Cloning，Expression and Functional Identification of BrMLP328 Gene in Brassica rapa subsp. pekinensis

doi:10.13560/j.cnki.biotech.bull.1985.2023-1082

Abstract

Abstract:

【Objective】 The BrMLP328 gene of Chinese cabbage （Brassica rapa subsp. pekinensis）was cloned, its expression pattern was analysed, and its function in regulating flowering was verified. This may lay the basis for further investigation into the mechanism of the role of this gene in regulating flower formation in Chinese cabbage.【Method】BrMLP328 was cloned by RT-PCR and analysed by bioinformatics. RT-qPCR was used to determine the relative expression of the BrMLP328. An overexpression vector was constructed and transformed into wild-type Arabidopsis thaliana by flower-dipping method, and the difference in flowering time between the T₂ generation plants and the wild type was compared.【Result】The coding sequence（CDS）of BrMLP328 has a total length of 456 bp, encoding 151 amino acids. The relative molecular weight of the protein was 17 493.82 Da and was localized in the nucleus. The expression of BrMLP328 was the highest in the stems of Chinese cabbage, followed by roots and lowest in flower buds. The expression in the growing point of the stem tip showed an elevated level after vernalization, after which it declined rapidly at the stage of flower bud differentiation and was maintained at a very low level. Compared with the wild type, the flowering time of the BrMLP328 of T₂ generation of transgenic plants delayed by 1.46-3.09 d.【Conclusion】BrMLP328 was cloned from Chinese cabbage. The expression of BrMLP328 was different in different tissues and different flowering stages, and the gene could delay flowering.

Key words: Brassica rapa subsp. pekinensis, BrMLP328, flowering transformation, gene cloning, functional verification

DU Ze-guang, REN Shao-wen, ZHANG Feng-qin, LI Mei-lan, LI Gai-zhen, QI Xian-hui. Cloning，Expression and Functional Identification of BrMLP328 Gene in Brassica rapa subsp. pekinensis[J]. Biotechnology Bulletin, 2024, 40(4): 122-129.

Figures/Tables 9

Table 1 Primer sequences used in the experiment

用途 Purpose	名称 Name	序列 Sequence（5'-3'）
基因克隆 Gene cloning	026653-1	F：ATGGCGACGTCGGGAACA R：TTAAGCTTTGACGACATGATTGTCC
大白菜RT-qPCR Fluorescent quantitative PCR in Chinese cabbage	ACTIN	F：CGGTGTCATGGTTGGGAGA R：CGTGCTCGATGGGGTACTTC
大白菜RT-qPCR Fluorescent quantitative PCR in Chinese cabbage	026653-2	F：CCACCACATCCAAGGTGTCA R：AACACCTCCGGTTTCCCATC
阳性鉴定 Positive identification	026653-3	F：GACGCACAATCCCACTATCC R：TCGGTTCGCTTCTCCCAGAT
拟南芥RT-qPCR Fluorescent quantitative PCR in Arabidopsis thaliana	ACTII	F：CACACTGGAGTGATGGTTGG R：ATTGGCCTTGGGGTTAAGAG

Table 2 Primary online software employed in the experiment

软件名称 Software name	软件网址 Software website
ProtParam	http://web.expasy.org/protparam/
ProtScale	http://web.expasy.org/protscale/
SOPMA	http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopm.html
SWISS-MODEL	http://swissmodel.expasy.org/
Plant-mPLoc	http://www.csbio.sjtu.edu.cn/bioinf/plantmulti/

Fig. 1 Electrophoresis map of PCR product of gene Bra026653 1: PCR product ; M: DL10000 marker

Fig. 2 Bioinformatics analysis of BrMLP328 A: Multiple alignment of BrMLP328 amino acid sequences. B: Phylogenetic tree of BrMLP328 homologous proteins. C: Analysis of hydrophilicity and hydrophobicity of BrMLP328. D: Prediction of secondary structures of BrMLP328 proteins. E: Prediction of tertiary structures of BrMLP328 proteins

Fig. 3 Expressions of BrMLP328 in different organs（A）and different developmental stages（B）of stem tip growth point

Fig. 4 Screening and positive identification of T1 resistant plants A: Screening of resistant plants in T1 generation. B: Positive identification of resistant plants in T1 generation.（M: DL2000 marker. Label 1-3 is the number of resistant seedlings. “-” is the pure water blank control, “+” is the plasmid control and “WT” is the wild type plant control.）

Fig. 5 T2 transgenic plants and the wild type Arabidopsis thaliana

Table 3 Statistics of flowering time between T2 transgenic plants and the wild type

T₁代植株编号 Number of T₁ plant	T₂代植株开花时间 Days of flowering after sowing of T₂ plant/d	比野生型植株延迟开花天数 Average number of days in flowering later than the wild type/d
WT	21.23 ± 0.45	-
Bra026653-1	24.32 ± 1.62	3.09
Bra026653-2	22.69 ± 2.38	1.46
Bra026653-3	23.26 ± 0.94	2.03

Fig. 6 RT-qPCR validation of BrMPL328 in T2 transgenic plants

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