蓖麻GeBP转录因子的全基因组鉴定与GeBP2基因的克隆、表达分析

doi:10.3969/j.issn.1004-1524.20230833

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (8): 1731-1740.DOI: 10.3969/j.issn.1004-1524.20230833

蓖麻GeBP转录因子的全基因组鉴定与GeBP2基因的克隆、表达分析

朱贵爽(), 李艳肖, 张安宁, 孙浩楠, 徐兴源, 李志刚, 向殿军()

内蒙古民族大学农学院,内蒙古通辽 028000

收稿日期:2023-07-05 出版日期:2024-08-25 发布日期:2024-09-06
作者简介:*向殿军,E-mail: Xiangdianjun00@126.com
朱贵爽(1999—),女,内蒙古通辽人,硕士研究生,研究方向为作物遗传改良与种质创新。E-mail: 1724864362@qq.com
通讯作者: 向殿军
基金资助:
国家自然科学基金(32060492);内蒙古自治区自然科学基金(2022MS03057);内蒙古自治区自然科学基金(2023MS03032)

Identification of RcGeBP transcription factor and cloning and expression analysis of RcGeBP2 gene in castor

ZHU Guishuang(), LI Yanxiao, ZHANG Anning, SUN Haonan, XU Xingyuan, LI Zhigang, XIANG Dianjun()

College of Agriculture, Inner Mongolia Minzu University, Tongliao 028000, Inner Mongolia, China

Received:2023-07-05 Online:2024-08-25 Published:2024-09-06
Contact: XIANG Dianjun

摘要/Abstract

摘要：

GeBP基因家族编码的转录因子可通过调节表皮毛的生长与分化影响植物的发育,增强植株在不利环境中的存活能力。为探究GeBP基因在蓖麻基因组中的分布与特征基因RcGeBP2的功能,本研究鉴定了蓖麻的全部GeBP基因,采取RT-PCR技术克隆RcGeBP2基因,分析其分子特征包括生物信息学、亚细胞定位和表达模式。试验表明,蓖麻的基因组中共有4个RcGeBP基因不均匀分布于3条染色体上且RcGeBP基因编码的蛋白质均为亲水性蛋白,高级结构以无规则卷曲为主。GeBP的进化树构建显示,蓖麻GeBP蛋白与番茄GeBP蛋白亲缘关系最近,蓖麻的GeBP基因分别与番茄和拟南芥的GeBP基因发生了3次和2次基因组间的复制。Ka/Ks结果显示,GeBP基因在物种间的进化是钝化的。启动子顺式作用元件预测结果显示,RcGeBP启动子区域含多个逆境响应和激素诱导类元件。RcGeBP基因的FPKM表达值显示,这些基因拥有组织表达特性且表达受到干旱和盐胁迫的激活。RcGeBP2蛋白质亚细胞定位结果显示,其定位在细胞核。综上,该研究对探明蓖麻RcGeBP基因家族参与盐和干旱胁迫的交叉调控具有一定的参考价值。

关键词: 蓖麻, GeBP, 基因克隆, 表达, 亚细胞定位

Abstract:

The GeBP gene family encodes a typical transcription factor that affects plant development and enhances plant survival in adverse environments by regulating the growth and differentiation of epidermal hairs. In order to explore the distribution of GeBP gene in the castor genome and the function of the characteristic gene RcGeBP2, all the GeBP genes of castor were identified in this paper. The RcGeBP2 gene was cloned by RT-PCR and its molecular characteristics were analyzed, including bioinformatics, subcellular localization and expression pattern.The results showed that four RcGeBP genes were unevenly distributed on three chromosomes in the genome of castor. The proteins encoded by RcGeBP genes were hydrophilic proteins, and the advanced structure was mainly random coil. The phylogenetic tree construction of GeBP showed that the GeBP protein of castor had the closest relationship with the GeBP protein of tomato. The GeBP gene of castor had three and two genome duplications with the GeBP gene of tomato and Arabidopsis thaliana, respectively. The results of Ka/Ks showed that the evolution of GeBP gene among species was passivated. The results of cis-acting element prediction showed that the RcGeBP promoter region contained multiple stress response and hormone-inducible elements.The FPKM expression value of RcGeBP genes showed that these genes had tissue expression characteristics and their expression was activated by drought and salt stress. The subcellular localization of RcGeBP2 protein showed that it was localized in the nucleus. In summary, this study has certain reference value for exploring the cross-regulation of castor RcGeBP gene family involved in salt and drought stress.

Key words: castor, GeBP, gene cloning, expression, subcellular localization

中图分类号:

朱贵爽, 李艳肖, 张安宁, 孙浩楠, 徐兴源, 李志刚, 向殿军. 蓖麻GeBP转录因子的全基因组鉴定与GeBP2基因的克隆、表达分析[J]. 浙江农业学报, 2024, 36(8): 1731-1740.

ZHU Guishuang, LI Yanxiao, ZHANG Anning, SUN Haonan, XU Xingyuan, LI Zhigang, XIANG Dianjun. Identification of RcGeBP transcription factor and cloning and expression analysis of RcGeBP2 gene in castor[J]. Acta Agriculturae Zhejiangensis, 2024, 36(8): 1731-1740.

图/表 13

表1 试验所用引物

Table 1 Primers used in the test

引物名称 Primer name	引物序列 Primer sequences(5'→3')	用途 Usage
RcGeBP2-F1	ATGGACTCCAATCTCAACCCACTCC	基因克隆Gene cloning
RcGeBP2-R1	TCATAACCTAACAGAACCCTCAAGC
RcGeBP2-F2	CTTCACTGTTGATACATATGGACTCCAATCTCAACCC	亚细胞定位Subcellular localization
RcGeBP2-R2	ATCCGGTACCCCCGGGTAACCTAACAGAACCCTCAAG

表2 RcGeBP蛋白质的基本理化性质

Table 2 Basic physicochemical properties of RcGeBP protein

基因登录号 GenBank No.	基因 Gene	氨基酸数目 Number of amino acid	分子量 Molecular weight/u	等电点 pI	总平均疏水性 Grand average of hydropathicity	不稳定系数 Instability index	脂肪系数 Aliphatic index	带正电氨基酸 Positively charged amino acids (Arg+Lys)	带负电氨基酸 Negatively charged amino acids (Asp+Glu)	亚细胞位置 Subcellular localization
Rc05T009423	RcGeBP1	391	43637.68	5.32	-1.155	46.84	57.16	71	82	细胞核 Nucleus
Rc05T012306	RcGeBP2	286	32508.92	9.54	-0.706	58.59	77.31	42	33	细胞核 Nucleus
Rc09T020714	RcGeBP3	383	42740.10	5.00	-1.140	52.29	64.13	60	79	细胞核 Nucleus
Rc10T024212	RcGeBP4	415	46173.80	4.62	-0.770	62.28	74.75	46	76	细胞核 Nucleus

表3 RcGeBP蛋白质的二级结构预测

Table 3 Secondary structure prediction of RcGeBP protein %

蛋白质名称 Protein name	α螺旋 α-Helix	β折叠 β-Sheet	无规卷曲 Random coil	延伸链 Extended strand
RcGeBP1	40.41	3.07	53.96	2.56
RcGeBP2	39.16	4.20	48.25	8.39
RcGeBP3	40.99	2.35	51.17	5.48
RcGeBP4	37.59	2.65	46.99	12.77

图1 RcGeBP蛋白质的三级结构预测与拉式构象模型

Fig.1 Tertiary structure prediction and pull conformation model of RcGeBP protein

图2 蓖麻RcGeBP基因在染色体上的位置

Fig.2 Location of RcGeBP gene on chromosome in castor

图3 蓖麻RcGeBP基因的结构与推导氨基酸的保守基序和结构域 A,氨基酸保守基序;B,氨基酸结构域;C,蓖麻RcGeBP基因的结构。

Fig.3 The structure and deduced amino acid conserved motifand domain of RcGeBP gene in castor A, Deduced amino acid conserved motif of RcGeBP gene in castor; B, Domain of RcGeBP gene in castor; C,The structure of RcGeBP gene in castor.

图4 GeBP基因在物种间的进化树

Fig.4 Phylogenetic tree of GeBP gene among species

图5 蓖麻GeBP基因同番茄和拟南芥GeBP基因的共线性分析

Fig.5 The collinearity analysis of RcGeBP gene with GeBP genes in tomato and Arabidopsis thaliana

表4 共线基因对的选择压力分析

Table 4 Selection pressure analysis of collinear gene pairs

共线基因名称 Collinear gene name	非同义替换率 Ka	同义替换率 Ks	非同义替换率/ 同义替换率 Ka/Ks
RcGeBP1_Solyc05g051330.1	0.651	-	-
RcGeBP2_Solyc07g052830.2	0.390	2.308	0.169
RcGeBP3_Solyc07g052900.1	0.530	2.510	0.211
RcGeBP1_AEE85025.1	0.449	-	-
RcGeBP1_AEE82001.1	0.336	1.702	0.198

图6 蓖麻RcGeBP启动子的顺式作用元件

Fig.6 The cis-acting elements of castor RcGeBP promoter

图7 蓖麻RcGeBP基因的表达谱分析 A、B分别代表RcGeBP基因的组织表达和胁迫表达;S和P分别表示盐胁迫和干旱胁迫,数字表示胁迫时间。

Fig.7 Expression profile analysis of RcGeBP gene in castor A and B represent the tissue expression and stress expression of RcGeBP gene, respectively. S and D represent salt stress and drought stress, respectively, and the number represents the stress time.

图8 RcGeBP基因的克隆 M,DL 2 000 marker;1和2,RcGeBP2扩增产物。

Fig.8 Cloning of RcGeBP gene M, DL 2 000 maker; 1 and 2, The amplification products of RcGeBP2.

图9 RcGeBP蛋白质的亚细胞定位分析 GFP,绿色荧光;CHL,叶绿体自发荧光;DIC,明场;Merge,叠加场。

Fig.9 Subcellular localization analysis of RcGeBP protein GFP, Green fluorescence; CHL,Chloroplast autofluorescence; DIC, Bright field; Merge, Superposition field.

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蓖麻GeBP转录因子的全基因组鉴定与GeBP2基因的克隆、表达分析

Identification of RcGeBP transcription factor and cloning and expression analysis of RcGeBP2 gene in castor

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