Cry2Aa基因优化设计及功能验证  

Optimization and Functional Verification of Cry2Aa Gene

作　　者：翁绿水[1,2] 陈芬[1] 肖国樱[1] 

机构地区：[1]中国科学院亚热带农业生态研究所亚热带农业生态过程重点实验室,长沙410125 [2]中国科学院大学,北京100049

出　　处：《农业生物技术学报》2013年第11期1261-1269,共9页Journal of Agricultural Biotechnology

基　　金：转基因生物新品种培育科技重大专项(No.2011ZX08001-003)

摘　　要：原核生物苏云金杆菌（Bacillus thuringiensis）的杀虫蛋白基因Cry2Aa不仅GC含量低，而且包含影响在真核生物中高效表达元件的结构，如果不加以改造而直接转入水稻中，不仅表达量低而且抗虫效果差。本研究首先通过同义密码子替换，提高了优化基因中GC含量和水稻（Oryza sativa L.）偏爱密码子的比例，消除原始基因中的Poly（A）加尾识别信号序列和内含子切割识别序列，然后在优化基因的5＇端添加转运信号肽序列PR1a和3＇端添加内质网定位序列KDEL，采用Mfold软件优化mRNA二级结构，最终获得的优化基因Cry2Aa#的GC含量达到57.3%，碱基改变了25.87%，密码子改变了67.51%；消除了在真核生物中影响mRNA稳定的序列，密码子使用频率与水稻基因组基本保持一致；另外，消除了优化基因中存在的9个茎环等mRNA二级结构，其总自由能也相应降低，相比优化之前降低了23.9 kcal/mol。利用优化基因Cry2Aa#构建植物表达载体，转化水稻成熟胚诱导愈伤组织获得转基因植株，其分蘖期叶片中Cry2Aa#蛋白的表达量为8.62~18.88 μg/g鲜重，对稻纵卷叶螟（Cnaphalocrocis medinalis）的抗性好，说明优化策略可行。本研究为外源基因优化设计提供重要参考数据，为水稻抗螟虫育种提供了优良抗虫基因资源。Cry2Aa gene from prokaryotic organism Bacillus thuringiensis consists of low GC content and some elements that affect gene expression in eukaryotic organisms, and the introduction of the unmodified gene into rice resulted in low levels of protein expression and insect resistance. In this paper, the proportion of rice （Oryza sativa L.） preferred codons and GC content in optimized Cry2Aa gene were increased, the potential Poly（A） addition signal sequences and the intron-exon boundary sequences were eliminated by synonymous codon substitution; then the optimized Cry2Aa gene was added with PR1a signal peptide sequence at 5＇ end and KDEL sequence at 3＇ end, and the secondary structure of its mRNA was optimized by using Mfold software. As a result, the GC content of the optimized gene Cry2Aa# was 57.29%，25.87% of the total nucleotides and 67.51% of the total codons were replaced in the optimized Cry2Aa# gene; the sequences which had been shown to destabilize mRNA in eukaryotic organisms were eliminated, codon usage frequency in the optimized Cry2Aa# gene kept consistent with the rice genome; In addition, nine stable mRNA stem-loop structures were eliminated, and the free energy of mRNA of the optimized Cry2Aa# gene decreased by 23.9 kcal/mol comparing with the free energy before optimization. The plant expression vector containing the optimized Cry2Aa# gene was constructed, and the optimized Cry2Aa# gene was transformed into rice via Agrobacterium-mediated method; the results of ELISA showed that the Cry2Aa# protein concentration ranged from 8.62 to 18.88 μg/g fresh weight （FW） of leaf at tillering stage in different transgenic lines; the results of bioassay showed that the transgenic lines were well resistant to rice leaf roller （Cnaphalocrocis medinalis）; that indicated the approaches for optimization of Cry2Aa# gene were viable. This study not only validats the methods for gene optimization, but also provides an excellent insect-resistant genetic resource for rice breeding.

关 键 词：Cry2Aa 偏爱密码子 密码子优化 水稻 

分 类 号：S332.3[农业科学—作物遗传育种]