大白菜BrMLP328的克隆、表达及功能验证

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

摘要/Abstract

摘要：

【目的】克隆大白菜BrMLP328基因，对其表达模式进行分析，并验证对花期调控的功能，为进一步探究该基因在大白菜成花调控过程的作用机理奠定基础。【方法】运用RT-PCR克隆BrMLP328，并进行生物信息学分析；利用RT-qPCR测定该基因的相对表达量；构建过表达载体并通过蘸花法转化野生型拟南芥，比较T₂代植株与野生型的开花时间差异。【结果】BrMLP328的CDS全长为456 bp，编码151个氨基酸，蛋白相对分子质量为17 493.82 Da，定位于细胞核。BrMLP328在大白菜茎中的表达量最高，根中次之，花蕾中最低；茎尖生长点中的表达量表现为春化后升高，之后在花芽分化阶段迅速下降，并维持在很低的水平。过表达BrMLP328的拟南芥开花时间比野生型延迟了1.46-3.09 d。【结论】从大白菜中克隆得到BrMLP328基因，其表达量在不同组织及不同成花阶段有所不同，该基因能够延迟开花。

关键词: 大白菜, BrMLP328, 成花转变, 基因克隆, 功能验证

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

杜泽光, 任少文, 张凤勤, 李梅兰, 李改珍, 齐仙惠. 大白菜BrMLP328的克隆、表达及功能验证[J]. 生物技术通报, 2024, 40(4): 122-129.

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.

图/表 9

表1 试验中用到的引物序列

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

表2 本试验使用的主要在线软件

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/

图1 Bra026653基因PCR产物电泳图 1：PCR产物，M：DL10000 marker

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

图2 BrMLP328的生物信息学分析 A：BrMLP328的氨基酸序列多重比对；B：BrMLP328与其他同源蛋白的亲缘关系树；C：BrMLP328的亲疏水性分析；D：BrMLP328的二级结构预测；E：BrMLP328的三级结构预测

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

图3 大白菜不同器官（A）和成花不同阶段茎尖生长点（B）中BrMLP328的表达

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

图4 T1代抗性植株的筛选及阳性鉴定 A：T1代抗性植株筛选；B：T1代抗性植株阳性鉴定（M：DL2000 marker，1-3：抗性植株，WT：野生型对照，-：阴性对照，+：阳性质粒对照）

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.）

图5 T2代转基因植株与野生型拟南芥

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

表3 T2代转基因植株与野生型的开花时间统计

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

图6 T2代转基因植株BrMPL328的RT-qPCR验证

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

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