CN115029372A - 一种基于优化起始密码子aug附近核苷酸序列提高翻译起始效率的方法 - Google Patents
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Abstract
本发明公开了一种基于优化起始密码子AUG附近核苷酸序列提高翻译起始效率的方法,该方法适用于植物细胞,要求在起始密码子AUG附近增加“AAAA”和“GCG”核苷酸,便能显著提高翻译起始效率。本发明操作简单、效率高、可重复性强,可通过在翻译水平上提高蛋白产量。
Description
技术领域
本发明属于生物分子检测技术领域,尤其涉及一种基于优化起始密码子AUG附近核苷酸序列提高翻译起始效率的方法。
背景技术
基因功能研究或者基因工程往往需要大量地表达候选蛋白。目前,在植物中,最常用的方法是用强启动子驱动更多mRNA产生从而提高蛋白的表达量。利用这种方法来提高蛋白表达量有时并不稳定,其原因在于过量mRNA的存在会引起RNA干涉现象从而导致mRNA的降解。如何基于基因自身启动子而提高蛋白表达无疑具有十分重要的意义。核糖体是将mRNA翻译成蛋白质的细胞器。根据扫描模型(Hinnebusch,2014),核糖体43亚基将从mRNA的5’端扫描mRNA以发现起始密码子AUG,并在该位置组装形成具有功能的核糖体,从而启动翻译。在动物细胞中,序列GCCRCCAUGG(R表示核苷酸A或者G,AUG为起始密码子)通过显著提高翻译起始效率而增加蛋白表达量(Kozak,1986,1987)。在植物细胞中,已有研究结果表明起始密码子AUG附近的核苷酸序列能够影响翻译起始的效率(Kim et al.,2014),但该序列的具体信息并未被特征化;另外,起始密码子AUG的下游核苷酸在多大程度上影响翻译起始效率,也不清楚。
发明内容
本发明的目的在于针对现有技术的不足,提供一种基于优化起始密码子AUG附近核苷酸序列提高翻译起始效率的方法。
本发明采用的技术方案具体如下:
一种基于优化起始密码子AUG附近核苷酸序列提高翻译起始效率的方法,该方法是:在构建载体时,通过在候选基因的起始密码子AUG前添加“AAAA”和/或在候选基因的起始密码子AUG后添加“GCG”核苷酸从而提高目的基因的翻译起始效率。
进一步地,所述目的基因为编码蛋白的基因。
进一步地,所述载体为植物表达载体。
本发明的有益效果是:本发明在构建载体时,通过在起始密码子AUG前后增添“AAAA”和/或“GCG”核苷酸,能显著提高目的基因的蛋白表达量。
附图说明
图1为起始密码子AUG附近的核苷酸序列分析;起始密码子AUG对应的位置分别为+1,+2,+3位,其上游紧邻AUG的位置为-1位,下游紧邻AUG的为+4位;图中,从下至上依次为A、U、G、C出现的频率;每个位置处左列为植物精细胞表达的mRNA、右列为植物体细胞表达的mRNA统计结果;
图2为评估上下游序列对翻译起始效率的影响;其中,A为PC、OUC、ODC、4A、GCG、4A-GCG 6种转基因系中H2B基因的起始密码子AUG前后(-18至+18位)的核苷酸序列;B-G为PC、OUC、ODC、4A、GCG、4A-GCG 6种转基因系的荧光图,H是PC、OUC、ODC、4A、GCG、4A-GCG 6种转基因系的荧光强度统计对比图。
具体实施方式
本发明提供了一种基于优化起始密码子AUG附近核苷酸序列提高翻译起始效率的方法,可以简单有效地提高候选基因的蛋白表达量。这种方法是基于提高核糖体在起始密码子处的翻译起始效率来完成的。本方法针对的是植物材料和植物表达载体,因此,仅适用于植物细胞。
本发明需要在表达的候选基因的起始密码子AUG前后添加“AAAA”和“GCG”核苷酸。添加该序列可以在构建载体时完成。具体做法:在合成引物时,人为引入上述序列;引物合成后,通过PCR再次扩增候选基因,连入表达载体,便能将“AAAAAUGGCG”(SEQ ID NO.1)置换原来的起始密码子AUG核苷酸。
实施例1:起始密码子AUG前后的核苷酸序列分析。
为解析起始密码子AUG前后核苷酸的分布特征,选择了1000个在植物精细胞表达和1000个在植物体细胞表达的mRNA起始密码子AUG附近(-18至+18位)的核苷酸序列进行序列分析。在每一个位置上,如果核苷酸的分布没有偏向性,则出现的频率应为25%左右(即四种核苷酸A,U,G,C,应该等概率出现)。但分析结果如图1所示,表明无论是在精细胞还是体细胞中,在-1至-4位,核苷酸A出现的频率明显偏高,为40%-60%左右;同时在+4至+6位,GCG出现的频率为40-60%。该分析表明AUG起始密码子附近的核苷酸呈现明显的偏向分布,尤其是-1至-4位,+4至+6位。
实施例2:4个腺嘌呤核苷酸的存在显著提高了蛋白表达量。
评估起始密码子AUG前后的核苷酸对翻译起始效率的影响。为探究起始密码子AUG前后的核苷酸是否影响翻译起始效率,构建了PC,OUC,ODC转基因系。这些转基因系利用精细胞特异表达的启动子proHTR10,驱动融合基因H2B-GFP的表达。但在不同的载体中,H2B基因的起始密码子AUG前后的核苷酸被替换成如图2A所示,表达的融合蛋白H2B-GFP会定位于细胞核,其GFP的荧光强度能够反映融合蛋白H2B-GFP的蛋白量。在PC转基因系中,AUG起始密码子前18个和后15个核苷酸被置换成各位置出现频率最低的核苷酸,序列为SEQ IDNO.2:CCCCCCCCCCCCCGCCGUAUGCUCCGCCGCCGCCGC(AUG为起始密码子),其候选基因的蛋白表达量呈现低丰度,对应的荧光强度值为12.3(来自于11个转基因系的统计结果)。而在OUC(SEQ ID NO.3:AAAAAAAAAAAAAAAAAAAUGCUCCGCCGCCGCCGC),ODC(SEQ ID NO.4:CCCCCCCCCCCCCGCCGUAUGGCGAAGGCAGAUAAA),荧光强度值分别为102.0(来自于12个转基因系的统计结果)和33.2(来自于10个转基因系的统计结果)。表明AUG前18个腺嘌呤核苷酸显著提高了翻译起始效率,而后15个核苷酸的优化有助于提高翻译起始效率。
进一步通过构建4A(SEQ ID NO.5:CCCCCCCCCCCCCGAAAAAUGCUCCGCCGCCGCCGC),GCG(SEQ ID NO.6:CCCCCCCCCCCCCGCCGUAUGGCGCGCCGCCGCCGC),4A-GCG(SEQ ID NO.7:CCCCCCCCCCCCCGAAAAAUGGCGCGCCGCCGCCGC)转基因系,测量出其荧光强度分别为88.2(来自于12个转基因系的统计结果),32.2(来自于11个转基因系的统计结果),103.7(来自于13个转基因系的统计结果),表明AUG起始密码子上游的-1至-4位的腺嘌呤核苷酸是提高翻译起始效率的主效位点,同时,起始密码子AUG的下游效应位点为GCG。因此,对比于动物中的kozak序列(GCCRCCAUGG),植物中有效提高翻译起始的序列为AAAAAUGGCG(AUG为起始密码子)。这些结果表明在植物中表达蛋白时,可以在候选基因的起始密码子AUG前增加一段序列“AAAA”及起始密码子AUG后增加“GCG”来显著提高蛋白表达量。
显然,上述实施例仅仅是为清楚地说明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其他不同形式的变化或变动。这里无需也无法把所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明的保护范围。
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Claims (3)
1.一种基于优化起始密码子AUG附近核苷酸序列提高翻译起始效率的方法,其特征在于,该方法是:在构建载体时,通过在候选基因的起始密码子AUG前添加“AAAA”和/或在候选基因的起始密码子AUG后添加“GCG”核苷酸从而提高目的基因的翻译起始效率。
2.如权利要求1所述基于优化起始密码子AUG附近核苷酸序列提高翻译起始效率的方法,其特征在于,所述候选基因为编码蛋白的基因。
3.如权利要求1所述基于优化起始密码子AUG附近核苷酸序列提高翻译起始效率的方法,其特征在于,所述载体为植物表达载体。
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刘庆坡 等: ""起译密码子AUG侧翼序列对水稻基因表达水平的影响"", 《中国农业科学》, vol. 37, no. 5, pages 625 - 629 * |
张莉: ""起始密码子上游核苷酸影响拟南芥精细胞翻译起始效率的研究"", 《中国优秀硕士学位论文全文数据库 基础科学辑》, no. 2, pages 8 - 10 * |
曹媛: ""拟南芥精细胞中的翻译起始调控研究"", 《中国优秀硕士学位论文全文数据库 基础科学辑》, no. 6, pages 38 - 41 * |
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Application publication date: 20220909 |