苹果U6启动子的克隆及功能分析

doi:10.3864/j.issn.0578-1752.2019.23.016

摘要/Abstract

摘要：

【目的】U6启动子是CRISPR/Cas9基因组编辑载体系统中驱动sgRNA转录的重要元件,其可能存在物种特异性因子,且长度不同转录活性存在差异。迄今在苹果（Malus×domestica）上对U6启动子尚缺乏研究。因此,筛选出转录活性高且片段大小合适的苹果U6启动子,可以优化苹果CRISPR/Cas9基因编辑体系。【方法】利用软件DNAMAN以及启动子元件在线分析网站PLACE和plant CARE对苹果U6启动子进行比对分析;克隆并构建U6启动子驱动萤火虫荧光素酶基因（Firefly luciferase,LUC）的融合表达载体,利用农杆菌介导的瞬时转化法分别转染苹果愈伤组织和本氏烟草（Nicotiana benthamiana）叶片;通过检测荧光素酶活性对各U6启动子进行转录活性比较。【结果】苹果基因组中共检索到6条U6 snRNA（E-value<3e ^-40）,分别位于第6、7、9、10、15和17号染色体上,取5′端27 bp snRNA及其上游1 500 bp作为候选U6启动子。序列比对结果显示,苹果U6启动子与拟南芥相同,均具有两个保守的元件,包括上游序列元件（Upstream sequence element,USE）和TATA-Like box。瞬时转化后荧光素酶活性检测结果显示,10号染色体上的U6启动子转录活性最高,10号染色体上5′端截短的U6启动子（长度分别为1 500、959、275和116 bp）中275 bp的启动子活性最强。另外,在苹果愈伤组织中,苹果U6启动子的转录活性要显著高于拟南芥U6启动子。【结论】从苹果基因组克隆6条U6启动子,并筛选出一条转录活性高且片段长度较短的U6启动子。

关键词: 苹果, U6启动子, 荧光素酶, 本氏烟草, 愈伤组织

Abstract:

【Objective】U6 promoter is an important element for the transcription of sgRNA in the CRISPR/Cas9 genome editing system. There may be species-specific factors in U6 promoter, and the activity of U6 promoter would be altered when its length changed. So far, in apple (Malus×domestica), the transcriptional characterization of U6 promoters has not been reported. Therefore, selecting an apple U6 promoter with high transcriptional activity and suitable fragment size would provide a basis for optimizing apple CRISPR/Cas9 gene editing technology system. 【Method】 DNAMAN, promoter cis element online predicting website PLACE and plant CARE were used to do the comparative analysis of apple U6 promoters; U6 promoters were cloned and constructed into firefly luciferase vector; the apple callus and tobacco (Nicotiana benthamiana) leaves were transformed via Agrobacterium-mediated transient transformation; the transcriptional activity of U6 promoter was determined according to the luciferase activity. 【Result】 There were six alternative U6 promoters in apple genome (E-value<3e ^-40), which were located on chr 6, chr 7, chr 9, chr 10, chr 15 and chr 17, respectively. 27 bp snRNA at 5′ end and its upstream 1 500 bp were selected as candidate U6 promoter. Sequence analysis results showed that the upstream sequence element (USE) and TATA-like elements were contained in six U6 promoters of apple, the same as Arabidopsis. After transient transformation, the luciferase activity assay showed that, the U6 promoter on chromosome 10 had the highest transcriptional activity. Among all U6 promoters which were shortened at 5′ end (1 500 bp, 959 bp, 275 bp, and 116 bp) on chromosome 10, the 275 bp one had the highest transcriptional activity. In addition, compared with the Arabidopsis U6 promoter, in apple callus, the transcriptional activity of the apple U6 promoter was higher. 【Conclusion】Six U6 promoters were cloned from the apple genome, and a U6 promoter with high transcriptional activity and suitable sequence fragment was obtained.

Key words: Malus×domestica, U6 promoter, luciferase, Nicotiana benthamiana, callus

卞书迅,韩晓蕾,袁高鹏,张利义,田义,张彩霞,丛佩华. 苹果U6启动子的克隆及功能分析[J]. 中国农业科学, 2019, 52(23): 4364-4373.

BIAN ShuXun,HAN XiaoLei,YUAN GaoPeng,ZHANG LiYi,TIAN Yi,ZHANG CaiXia,CONG PeiHua. Cloning and Functional Analysis of U6 Promoter in Apple[J]. Scientia Agricultura Sinica, 2019, 52(23): 4364-4373.

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引物名称 Prime name	上游引物 Forward sequence (5′-3′)	下游引物 Reverse sequence (5′-3′)
MdU6-6P	F：ggtaccTTTGGAGTTGAAGGATTT	R;aagcttAATTTTATCGGATGTCCC
MdU6-7P	F：ggtaccCCCCCGTTTGGATGACCCA	R：aagcttAATTTTATCGGATGTCCC
MdU6-9P	F：aagcttATGCTTCTCCCATGGAAAT	R：ggatccAATTTTATCGGATGTCCC
MdU6-10P	F：ggtaccTGGTGACATTGAGGTTCT	R：aagcttAATTTTATCGGATGTCCC
MdU6-15P	F：ggtaccTTTGGCGTTGCATTAG	R：ggatccAATTTTATCGGATGTCCC
MdU6-17P	F：ggtaccCTCCCCGGAAATGAC	R：ggatccAATTTTATCGGATGTCCC
MdU6-10-2P	F：ggtaccTGATATGTGGTGTTTCTAGG	R：aagcttAATTTTATCGGATGTCCC
MdU6-10-3P	F：ggtaccGGCGAAAGGTTTATGTTC	R：aagcttAATTTTATCGGATGTCCC
MdU6-10-4P	F：ggtaccCTCCTGACTAGTAAAGAAGG	R：aagcttAATTTTATCGGATGTCCC
AtU6-1	F;ggtaccAGAAATCTCAAAATTCCGGCAG	R：aagcttCAATCACTACTTCGTCTCTAACCATAT

基因名称 Gene names	基因ID Gene ID	染色体定位 Chromosome：Location
MdU6-06	MD06G1040500	Chr06: 5202556..5202455
MdU6-07	MD07G1079000	Chr07: 7617645..7617544
MdU6-09	MD09G1120900	Chr09: 9346234..9346335
MdU6-10	MD10G1255200	Ch10: 34716072..34715971
MdU6-15	MD15G1130400	Chr15: 9427466..9427365
MdU6-17	MD17G1111900	Chr17: 9588121..9588222