蒺藜苜蓿MtCIM基因结构和功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0484

摘要/Abstract

摘要：

【目的】CIM（cytokinin induce messager）属于Expansin蛋白家族，该蛋白是一类在植物生长和发育过程中起关键作用的蛋白质。为分析蒺藜苜蓿MtCIM基因结构、表达、亚细胞定位及在植物生长和发育过程中的功能。【方法】利用生物信息学工具对其进行深入分子量、理论等电点、不稳定系数及蛋白质结构预测和分析；同时，利用RT-qPCR方法，对不同激素处理（IAA、ABA、GA₃）下的基因表达进行了检测；并通过融合蛋白瞬时表达及酵母表达方法鉴定其亚细胞定位及转录自激活情况；对MtCIM转基因植株进行低磷环境的适应分析。【结果】生物信息学分析显示，CIM蛋白的相对分子量为29.563 kD，理论等电点为5.54，不稳定系数为41.62，其蛋白质性质不稳定。二级及三级结构预测表明，该蛋白主要由无规则卷曲构成。外施IAA能够显著提高MtCIM的表达，外施ABA和GA₃后，MtCIM表达呈现先升高后降低的趋势。亚细胞定位结果显示，其定位于细胞质中。对MtCIM进行转录自激活活性分析显示，蒺藜苜蓿MtCIM不存在自激活活性。对获得的20株转基因株系进行分析表明，转基因株系叶片细胞壁更厚；转基因植株对低磷环境具有更好的适应能力。【结论】对蒺藜苜蓿MtCIM的全面分析，揭示了其作为Expansin蛋白家族成员在植物生长和发育中的关键作用，并提高了植物在低磷环境中适应能力。

关键词: 蒺藜苜蓿, MtCIM基因, 自激活检测, 表达分析, 亚细胞定位, 低磷响应

Abstract:

【Objective】CIM（cytokinin induce messager）belongs to the Expansin protein family, which plays a critical role in plant growth and development. To analyze the structure, expression, subcellular localization, and function of MtCIM during Medicago truncatula growth and development. 【Method】Bioinformatics tools were employed to conduct a comprehensive study of molecular weight, theoretical isoelectric point, instability index, and protein structure prediction and analysis. Meanwhile, RT-qPCR method was used to measure the gene expressions under different hormone treatments（IAA, ABA and GA₃）. Fusion protein transient expression and yeast expression methods were used to identify its subcellular localization and transcriptional self-activation, respectively. Analysis of MtCIM-transgenic plants was performed in terms of their adaptation to a low phosphorus environment. 【Result】Bioinformatics analysis showed that the CIM protein had a relative molecular weight of 29.563 kD, theoretical isoelectric point of 5.54, and instability index of 41.62, indicating its protein property were unstable. The secondary and tertiary structure predictions showed that the protein was mainly composed of irregular curls. Exogenous IAA significantly increased the expression of MtCIM, while the expression of MtCIM showed a trend of rising and then decreasing after exogenous ABA and GA₃. The subcellular localization results showed that it was located in the cytoplasm. Upon analysis of transcriptional self-activation, it was found that there existed no self-activation activity in MtCIM. Analysis of the 20 obtained transgenic plants revealed that the cell walls of the leaves from these transgenic lines were thicker. Moreover, the transgenic plants demonstrated superior adaptability to low-phosphorus environments. 【Conclusion】A comprehensive analysis of the MtCIM gene in M. truncatula revealed its key role as a member of the Expansin protein family in plant growth and development. The phenotype of the transgenic plants demonstrated that MtCIM enhanced the adaptability of plants in low-phosphorus environments. These findings provide new insights for further understanding the role of the Expansin protein family in plant growth and development.

Key words: Medicago truncatula, MtCIM gene, self-activation testing, expression analysis, subcellular localization, low phosphorus response

谢宏, 周丽莹, 李舒文, 王梦迪, 艾晔, 晁跃辉. 蒺藜苜蓿MtCIM基因结构和功能分析[J]. 生物技术通报, 2024, 40(1): 262-269.

XIE Hong, ZHOU Li-ying, LI Shu-wen, WANG Meng-di, AI Ye, CHAO Yue-hui. Structural and Functional Analysis of MtCIM Gene in Medicago truncatula[J]. Biotechnology Bulletin, 2024, 40(1): 262-269.

图/表 9

表1 引物序列

Table 1 Primer sequences

引物名称 Prime name	引物序列 Prime sequence（5'-3'）
T7	TAATACGACTCACTATAGG
3-BD	TAAGAGTCACTTTAAAATTTGTAT
pGBKT7-CIM-F	GCTATTTCTACTGATTTTTCATGGCTCTTACACTTGAACA
pGBKT7-CIM-R	CAAAATCATGTCAAGGTCTTTTAAAAATTCACAATTGACC
3302Y-F	TGACGCACAATCCCACTATCCTT
3302Y-R	CCGTCCAGCTCGACCAGGAT
3302Y-MtCIM-F	GAACACGGGGGACTCTTGACCATGGCTCTTACACTTGAACA
3302Y-MtCIM-R	ACGCGTACTAGTCAGATCTACAAAATTCACAATTGACCTAT
MtActin-F	TGATCTGGCTGGTCGTGACCTTA
MtActin-R	ATGCCTGCTGCTTCCATTCCTAT
MtCIM-RT-F	TGGTGACGGAAGCGAAGGTGGTG
MtCIM-RT-R	CTTAACAGGGTTGCCTGAACATA

图1 MtCIM蛋白结构预测 A：蛋白二级结构；B：蛋白三级结构

Fig. 1 Prediction of MtCIM protein structure A: Protein secondary structure. B: Protein tertiary structure

图2 MtCIM蛋白信号肽预测

Fig. 2 Signal peptide prediction of MtCIM protein

表2 MtCIM启动子的主要顺式作用元件

Table 2 The main cis-acting elements of MtCIM promoter

顺式元件Cis-element	基序Motif（5'-3'）	位置Position	功能Function
ABRE	CACGTG	348	脱落酸响应元件ABA response element
	ACGTC	349
	ACGTC	708
P-box	CCTTTTG	53	赤霉素响应元件Gibberellin response element
TC-rich repeats	GTTTTCTTAC	103	防御和胁迫反应相关的元件Defense and stress response related elements
TGA-element	AACGAC	510	生长素响应元件Auxin response element
TCT-motif	TCTTAC	103	光响应元件Light response element
ARE	AAACCA	567	厌氧诱导元件Anaerobic induction element
TATA-box	TATTTAAA	21	转录起始点-30附近的核心启动子元件 Core promoter element near trabscription starting point -30
	TATAA	148
	TATTA	633
	TATA	502
	TATATA	87
	TATA	275
G-BOX	CACGTG	348	参与光反应的顺式作用元件Cis-acting elements involved in photoreactions
G-BOX	CACGTG	707

图3 MtCIM的亚细胞定位

Fig. 3 Subcellular localization of MtCIM

图4 不同激素处理下MtCIM在蒺藜苜蓿中的表达分析不同小写字母表示差异显著（P<0.05）

Fig. 4 MtCIM expression analysis in Medicago truncatula under different hormone treatments Different lowercase letters indicate a significant difference at the 0.05 level

图5 pGBKT7-CIM转录自激活检测

Fig. 5 Transcriptional self-activation testing of pGBT7-CIM

图6 番红固绿染色法对叶片结构的观察 A：野生型植株；B：转基因植株；C：转基因植株

Fig. 6 Observation of leaf structure by saffred solid green staining method A: Wild-type plants; B: transgenic plants; C: transgenic plants

图7 全磷和低磷处理下转基因和野生型植株差异 CIM：转基因；WT：野生型；A、C：全磷条件；B、D：低磷条件

Fig. 7 Difference between transgenic and wild type plants under total phosphorus and low phosphorus treatment CIM: Transgenic plants. WT: Wild types. A, C: Total phosphorus. B, D: Low phosphorus

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