甜菜磷脂酰肌醇转运蛋白基因SbSEC14的克隆及低温胁迫下的表达分析

doi:10.11924/j.issn.1000-6850.casb20191100883

中国农学通报 ›› 2020, Vol. 36 ›› Issue (32): 39-48.doi: 10.11924/j.issn.1000-6850.casb20191100883

所属专题：生物技术；园艺

甜菜磷脂酰肌醇转运蛋白基因SbSEC14的克隆及低温胁迫下的表达分析

邹锋康¹(), 贾海伦¹^,²^,³, 丁广洲¹^,²^,³(), 陈丽¹^,²^,³

¹黑龙江省甜菜工程技术研究中心,哈尔滨 150080
²国家糖料改良中心,哈尔滨 150080
³黑龙江省高校甜菜遗传育种重点实验室,哈尔滨 150080

收稿日期:2019-11-27 修回日期:2020-01-25 出版日期:2020-11-15 发布日期:2020-11-19
通讯作者: 丁广洲
作者简介:邹锋康,男,1992年出生,陕西渭南人,硕士研究生,研究方向:植物抗性基因。通信地址:150080 黑龙江省哈尔滨市南岗区学府路74号黑龙江大学农作物研究院,Tel:0451-86609312,E-mail: 614673407@qq.com。
基金资助:
黑龙江省经济作物产业技术协同创新体系项目“甜菜育种岗位”(2018711);国家糖料产业技术体系岗位科学家项目“甜菜种质资源收集与评价”(CARS-170102)

Phosphatidylinositol Transporters Gene SbSEC14 C in Sugarbeet: Cloning and Expression Analysis Under Low Temperature Stress

Zou Fengkang¹(), Jia Hailun¹^,²^,³, Ding Guangzhou¹^,²^,³(), Chen Li¹^,²^,³

¹Sugar Beet Engineering Research Center of Heilongjiang Province, Harbin 150080
²National Sugar Crops Improvement Center, Harbin 150080
³Key Laboratory of Sugarbeet Genetics and Breeding in Heilongjiang University, Harbin 150080

Received:2019-11-27 Revised:2020-01-25 Online:2020-11-15 Published:2020-11-19
Contact: Ding Guangzhou

摘要/Abstract

摘要：

磷脂酰肌醇转运蛋白(PITPs)广泛存在于真核生物细胞中,能够在体外膜脂质双层之间调控磷脂酰肌醇(PtdIns)或者磷脂酰胆碱(PtdCho)单体的独立转运。参与磷酸肌醇代谢、膜运输、极性生长、信号转导、逆境胁迫、胞浆运动和细胞周期调节等多种重要的生命过程,在植物的逆境响应以及发育调节中具有重要的作用。为了研究甜菜磷脂酰肌醇转运蛋白基因及其在低温胁迫下的表达情况。本研究以甜菜基因组数据库中一条预测的磷脂酰肌醇转运蛋白基因CRS1为模板,用基因克隆的方法得到一条全长765 bp,开放阅读框596 bp,编码198个氨基酸的甜菜SEC14基因,命名为SbSEC14。理化性质分析表明,该蛋白质为不稳定亲水蛋白;蛋白质二、三级结构分析表明,该蛋白质α-螺旋所占的比例最高,为47.47%,β-转角所占比例最低,为5.56%;蛋白质保守结构分析表明,该蛋白质有典型的SEC14结构域;蛋白质系统进化树分析表明,甜菜SbSEC14基因与菠菜、藜麦的磷脂酰肌醇运转蛋白基因亲缘关系最近;实时荧光定量结果表明,SbSEC14基因在甜菜中组成型表达,在甜菜叶中的表达量最高,在甜菜根中的表达量最低,在叶中表达量约为根中的2.5倍。甜菜幼苗4℃处理0、2、6、12、24 h,该基因在植株处理0~2 h时表达量呈上升趋势,在植株处理2~6 h时表达量呈下降趋势,在植株处理6~24 h时表达量趋于平稳状态。因此,预测该基因与甜菜抗低温胁迫相关。

关键词: 甜菜, 磷脂酰肌醇运转蛋白, SEC14, 低温, 非生物胁迫

Abstract:

Phosphatidylinositol transporters (PITPs) are widely present in eukaryotic cells and can regulate the independent transport of phosphatidylinositol (PtdIns) or phosphatidylcholine (PtdCho) monomers between lipid bilayers in vitro. They are involved in many important life processes such as phosphoinositide metabolism, membrane transport, polar growth, signal transduction, stress, cytoplasmic movement, and cell cycle regulation. They play an important role in plant stress response and development regulation. In order to study the sugarbeet phosphatidylinositol transporter gene and its expression under low temperature stress, in this study, using a predicted phosphatidylinositol transporter gene CRS1 from sugarbeet genome database as a template, a beet SEC14 gene with a length of 765 bp, an open reading frame of 596 bp, and a coding of 198 amino acids was obtained by gene cloning. It was named SbSEC14. The analysis of the physicochemical properties showed that the protein was an unstable hydrophilic protein. The secondary and tertiary structure analysis of the protein showed that the protein's α-helix accounted for the highest proportion, 47.47%, and the β-turn angle accounted for the lowest, 5.56%. The conserved structural analysis of the protein showed that the protein had a typical SEC14 domain. The phylogenetic tree analysis of the protein showed that the beet SbSEC14 gene was most closely related to spinach and quinoa's. Real-time fluorescence quantitative results showed that the constitutive expression of SbSEC14 gene appeared in sugarbeet, the highest expression was in sugarbeet leaves, the lowest expression was in sugarbeet roots, and the expression in leaves was about 2.5 times that of roots. Sugarbeet seedlings were treated at 4℃ for 0, 2, 6, 12, and 24 h, the expression level of this gene showed an upward trend when the plants were treated for 0-2 h, the expression level was decreased when the plants were treated for 2-6 h and the expression level tended to be steady for 6-24 h. Therefore, it is predicted that this gene is related to the resistance of sugarbeet to low temperature stress.

Key words: sugarbeet, phosphatidylinositol transporter, SEC14, low temperature, abiotic stress

中图分类号:

S566.3

邹锋康, 贾海伦, 丁广洲, 陈丽. 甜菜磷脂酰肌醇转运蛋白基因SbSEC14的克隆及低温胁迫下的表达分析[J]. 中国农学通报, 2020, 36(32): 39-48.

Zou Fengkang, Jia Hailun, Ding Guangzhou, Chen Li. Phosphatidylinositol Transporters Gene SbSEC14 C in Sugarbeet: Cloning and Expression Analysis Under Low Temperature Stress[J]. Chinese Agricultural Science Bulletin, 2020, 36(32): 39-48.

图/表 16

引物	引物序列(5'-3')	用途
Sbsec14	R:GCTAGTTTTGGAAGTGAAGGAAAG	基因克隆
Sbsec14	F:ATGCTCTCAAGTCATAGATCACCT	基因克隆
GAPDH	R:GTTGGAACACGGAA AGCC	内参基因
GAPDH	F:TGGAGAGGTGGAAGG	内参基因
qSbsec14	R:GAGGAAACCGTGCCTGCCATCT	荧光定量
qSbsec14	F:TATTATCCAGCGAGGGGCTACAA	荧光定量

表1. SbSEC14基因克隆与表达所用引物

图1. 甜菜幼苗RNA电泳检测

图2. 甜菜SbSEC14基因的PCR扩增 M:D2000 plus DNA Ladder;SbSEC14:目的条带

图3. 甜菜Sbsec14基因的cDNA序列及其推导的氨基酸序列（*终止密码子）方框部分为特异性引物在基因序列中的位置。

图4. 甜菜SbSEC14蛋白序列的信号肽预测

图5. 甜菜SbSEC14蛋白氨基酸的疏水性/亲水性预测

图6. 甜菜SbSEC14蛋白的二级结构预测

图7. 甜菜SbSEC14蛋白的三级结构预测

图8. 甜菜SbSEC14蛋白的磷酸化位点预测

图9. 甜菜SbSEC14蛋白的保守结构域

图10. 甜菜SbSEC14蛋白与其他物种的SEC14蛋白的氨基酸序列比对

图11. 甜菜SbSEC14蛋白与其他物种SEC14蛋白的系统进化树分析

图12. 甜菜SbSEC14基因根、叶柄、叶的相对表达量

图13. 4℃处理不同时间甜菜根中SbSEC14基因的表达量

图14. 4℃处理不同时间甜菜叶柄中SbSEC14基因的表达量

图15. 4℃处理不同时间甜菜叶中SbSEC14基因的表达量

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甜菜磷脂酰肌醇转运蛋白基因SbSEC14的克隆及低温胁迫下的表达分析

Phosphatidylinositol Transporters Gene SbSEC14 C in Sugarbeet: Cloning and Expression Analysis Under Low Temperature Stress

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