Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (23): 4364-4373.doi: 10.3864/j.issn.0578-1752.2019.23.016

• SPECIAL FOCUS: MOLECULAR BIOLOGY OF APPLE • Previous Articles     Next Articles

Cloning and Functional Analysis of U6 Promoter in Apple

BIAN ShuXun,HAN XiaoLei,YUAN GaoPeng,ZHANG LiYi,TIAN Yi,ZHANG CaiXia(),CONG PeiHua()   

  1. Institute of Pomology, Chinese Academy of Agricultural Sciences/Key Laboratory of Fruit Germplasm Resources Utilization, Ministry of Agriculture/National Apple Breeding Center, Xingcheng 125100, Liaoning
  • Received:2019-07-31 Accepted:2019-09-19 Online:2019-12-01 Published:2019-12-01
  • Contact: CaiXia ZHANG,PeiHua CONG E-mail:cxzhang-bj@163.com;congph@163.com

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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

Table 1

Primer sequences used in this study"

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

Fig. 1

Construction of LUC reporter gene fusion expression vector driven by U6 promoters"

Table 2

Mapping of U6 snRNA chromosome in apple genome"

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

Fig. 2

Sequence analysis of MdU6 promoters"

Fig. 3

PCR amplification and identification of the MdU6 promoters"

Fig. 4

Transient expression of MdU6-Ps::LUC in N. benthamiana leaves"

Fig. 5

Transient expression of MdU6-9P::LUC and MdU6-10P::LUC in apple callus Different lowercase letters indicate significant difference (P<0.05). The same as below"

Fig. 6

Schematic diagram of different MdU6-10-Ps truncated at the 5′ end"

Fig. 7

PCR amplification and identification of truncated MdU6 promoters"

Fig. 8

Transient expression of MdU6-10-Ps::LUC in N. benthamiana leaves"

Fig. 9

Transient expression of MdU6-10-Ps::LUC in apple callus"

Fig. 10

Transient expression of AtU6-1P::LUC and MdU6-10-3P::LUC in apple callus"

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