Cloning，Expression and Functional Identification of PRR5 Gene in Pakchoi

doi:10.13560/j.cnki.biotech.bull.1985.2023-0338

Abstract

Abstract:

Cloning BrcPRR5 and studying its expression pattern and function in different tissues and different developmental stages may lay a foundation for understanding the effect of PRR5 on flowering transformation of pakchoi. The homologous gene BrcPRR5 of PRR5 was cloned by RT-PCR and analyzed by bioinformatics. The relative expressions of the gene in different tissue sites and different developmental stages were determined by fluorescence quantitative PCR. The overexpression vector was constructed and transformed into Arabidopsis for functional verification. The results showed that the full-length CDS of BrcPRR5 was 1 701 bp, encoding 566 amino acids. Through multiple amino acid sequence alignment with homologous proteins of other species, the obtained sequence belonged to PRR5 homologous gene of pak choi. The expression of BrcPRR5 in the stem and flower was higher than that in the leaf and pod, and the highest expression was in S0 stem tip, indicating that the up-regulated expression of S0 promoted floral transformation during flowering transition of pakchoi. The flowering stage of overexpressed transgenic plants was earlier, and the plant height and stem diameter of transgenic plants were significantly better than those of wild type. BrcPRR5 may promote plant bolting and flowering earlier.

Key words: pakchoi, BrcPRR5, flowering transformation, gene cloning

HOU Rui-ze BAO Yue CHEN Qi-liang MAO Gui-ling WEI Bo-lin HOU Lei-ping LI Mei-lan. Cloning，Expression and Functional Identification of PRR5 Gene in Pakchoi[J]. Biotechnology Bulletin, 2023, 39(10): 128-135.

Figures/Tables 9

Table 1 Primers of Bra009768

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	引物用途 Primer purpose
C-Bra009768	F：ATGGGAGAAGTGAGCGACGAAG	基因克隆
C-Bra009768	R：TCATTGTGGAGCTTCTTGTGTTGAG	Gene clone
G-Bra009768	F：GAGAACACGGGGGACTCTAGAATGGGAGAAGTGAGCGACGAAG	基因克隆 Gene clone
G-Bra009768	R：ATAAGGGACTGACCACCCGGGTCATTGTGGAGCTTCTTGTGTTGAG
N-YZ	F：GACGTTCCAACCACGTCTTC	菌液PCR引物
N-YZ	R：CCAGACTGAATGCCCACAGG	Bacterial liquid PCR primer

Table 2 Primers of real time quantitative PCR

引物名称 Gene name	引物序列 Primer sequence（5'-3'）	引物用途 Primer purpose
ACTIN	F：GTTGCTATCCAGGCTGTTCT	大白菜内参引物
ACTIN	R：AGCGTGAGGAAGAGCATAAC	Internal primers in Brassica pekinensis
Bra009768	F：AGTTACAGAGTCGCTGCT	荧光定量引物
Bra009768	R：TATTAACCGAGTCCTGTGTTG	Fluorescent quantitative primer
ACT11	F：CACACTGGAGTGATGGTTGG	拟南芥内参引物
ACT11	R：ATTGGCCTTGGGGTTAAGAG	The internal reference gene of Arabidopsis

Fig. 1 Cloning of Bra009768 and detection results of its broth A: Cloning of Bra009768（M: DNA marker; 1-3: Bra009768）. B: PCR detection of bacterial liquid（M: DNA marker; 1-12: Bra009768; 13-14: negative control）

Fig. 2 Bioinformatics analysis of Bra009768 A: Multiple alignment of Bra009768 amino acid sequences. B: Phylogenetic tree of Bra009768 homologous proteins. C, D: Prediction of secondary and tertiary structures of Bra009768 proteins

Fig. 3 Expressions of Bra009768 in different organs（A）and different developmental stages（B）of pakchoi

Fig. 4 Selection of transgenic Arabidopsis thaliana resistant plants A: Screening of resistant plants in T1 generation. B: Screening of resistant plants in T2 generation

Table 3 Phenotypic statistics of T1 and T2 transgenic plants

植株 Plant	抽薹时间 Bolting time/d	侧薹数 Number of side bolting	株高 Plant height/cm	茎粗 Stem thick/mm
Col-1	13.75±0.82	11.50±0.31	25.90±0.88	0.45±0.04
T-1	12.50±2.38	11.25±2.12	25.75±3.13	0.54±0.12
Col-2	17.20±1.25	7.50±0.21	22.30±0.91	0.53±0.02
T-2	15.72±0.78	8.10±0.13	29.80±0.53	0.94±0.06

Fig. 5 Phenotypic observation of transgenic plants and RT-qPCR validation of BrcPRR5 A: Vegetative growth period. B: Bolting stage. C: Budding stage. D-E: Mature stage（left: wild type（Col）; right: transgenic plants）. F: RT-qPCR detection of transgenic plants

Fig. 6 Model for the effect of the PRR9/PRR5 sub-group on flowering time and plant biomass

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