芥蓝BoaBKI1基因的克隆及表达分析

doi:10.16036/j.issn.1000-2650.202112051

摘要/Abstract

摘要： 【目的】克隆芥蓝中的BoaBKI1基因,进行生物信息学与表达分析。【方法】采用改良CTAB法提取芥蓝总RNA,并反转录为cDNA;设计引物,克隆BoaBKI1基因;使用生物信息学方法分析BoaBKI1基因序列;用半定量PCR进行BoaBKI1基因的时空表达分析。【结果】成功克隆到BoaBKI1基因,序列分析显示其开放阅读框为1 026 bp,编码20种氨基酸;理论分子量为37.30 kD,等电点为9.67,分子式为C₁₆₀₃H₂₅₈₆N₄₇₆O₅₃₄S₇。同源性序列比对和进化树分析发现芥蓝BoaBKI1与同属植物甘蓝、欧洲油菜的序列一致性高达99%。半定量PCR分析发现,不同发育时期中,BoaBKI1基因在芥蓝真叶期表达量最高,萌动种子期最低;花蕾中花瓣表达水平最高,萼片表达水平最低;开放花朵中雌蕊表达量明显高于其他器官;随花朵开放,BoaBKI1基因在各个花组织中表达水平均出现上调,雌蕊和雄蕊趋势最显著。【结论】上述结果可为研究芥蓝BoaBKI1的功能与调控机制提供理论依据。

关键词: 芥蓝, BoaBKI1, 克隆, 表达分析

Abstract: 【Objective】 This paper intends to clone BoaBKI1 gene in Chinese kale （Brassica oleracea var. alboglabra）,and perform bioinformatics and spatio-temporal expression analysis on it. 【Method】 The total RNA of Chinese kale （Brassica oleracea var. alboglabra） was extracted by modified CTAB method and reverse transcribed into cDNA. The primers were designed to clone BoaBKI1 gene. The sequence analysis of BoaBKI1 gene was performed by bioinformatics method. Spatio-temporal expression analysis of BoaBKI1 gene was performed by semi-quantitative PCR. 【Result】 The BoaBKI1 gene was cloned in Chinese kale. Sequence analysis showed that its open reading frame was 1 026 bp,which encoded 341 amino acids;its theoretical molecular weight was 37.30 kD,the isoelectric point was 9.67,and the molecular formula was C₁₆₀₃H₂₅₈₆N₄₇₆O₅₃₄S₇. The results of the homology sequence alignment and phylogenetic tree analysis showed that the BoaBKI1 gene was conserved during evolution and has a sequence identity of up to 99% with Cabbage and European rape,which all belongs to the Brasscia genus. Semi-quantitative PCR analysis results showed that among different developmental stages,the highest expression of BoaBKI1 was observed in the true leaf stage,and the lowest one was in the germinating seed stage. The highest expression level of BoaBKI1 in flower buds was observed in the petals,and the lowest was in the sepals. The expression in the open flowers was significantly higher in the pistils than in other organs. With the flower opening,the expression levels of BoaBKI1 gene increased in each flower tissues,and pistils and stamens showed the most up-regulated trends. 【Conclusion】 These findings can provide a theoretical basis for studying the function and regulatory mechanism of BoaBKI1 in Chinese kale.

Key words: Chinese kale （Brassica oleracea var. alboglabra）, BoaBKI1, cloning, expression analysis

中图分类号:

S635

梁莎, 黄文莉, 李香香, 王依霖, 张晨璐, 张芬, 孙勃. 芥蓝BoaBKI1基因的克隆及表达分析[J]. 四川农业大学学报, 2022, 40(2): 163-171.

LIANG Sha, HUANG Wenli, LI Xiangxiang, WANG Yilin, ZHANG Chenlu, ZHANG Fen, SUN Bo. Cloning and Expression Analysis of BoaBKI1 Gene in Chinese Kale (Brassica oleracea var. alboglabra)[J]. Journal of Sichuan Agricultural University, 2022, 40(2): 163-171.

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