全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用:99美元

查看量下载量

相关文章

更多...

杀虫基因Cry1Ab的合成、表达及其抗虫性鉴定

DOI: 10.7668/hbnxb.2014.06.023, PP. 131-135

Keywords: Cry1Ab,人工合成,原核表达,玉米螟,抗虫鉴定

Full-Text   Cite this paper   Add to My Lib

Abstract:

对苏云金芽孢杆菌Cry1Ab野生型基因的编码区序列进行改造,以获得密码子优化的抗虫基因,构建原核表达载体后,将重组载体转入大肠杆菌进行诱导表达并对诱导蛋白进行抗虫试验。首先根据植物密码子的偏好性及使用频率,优化、改造并人工合成了Cry1Ab基因,序列分析表明:改造后的Cry1Ab基因全长1848bp,与原始序列同源性为66%,G+C含量由原来的37.34%提高到63.04%。将改造后的Cry1Ab基因与原核表达载体pET28b(+)连接,转化大肠杆菌BL21(DE3),阳性菌液进行诱导表达后,SDS-PAGE分析结果显示:Cry1Ab蛋白在BL21(DE3)细胞中得到高效表达,分子量为70kDa。喂虫试验结果表明:诱导表达的蛋白对玉米螟具有很强的毒性,幼虫死亡率达到93.33%,而且,存活的玉米螟生长发育也受到明显抑制。该抗虫基因可以作为培育转基因抗虫作物的候选基因。

References

[1]  Gonin P,Pirzadeh B,Gagnon C A,et al.Seroneutralization of porcine reproductive and respiratory syndrome virus correlates with antibody response to the GP5 major envelope glycoprotein[J].Journal of Veterinary Diagnostic Investigation,1999,11(1):20-26.
[2]  Vaeck M,Reynaerts A,Hofte H,et al.Transgenic plants protected from insect attack [J].Nature,1987(6125),328:33-37.
[3]  Adang M J,Staver M J,Rocheleau T A,et al.Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp.kurstaki HD-73 and their toxicity to Manduca sexta [J].Gene,1985,36(3):289-300.
[4]  Perlak F J,Deaton R W,Armstrong T A,et al.Inset resistant cotton plants[J].Bio Technology,1990,8(10):939-943.
[5]  Goodall G J,Filipowicz W.The AU-rich sequences present in the introns of plant nuclear pre-mRNAs are required for splicing[J].Cell,1989,58(5):473-483.
[6]  Joshi C P.Putative polyadenylation signals in nuclear genes of higher plants:acompilation and analysis[J].Nucleic Acids Research,1987,15(23):9627-9640.
[7]  Shaw G,Kamen R.A conserved AU sequence from the 3'' untranslated region of GM-CSF mRNA mediates selective mRNA degradation[J].Cell,1986,46(5):659-667.
[8]  Adang M J,Brody M S,Cardineau G.The reconstruction and expression of a Bacillus thuringiensis cry3A gene in protoplasts and potato plants[J].Plant Molecular Biology,1993,21(6):1131-1145.
[9]  Perlak F J,Fuchs D A,Dean S L,et al.Modification of the coding sequence enhances plant expression of insect control protein genes[J].Proceedings of the National Academy of Sciences of the United States of America,1991,88:3324-3328.
[10]  郭三堆,倪万潮,徐琼芳.携带编码杀虫蛋白质融合基因的表达载体及其转基因植物[P].中国专利,95119563.8,1996-11-06.
[11]  刘允军.人工改造的 crylAc、 crylIe 基因在大肠杆菌、转基因烟草和玉米中的表达[D].北京:中国农业大学,2004.
[12]  王悦冰,郎志宏,张 杰,等.利用ubi1内含子增强Bt Cry1Ah 基因在转基因玉米中的表达[J].科学通报,2008,53(17):2041-2046.
[13]  赖锦盛,董永彬,宋伟彬,等.人工合成用于转基因抗虫植物的Bt杀虫基因[P].中国专利,200910082840.6,2009-11-18.
[14]  董 伟,董晓慧,楚雍烈,等.人杀菌渗透增强性蛋白N末端片段在大肠杆菌中的表达[J].西安交通大学学报:医学版,2006,27(1):11-14.
[15]  戴双双,何凤田,杨朝辉,等.人可溶性APRIL基因的克隆、表达及生物学活性检测[J].中国生物化学与分子生物学报,2005,21(1):61-64.
[16]  杨艳华,戴 莉,陈克平.Bt转基因抗虫水稻的研究进展及其对家蚕的风险评估[J].河南农业科学,2013,42(2):1-6.
[17]  Jansens S,van Vliet A,Dickburt C,et al.Transgenic corn expressing a Cry9C insecticidal protein from Bacillus thuringiensis protected from European corn borer damage [J].Crop Science,1997,37(5):1616-1624.
[18]  胡晓婷,宋福平,冯书亮,等.Bt分子生物学研究进展[J].华北农学报,2007,22(Z2):6-9.
[19]  Fishhoff D A,Bowdish K S,Perlak F J.Insect transgenic tomato[J].Biotechnology,1987,5:807-813.
[20]  Koziel M G,Beland G L,Bowman C,et al.Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis [J].Biotechnology 1993,11(2):194-200.
[21]  李圣彦,郎志宏,朱 莉,等.利用密码子优化提高Bt CrylAh 基因在转基因玉米(Zea mays L.)中的表达[J].中国农业科技导报,2011,13(6):20-26.

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133