words:

sweet sorghum (Sorghum bicolor L. Moench), cry1Ah, Agrobacterium tumefaciens transformation, insect-resistance

朱莉, 郎志宏, 李桂英, 何康来, 岳同卿, 张杰, 黄大昉. 农杆菌介导甜高粱转Bt cry1Ah的研究[J]. 中国农业科学, 2011, 44(10): 1989-1996.

ZHU Li, LANG Zhi-Hong, LI Gui-Ying, HE Kang-Lai, YUE Tong-Qing, ZHANG Jie, HUANG Da-Fang. Introduction of Bt cry1Ah Gene into Sweet Sorghum (Sorghum bicolor L. Moench) by Agrobacterium tumefaciens-Mediated Transformation[J]. Scientia Agricultura Sinica, 2011, 44(10): 1989-1996.

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参考文献

[1]刘公社, 周庆源, 宋松泉, 景海春, 谷卫彬, 李晓峰, 苏蔓, Ramachandran Srinivasan. 能源植物甜高粱种质资源和分子生物学研究进展. 植物学报, 2009, 44(3): 253-261.

Liu G S, Zhou Q Y, Song S Q, Jing H C, Gu W B, Li X F, Su M, Srinivasan R. Research advances into germplasm resources and molecular biology of the energy crop sweet sorghum. Chinese Bulletin of Botany, 2009, 44(3): 253-261. (in Chinese)

[2]Antonopoulou G, Gavala H N, Skiadas I V, Angelopoulos K, Lyberatos G. Biofuels generation from sweet sorghum: Fermentative hydrogen production and anaerobic digestion of the remaining biomass. Bioresource Technology, 2008, 99: 110-119.

[3]Zhao Z Y, Cai T, Taglini L, Miller M, Wang N, Pang H, Rudert M, Schroeder S, Hondred D, Seltzer J, Pierce D. Agrobacterium-mediated sorghum transformation. Plant Molecular Biology, 2000, 44: 789-798.

[4]Nguyen T V, Thu T T, Claeys M, Angenon G. Agrobacterium- mediated transformation of sorghum (Sorghum bicolor (L.) Moench) using an improved in vitro regeneration system. Plant Cell Tissue and Organ Culture, 2007, 91(11): 155-164.

[5]Gurel S, Gurel E, Kaur R, Wong J, Meng L, Tan H Q, Lemaux P G. Efficient, reproducible Agrobacterium-mediated transformation of sorghum using heat treatment of immature embryos. Plant Cell Reports, 2009, 28(3): 429-444.

[6]Lu L, Wu X R, Yin X Y, Morrand J, Chen X L, Folk W R, Zhang Z Y J. Development of marker-free transgenic sorghum [Sorghum bicolor (L.) Moench] using standard binary vectors with bar as a selectable marker. Plant Cell Tissue and Organ Culture, 2009, 99(1): 97-108.

[7]Howe A, Sato S, Dweikat I, Fromm M, Clemente T. Rapid and reproducible Agrobacterium-mediated transformation of sorghum. Plant Cell Reports, 2006, 25(8): 784-791.

[8]Girijashankar V, Sharma H C, Kiran K S, Swathisree V, Sivarama P L, Bhat B V, Monique R, Blanca S S, Lakshmi N M, Altosaar I, Seetharama N. Development of transgenic sorghum for insect resistance against the spotted stem borer (Chilo partellus). Plant Cell Reports, 2005, 24: 513-522.

[9]张明洲, 唐乔, 陈宗伦, 刘军, 崔海瑞, 舒庆尧, 夏英武, I Altosaar. 农杆菌介导Bt基因遗传转化高粱. 生物工程学报, 2009, 25(3): 418-423.

Zhang M Z, Tang Q, Chen Z L, Liu J, Cui H R, Shu Q Y, Xia Y W, Altosaar I. Genetic transformation of Bt gene into sorghum (Sorghum bicolor L.) mediated by Agrobacterium tumefaciens. Chinese Journal of Biotechnology, 2009, 25(3): 418-423. (in Chinese)

[10]Cheng X, Sardana R, Kaplan H, Altosaar I. Agrobacterium- transformed rice plants expressing synthetic cryIA (b) and cryIA (c) genes are highly toxic to striped stem borer and yellow stem borer. Proceedings of the National Academy of Sciences of the USA, 1998, 95: 2767-2772.

[11]De Maagd R A, Bosch D, Stiekema W. Bacillus thuringiensis toxin-mediated insect resistant in plants. Trends in Plant Sciences, 1999, 4(1):9-13.

[12]Schnepf H E, Whiteley H R. Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli. Proceedings of the National Academy of Sciences of the USA, 1981, 78: 2893-2897.

[13]Xue J, Liang G M, Crickmore N, Li H T, He K L, Song F P, Feng X, Huang D F, Zhang J. Cloning and characterization of a novel Cry1A toxin from Bacillus thuringiensis with high toxicity to the Asian corn borer and other lepidopteran insects. FEMS Microbiology Letter, 2008, 280(1): 95-101.

[14]Casas A M, Kononowirz A X, Haan T G, Zhang L, Tomer D J, Bressen R A, Hasegawa P W. Transgenic sorghum plants obtained after microprojectile bombardment of immature inflorescence. In Vitro Cellular and Developmental Biology-Plant, 1997, 33: 92-100.

[15]Pandey A K, Bhat B V, Balakrishna D, Seetharama N. Genetic transformation of sorghum (Sorghum bicolor (L.) Moench.). International Journal of Biotechnology and Biochemistry, 2010, 6(1): 45-53.

[16]Jeoung M J, Krishnaveni S, Muthukrishnan S, Trick H N, Liang G H. Optimization of sorghum transformation parameters using genes for green fluorescent protein and β-glucuronidase as visual markers. Hereditas, 2002, 137: 20-28.

[17]Gao Z S, Jayaraj J, Muthukrishnan S, Claflin L, Liang G H. Efficient genetic transformation of sorghum using a visual screening marker. Genome, 2005, 48(2): 321-333.

[18]Able J A, Rathus C, Godwin I D. The investigation of optimal bombardment parameters for transient and stable transgene expression in sorghum. In Vitro Cellular and Developmental Biology-Plant, 2001, 37: 341-348.

[19]Tadesse Y, Sagi L, Swennen R, Jacobs M. Optimization of transformation conditions and production of transgenic sorghum (Sorghum bicolor) via microparticle bombardment. Plant Cell Tissue and Organ Culture, 2003, 75: 1-18.

[20]Indra Arulselvi P, Michael P, Umamaheswari S, Krishnaveni S. Agrobacterium mediated transformation of Sorghum bicolor for disease resistance. International Journal of Pharma and Bio Sciences, 2010, 1(4): 272-281.

[21]Devi P B, Sticklen M B. In vitro culture and genetic transformation of sorghum by microprojectile bombardment. Plant Biosystems, 2003, 137(3): 249-254.

[22]Carvalho C H S, Zehr U B, Gunaratna N, Anderson J M, Kononowicz H H, Hodges T K, Axtell J D. Agrobacterium-mediated transformation of sorghum: Factors that affect transformation efficiency. Genetics and Molecular Biology, 2004, 27(2): 259-269.

[23]Jogeswar G, Ranadheer D, Anjaiah V, Kishor P B K. High frequency somatic embryogenesis and regeneration in different genotypes of Sorghum bicolor (L.) Moench from immature inflorescence explants. In Vitro Cellular and Developmental Biology-Plant, 2007, 43: 159-166.

[24]MacKinnon C, Gunderson G, Nabors M W. Plant regeneration by somatic embryogenesis from callus cultures of sweet sorghum. Plant Cell Reports, 1986, 5: 349-351.

[25]Ma H T, Gu M H, Liang G H. Plant regeneration from cultured immature embryos of Sorghum bicolor (L.) Moench. Theoretical and Applied Genetics, 1987, 73: 389-394.

[26]Rao A M, Padma Sree K, Kavi Kisho P B K. Enhanced plant regeneration in grain and sweet sorghum by asparagine, praline and cefotaxime. Plant Cell Reports, 1995, 15: 72-75.

[27]Zhao L M, Liu S J, Song S Q. Optimization of callus induction and plant regeneration from germinating seeds of sweet sorghum (Sorghum bicolor Moench). African Journal of Biotechnology, 2010, 9(16): 2367-2374.

[28]Raghuwanshi A, Birch R G. Genetic transformation of sweet sorghum. Plant Cell Reports, 2010, 29(9): 997-1005.

[29]Wang Y B, Lang Z H, Zhang J, He K L, Song F P, Huang D F. Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize (Zea mays L.). Chinese Science Bulletin, 2008, 53(20): 3185-3190.

[30]岳同卿, 郎志宏, 王延锋, 张杰, 何康来, 黄大昉. 转Bt cry1Ah基因抗虫玉米的获得及其遗传稳定性分析. 农业生物技术学报, 2010, 18(4): 638-644.

Yue T Q, Lang Z H, Wang Y F, Zhang J, He K L, Huang D F. Acquirement of the transgenic maize harboring Bt cry1Ah gene and analysis of its inheritable stability. Journal of Agricultural Biotechnology, 2010, 18(4): 638-644. (in Chinese)

[31]卢美贞, 崔海瑞, 姚艳玲, 金基强, 刘芦苇, 舒庆尧. 不同发育时期转基因高粱不同株系Cry1Ab蛋白含量的比较. 农业生物技术学报, 2006, 14(5): 826-827.

Lu M Z, Cui H R, Yao Y L, Jin J Q, Liu L W, Shu Q Y. Comparison on Cry1Ab protein content in different transgenic sorghum lines at different developmental stages. Journal of Agricultural Biotechnology, 2006, 14(5): 826-827. (in Chinese)

[32]Emani C, Sunilkumar G, Rathore K S. Transgene silencing and reactivation in sorghum. Plant Science, 2002, 162: 181-192.

[33]石春林, 朱祯, 徐鸿林, 肖桂芳, 冯平章. 转基因烟草中Bt毒蛋白基因的表达行为. 植物学报, 2000, 42(3): 269-273.

Shi C L, Zhu Z, Xu H L, Xiao G F, Feng P Z. Expression behavior of Bt toxin gene in transgenic tobaccos. Acta Botanica Sinica, 2000, 42(3): 269-273. (in Chinese)

[34]李汉霞, 张晓辉, 付雪林, 张余洋, 张俊红, 王涛涛, 叶志彪. 双Bt基因提高青花菜对菜粉蝶和小菜蛾抗性. 农业生物技术学报, 2010, 18(4): 654-662.

Li H X, Zhang X H, Fu X L, Zhang Y Y, Zhang J H, Wang T T, Ye Z B. Two different Bacillus thuringiensis toxin genes confer resistance to diamondback moth and cabbage worm in transgenic broccoli. Journal of Agricultural Biotechnology, 2010, 18(4): 654-662. (in Chinese)