普通白菜PRR5的克隆、表达及功能验证

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

摘要/Abstract

摘要：

克隆BrcPRR5，并研究其在不同组织和不同发育时期的表达模式及功能，为了解PRR5对普通白菜成花转变的影响奠定基础。运用RT-PCR方法克隆PRR5的同源基因BrcPRR5，并对其进行生物信息学分析，利用荧光定量PCR测定该基因在不同组织部位和不同发育时期的相对表达量，构建过表达载体并转化拟南芥进行基因的功能验证。结果表明，BrcPRR5的CDS全长为1 701 bp，编码566个氨基酸，通过与其他物种的同源蛋白进行氨基酸序列多重比对，确定得到的序列属于普通白菜PRR5同源基因。BrcPRR5在茎和花中的表达量高于叶和果荚中的表达量；在S0茎尖中表达量最高，说明其在普通白菜成花转变过程中表达量上调可能促进成花转变。超表达转基因植株开花期提前，株高和茎粗明显优于野生型。BrcPRR5可以促进植株提早抽薹开花。

关键词: 普通白菜, BrcPRR5, 成花转变, 基因克隆

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

侯瑞泽, 鲍悦, 陈启亮, 毛桂玲, 韦博霖, 侯雷平, 李梅兰. 普通白菜PRR5的克隆、表达及功能验证[J]. 生物技术通报, 2023, 39(10): 128-135.

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.

图/表 9

表1 Bra009768引物列表

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

表2 RT-qPCR验证引物

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

图1 Bra009768的克隆及其菌液检测结果 A：Bra009768的克隆（M：DNA marker；1-3：Bra009768）；B：菌液的PCR检测（M：DNA maker；1-12：Bra009768；13-14：阴性对照）

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）

图2 Bra009768的生物信息学分析A：Bra009768氨基酸序列多重比对；B：Bra009768同源蛋白的系统进化树；C、D：Bra009768蛋白质二级、三级结构预测

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

图3 Bra009768在普通白菜不同器官（A）及不同发育时期（B）的表达

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

图4 转基因拟南芥抗性植株筛选 A：T1代抗性植株筛选；B：T2代抗性植株筛选

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

表3 T1、T2转基因植株表型统计

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

图5 转基因植株的表型观察及BrcPRR5的RT-qPCR验证 A：营养生长期；B：抽薹期；C：现蕾期；D-E：成熟期（左：野生型（Col）；右：转基因植株）；F：转基因植株的RT-qPCR检测

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

图6 PRR9/PRR5亚群对开花时间和植株生物量影响的模型

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

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