Genetic Analysis of cryIAC Gene in Transgenic Maize Plants and Its Effects on Insect-resistance

doi:10.13560/j.cnki.biotech.bull.1985.2015.01.019

Abstract

Abstract: In order to analyse the genetic regularity of target gene cryIAc in transgenic and their descendant plants（lines）, and to study the effect of target gene on insect-resistant activity of transgenic plants at the same time, this research work was conducted. Firstly, cryIAc gene was transformed into maize inbred ‘Zheng 58’ and ‘Chang7-2’ separately by pollen-mediated transformation method. PCR, Southern, ELISA analysis and bioassay in the field of each generation transgenic plants were then conducted according to experimental requirements. Results showed that ⑴ A total of 24 and 41 transgenic plants were obtained through transformed cryIAc gene into maize inbred ‘Zheng 58’ and ‘Chang7-2’, respectively. ⑵ The molecular detection results of transgenic T₂, hybridization F₂ and backcross B₁plants suggested that the segregation of target gene in these lines followed 3: 1, 3: 1 and 1: 1 segregation ratio of the Mendel laws of heredity, respectively. ⑶ The molecular detection results of T₁ to T₄ transgenic plants（lines）showed that the target gene could stably inherited and expressed effectively, the expression amount of the target gene was from 9.8 to 14.3 ng/gleaf fresh weight. ⑷ Moreover, the results of bioassay in the field indicated that in the case of high insect-susceptibility of negative control line, the transgenic lines still showed highly insect-resistant activity. ⑸ In addition, the target gene integrated into genomics of transgenic plants could inherit to the next generation plants by hybridization. ⑹ At last, 5 high insect-resistant transgenic lines, SZ003, SZ005, SC001, SC004 and SC007, was gained by screening. The pollen-mediated transformation method was a effective and shortcut tool used in plant transformation, cryIAc gene could confer and enhance insect-resistant activity of transgenic plants（lines）tansfomated with it.

Key words: transgenic maize, cryIAc gene, genetic regularity, insect-resistance

Wang Jianjun, Yang Huizhen, Liu Jiao. Genetic Analysis of cryIAC Gene in Transgenic Maize Plants and Its Effects on Insect-resistance[J]. Biotechnology Bulletin, 2015, 31(1): 122-130.

References

[1] 林良斌, 官春云.毒蛋白基因与植物抗虫基因工程[J].生物工程进展, 1997, 17（2）：51-55.
[2] 袁胜亮, 李明, 周国娜, 等.浅析抗虫基因种类及抗虫原理[J].安徽农业科学, 2007, 35（31）：9963-9964.
[3] Fromm ME, Morrish F, Armstrong C, et al. Inheritance and expression of chimaeric genes in the progeny of transgenic maize plants[J]. Biotechnology, 1990, 8（9）：833-839.
[4] Gordon-Kamm WJ, Spencer TM, Mangano ML, et al. Transformation of maize cells and regeneration of frtile transgenic plants[J]. Plant Cell, 1990, 2（7）：603-618.
[5] Lauer J, Wedberg J. Grain yield of initial Bt corn hybrid introducti-ons to farmers in the northern Corn Belt[J]. Journal of Production Agriculture, 1999, 12（3）：373-376.
[6] Armstrong CL, Parker GB, Pershing JC, et al. Field evaluation of European corn borer control in progeny of 173 transgenic corn events expres-sing an insecticidal protein from Bacillus thuringiensis[J]. Crop Science, 1995, 35（2）：550-557.
[7] Labatte JM, Meusnier S, Migeon A, et al. Field evaluation of and modeling the impact of three control methods on the larval dynamis of Ostrinia nubilalis（Lepidoptera：Pyralidae）[J]. Journal of Economic Entomology, 1996, 89（4）：852-862.
[8] 丁群星, 谢友菊, 戴景瑞, 等.用子房注射法将Bt 毒蛋白基因导入玉米的研究[J].中国科学：B辑, 1993, 23（7）：707-713.
[9] 王国英, 杜天兵, 张宏, 等.用基因枪将Bt 毒蛋白基因转入玉米及转基因植株再生[J].中国科学：B辑, 1995, 25（1）：71-77.
[10] 袁英, 李晓辉, 孔祥梅, 等. Bt 基因转化玉米培育抗玉米螟自交系[J].分子植物育种, 2007, 5（4）：572-576.
[11] Budar F, Thia-Toongl L, Van MM, Hernalseens JP. Agrobacterium- mediated gene transfer results mainly in transgenic plants transmitting as a single Mendelian factor[J]. Genetics, 1986, 114（1）：303-313.
[12] Walters DA, Vetsch CS, Potts DE, Lundquist RC. Transformation and inheritance of a hygramycin phosphoto transferase gene in maize plants[J]. Plant Molecular Biology, 1992, 18（2）：189-200.
[13] Spencer TM, James VO, William GS, et al. Segregation of transgenes in maize[J]. Plant Molecular Biology, 1992, 18（2）：201-210.
[14] Christon P, Swain WF, Yang NS, Mccabe DE. Inheritance and expression of foreign gene in transgenic soybean plants[J]. Proc Nati Acad Sci USA, 1989, 86（19）：7500-7504.
[15] 王守才, 王国英, 丁群星, 等.转基因在玉米中的遗传分离与整合特性的研究[J].遗传学报, 1999, 26（3）：254-261.
[16] 王得元, 殷秋秒, 石尧清, 李乃坚.外源基因在作物基因组的整合特性及遗传分析[J].湖北农学院学报, 1999, 19（2）：113-115.
[17] 姚方印, 朱常香, 李广贤, 温孚江. Bt 水稻的抗虫性鉴定及转基因的遗传分析[J].中国农业科学, 2002, 35（2）：142-145.
[18] 朱卫红, 王庆东, 刘宗华, 等.玉米转Bt基因后代的抗虫性鉴定及其遗传分析[J].河南农业大学学报, 2004, 38（2）：135-138.
[19] 王国英, 张宏, 谢友菊, 等.玉米胚性愈伤组织转化及转Bt基因植株的抗虫性[J].农业生物技术学报, 1995, 3（3）：49-53.
[20] 李余良, 胡建广, 苏菁, 刘建华.子房注射法将Bt基因导入超甜玉米[J].玉米科学, 2005, 13（1）：41-43.
[21] Deroles SC, Gardner RC. Expression and inheritance of kanamycin resistance in a large number of transgenic Petunias generated by Agrobacterium-mediated transformation[J]. Plant Molecular Biology, 1988, 11（3）：355-364.
[22] Brandle JE, Mchugh SG, James L, et al. Instability of transgene exptession in field grown obacco carring the csrL-L gene for sulfonylurea herbicide resistance[J]. Biotechnology, 1995, 13（9）：994-998.
[23] Matzke MA, Matzke AJM. How and why do plants inactivation homologous（trans）gene?[J]. Plant Physiology, 1995, 107（3）：679-685.
[24] Richard AJ. Cosuppression, flower clolor pattern and matastable gene expression states[J]. Science, 1995, 268（5211）：686-691.
[25] 孙毅, 崔贵梅, 郝曜山, 等.超声波辅助花粉介导植物转基因方法.中国, 201110041484.0[P] . 2011-02-18.
[26] Sanbrook J, Fritsch EF, Maniatis T. Molecular cloning：A Laboratory Manual[M] . 2^nd ed. New York：Cold Spring Harbor Laboratory Press, 1989：19-27, 474-490.
[27] 杨文鹏, 王明春, 杨留启, 王伟.玉米总蛋白质提取技术和蛋白质样品浓度测定方法的比较[J].种子, 2010, 29（12）：18-23.
[28] 杜建中, 孙毅, 王景雪, 等.转水稻NibT基因玉米植株的获得及抗病性研究[J].西北植物学报, 2011, 31（5）：893-901.
[29] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 1976, 72（1-2）：248-254.
[30] 张红伟, 张红梅, 郑祖平, 等. Bt转基因玉米的获得及其对玉米螟的抗性分析[J].西北植物学报, 2004, 24（7）：1266-1270.
[31] Michael GK, Gary LB, Cindy B, et al. Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis[J]. Biotechnology, 1993, 11（2）：194-200.
[32] 李慧芬, 李旭刚, 刘翔, 等.转苏云金杆菌毒蛋白基因玉米植株的获得及其抗虫性分析[J].植物学报, 2002, 44（6）：684-688.
[33] 朱常香, 宋云枝, 张杰道, 等.抗虫、抗除草剂转基因玉米的获得及遗传研究[J].山东农业大学学报：自然科学版, 2002, 33（2）：120-125.
[34] Linn F, Heidmann I, Safdler H, Meyer P. Epigenetic changes in the expression of the maize A1 gene in Petunia hybrid：role of number of integrated gene copies and state of methylation[J]. Molecular Genetics and Genomics, 1990, 222（2-3）：329-336.
[35] Sangtong V, Moran L, Chikwamba R, et al. Expression and inheritance of the wheat Glu-1DX5 gene in transgenic maize[J]. Theoretical and Applied Genetics, 2002, 105（6-7）：937-945.
[36] 郭东全, 杨向东, 包绍君, 等.转CryIA和CpTI双价抗虫基因大豆的获得与稳定表达[J].中国农业科学, 2008, 41（10）：2957-2962.
[37] 王金凤, 李钊, 杜丽璞, 等.转PvPGIP2基因小麦的获得与纹枯病抗性鉴定[J].植物遗传资源学报, 2013, 14（1）：179-183.
[38] Steven RS, Larry RH. Insect bioassay for determining soil degrada-tion of Bacillus thuringiensis subsp. Kurstaki Cry1A（b）protein in corn tissue[J]. Environmental Entomology, 1996, 25（3）：659-664.
[39] Saxena D, Stotzky G. Bacillus thuringensis（Bt）toxin released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil[J]. Soil Biology & Biochemistry, 2001, 33（9）：1225-1230.
[40] 路兴波, 孙红炜, 杨崇良, 等.转基因玉米外源基因通过花粉漂移的频率和距离[J].生态学报, 2005, 25（9）：2450-2453.
[41] 梁革梅, 谭维嘉, 郭予元.棉铃虫对Bt 的抗性筛选及交互抗性研究[J].中国农业科学, 2000, 33（4）：46-53.
[42] 高翔, 别晓敏, 佘茂云, 叶兴国.安全型转基因植物培育技术研究进展[J].植物遗传资源学报, 2009, 10（4）：607-612.