Functional Validation and Expression Analysis of BrPIF5 Transcription Factor Gene in Brassica rapa

doi:10.7668/hbnxb.20195034

Abstract

Abstract:

The folding mode of Chinese cabbage leaf ball is the main character that determines the appearance shape,taste and stress resistance of commercial organs.In order to explore the internal molecular mechanism of the formation of the folding mode of Chinese cabbage,we cloned the full length sequence of the transcription factor BrPIF5 gene from overlaping and outward-curling Chinese cabbage as experimental materials,and conducted bioinformatics analysis,constructed the plant overexpression vector,and used Agrobacterium to mediate the transformation into tobacco to obtain positive transformation plants.The expression level of BrPIF5 gene in tobacco was detected by qRT-PCR.The results showed that the protein encoded by BrPIF5 gene was a hydrophilic protein with a continuous and complete open reading frame of 634 bp,containing 210 amino acids.The protein was composed of more α-helical structure and random curl,including an AP2/ERF domain.BrPIF5 protein and the other 9 gene family members contained a conserved motif 1,and the position was different from that of other gene family members,which was located in the front of the protein sequence.Phylogenetic tree showed that BrPIF5 gene had close evolutionary relationship with SoPIF15,BhPIF1,BoPIF4,AtPIF4 and BrPIF4 family members.The tobacco strain with overexpression of BrPIF5 was obtained by Agrobacterum-mediated genetic transformation,and the leaves of the tobacco positive transformation strain showed inward curling.qRT-PCR showed that the expression level of BrPIF5 gene in the overlaping Chinese cabbage was higher than that in the outward-curling Chinese cabbage,and the gene expression level in the positive tobacco plants was higher than that in the control.It was further proved that BrPIF5 gene controlled the inward curling of Chinese cabbage leaves,thus promoting the formation of leaf ball folding type.

Key words: Brassica rapa ssp.pekinensis, BrPIF5 gene, Expression analysis, Leaf development, PIF gene family

MENG Chuan, MA Xiaochao, WU Fang, WANG Qingfeng, MA Lei, WANG Hongle, WANG Mingqiu, LIU Xiaodong. Functional Validation and Expression Analysis of BrPIF5 Transcription Factor Gene in Brassica rapa[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(6): 18-26. doi: 10.7668/hbnxb.20195034.

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Figures/Tables 10

Fig.1 Leafy head of overlapping type cabbage and outward-curling type cabbage

Fig.2 Secondary structure prediction of BrPIF5 protein

Fig.3 Tertiary structure prediction of BrPIF5 protein

Fig.4 Conserved motif analysis of the PIF gene family members proteins

Fig.5 Gene sequence analysis of PIF gene family members

Fig.6 Sequence alignment and phylogenetic analysis of BrPIF5 and other PIF gene family members

Fig.7 Construction of BrPIF5 gene plant expression vector and vector map A.The constructed plant expression vector map;B.PCR validation of colonies:1.Digestion bands,2.DNA Marker.

Fig.8 PCR identification of transgenic lines +.The positive control;-.The negative control;O.The water control.

Fig.9 Phenotypic traits of transgenic strains and wild-type strains A.Wild-type N.benthamiana(frontal shot);B.Wild-type N.benthamiana(overhead shot);C.Transgenic N.benthamiana(frontal shot);D.Transgenic Nicotiana benthamiana(overhead shot).

Fig.10 The expression levels of the BrPIF5 gene in different plants Different lowercase letters indicate significant differences(P<0.05).

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