Cloning and Expression Analysis of BnaFIL Gene Promoter in Brassica napus L.

doi:10.7668/hbnxb.20192788

Abstract

Abstract:

To further understand the role of the promoter in the regulation of the expression of the Brassica napus FIL gene(BnaFIL),it used the DNA extracted from the leaves of Brassica napus as a template based on the genomic data of Brassica napus.For cloning,the length was 1 326 bp.Using PlantCARE online analysis software to perform bioinformatics sequence analysis on the promoter sequence,the results showed that the sequence contained some conserved DNA modules involved in the photoreaction and essential elements for core promoters such as CAAT-box and TATA-box.Expression related cis-acting regulatory elements CAT-box and photosensitive response elements.Replace the CaMV35S promoter on the pBI121 plant vector with the promoter sequence,fused the promoter with the GUS gene to obtain the pBnaFIL-GUS expression vector,and transfer the vector into Arabidopsis thaliana by the method of Agrobacterium inflorescence.Flower promoter recombinant plasmid-positive transgenic lines and late flower promoter recombinant plasmid-positive transgenic lines.After that,GUS staining analysis was performed on the transgenic Arabidopsis plants,and the expression effect of the promoter was tested.Finally,the GUS gene expression was found in different transgenic Arabidopsis plants.The results showed that there were differences in promoter expression between early-flowering materials and late-flowering materials.The promoters of early-flowering materials could drive gene expression better than those of late-flowering materials.It was inferred from this that the driving effect of the promoter regulates the early and late flowering of rape,resulting in different expression effects of the FIL gene in different materials.

Key words: Brassica napus, BnaFIL gene, Promoter, GUS staining, Expression vector

CHEN Jing, HU Rong, LIU Yong, QIN Yi, XIONG Xinghua. Cloning and Expression Analysis of BnaFIL Gene Promoter in Brassica napus L.[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(3): 53-59. doi: 10.7668/hbnxb.20192788.

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Figures/Tables 10

Tab.1 The primer sequences

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')	退火温度/℃ Annealing temperature
BnaFIL-P-F	ACATATTTTCAGAGGTTCGTC	52
BnaFIL-P-R	GATCTTCTTATTTTGGGAGTG
ScaⅠ-P-F	AAAAGTACTACATATTTTCAGAGGTTCGTC	57
BamHⅠ-P-R	CGCGGATCCGATCTTCTTATTTTGGGAGTG
P-pBI121-F	CATCGTTTGTTTCGGACCACT	57
P-pBI121-R	CACTTCCTGATTATTGACCCA

Fig.1 PCR amplification of the promoter of BnaFIL gene 1.Late flower material;2.Early flower material; M.DL2000 Marker.The same as Fig.3.

Fig.2 Analysis of cis-acting elements of the promoter of BnaFIL gene 1,2.SNP sites.

Tab.2 Promoter elements

所含元件 Contains components	功能 Function
ATCT-motif、BOX4	参与光响应的部分保守DNA模块
CAAT-BOX	在启动子及增强子区域共有的顺式作用元件
CAT-BOX	顺式作用的调控元件与分生组织表达有关
TATA-BOX	核心启动子元件
TCA-element	参与水杨酸反应的顺式作用元件
TCCC-motif、TCT-motif	光敏元件的一部分

Fig.3 Promoter amplification

Fig.4 Flow chart for construction of the plant expression vector

Fig.5 Double enzyme digestion identification of recombinant plasmid 1.Recombinant plasmid of late flowering material;2.Recombinant plasmid of early flower material;M.Trans 5k DNA Marker.

Fig.6 PCR detection of Agrobacterium tumefaciens A.Transforming early flowering promoter colony PCR; B.Transforming late flowering promoter colony PCR;M.DL2000 Marker.

Fig.7 Positive identification of transgenic Arabidopsis thaliana 1-3.Early-flowering recombinant plasmid resistant seedlings; 4-8.Late-flowering recombinant plasmid resistant seedlings; 9.Wild type Arabidopsis thaliana.

Fig.8 GUS staining results A.Wild-type Arabidopsis;B.Vector Arabidopsis containing pBI121;C.Transgenic Arabidopsis containing late-flowering promoter recombinant plasmid;D.Transgenic Arabidopsis containing early-flowering promoter recombinant plasmid.

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