唐古特白刺NtCBL1、NtCBL2基因克隆及表达分析

doi:10.12302/j.issn.1000-2006.202009025

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南京林业大学学报（自然科学版） ›› 2021, Vol. 45 ›› Issue (3): 93-99.doi: 10.12302/j.issn.1000-2006.202009025

唐古特白刺NtCBL1、NtCBL2基因克隆及表达分析

黎梦娟¹(), 朱礼明¹, 霍俊男¹, 张景波², 施季森³, 成铁龙¹^,^*()

1.南京林业大学生物与环境学院,南方现代林业协同创新中心,江苏南京 210037
2.中国林业科学研究院沙漠林业实验中心,内蒙古磴口 015200
3.南京林业大学,林木遗传与生物技术省部共建教育部重点实验室,江苏南京 210037

收稿日期:2020-09-11 修回日期:2021-02-22 出版日期:2021-05-30 发布日期:2021-05-31
通讯作者: 成铁龙
基金资助:
国家自然科学基金项目(31770715)

Cloning and expression analyses of NtCBL1,NtCBL2 gene of Nitraria tangutorum

LI Mengjuan¹(), ZHU Liming¹, HUO Junnan¹, ZHANG Jingbo², SHI Jisen³, CHENG Tielong¹^,^*()

1. Co-Innovation Center for the Sustainable Forestry in Southern China, College of Biology and the Environment, NanjingForestry University, Nanjing 210037, China
2. Experimental Center of Desert Forestry, Chinese Academy of Forestry, Dengkou015200, China
3. Key Laboratory of Forest Genetics & Biotechnology, Nanjing Forestry University, Nanjing 210037,China

Received:2020-09-11 Revised:2021-02-22 Online:2021-05-30 Published:2021-05-31
Contact: CHENG Tielong

摘要/Abstract

摘要：

【目的】唐古特白刺(Nitraria tangutorum)是土壤荒漠化和盐碱化防治的先锋植物,对高盐和干旱有极强的适应能力,植物CBL基因作为钙离子感受器,在植物逆境应答及发育过程中具有重要功能。以盐生植物唐古特白刺为材料,开展唐古特白刺CBL基因克隆及表达分析,以深入了解唐古特白刺的抗逆分子机制。【方法】根据唐古特白刺的转录组数据,设计特异性引物,克隆两个CBL基因,并对其进行生物信息学分析和亚细胞定位鉴定。使用实时荧光定量PCR技术分析盐胁迫条件下唐古特白刺CBL基在叶片中的表达模式。【结果】克隆鉴定了唐古特白刺的NtCBL1、NtCBL2基因,NtCBL1基因cDNA编码区长度为642 bp,可编码213个氨基酸,蛋白质分子质量为52.77 ku,分子式为C_{1 971}H_{3 302}N₆₄₂O₈₃₆S₁₀₆;NtCBL2基因cDNA编码区长度为681 bp,可编码226个氨基酸,蛋白质分子质量为26.10 ku,分子式为C_{1 179}H_{1 832}N₂₉₈O₃₅₆S₇。亚细胞定位预测和鉴定结果显示NtCBL1、NtCBL2蛋白均定位于细胞膜上。在胁迫、干旱迫、冷胁迫处理下,NtCBL1、NtCBL2基因均表现出不同程度的变化,而NtCBL1基因的变化更为明显,推测其在盐、干旱胁迫中发挥重要作用。【结论】从唐古特白刺中克隆出NtCBL1、NtCBL2基因,发现NtCBL1基因在盐胁迫下表达量明显上升,而NtCBL2基因变化不大。结果表明NtCBL1基因可能参与唐古特白刺的盐、干旱胁迫响应,而NtCBL2基因在冷胁迫下起一定作用。

关键词: 唐古特白刺, CBL基因, 盐胁迫, 干旱胁迫, 基因克隆, 基因表达

Abstract:

【Objective】 Nitraria tangutorum is a pioneer plant for the soil desertification and salinization control. It is adaptable to high salt and drought conditions. The CBL gene plays an important role in plant stress responses and development as it encodes a calcium ion receptor. Here, we aimed to clone and express CBL in N. tangutorum, which laid a foundation for the study of CBL gene family responses stress and the molecular mechanism of stress resistance of N. tangutorum. 【Method】 We designed specific primers based on N. tangutorum transcriptome data to clone two CBL genes that were further characterized using bioinformatics tools, and their subcellular localization was determined. We investigated the expression of the gene in N. tangutorum under CBL-base salt stress by using real-time quantitative PCR. 【Result】 The NtCBL1 and NtCBL2 genes of N. tangutorum were cloned and identified. The cDNA of NtCBL1 was 642 bp long and encoded 213 amino acids. The relative molecular weight of the protein was 52.77 ku, and the molecular formula was C_{1 971}H_{3 302}N₆₄₂O₈₃₆S₁₀₆. The cDNA of NtCBL2 was 681 bp long and encoded 213 amino acids. The relative molecular weight of the protein was 26.1 ku, and the molecular formula was C_{1 179}H_{1 832}N₂₉₈O₃₅₆S₇. We predicted that the NtCBL1 and NtCBL2 proteins localized at the cell membrane. Under the salt stress, drought stress and cold stress, the expression of NtCBL1 and NtCBL2 changed by various degrees, but NtCBL1 gene was significantly up-regulated under salt and drought stress, and NtCBL2 gene was significantly up-regulated under cold stress. 【Conclusion】 The NtCBL1 and NtCBL2 genes contribute to the adaptation of N. tangutorum to the salt stress. The NtCBL1 gene may be involved in the response of N. tangutorum to the salt as well as drought stress, and the NtCBL2 gene plays a certain role under cold stress.

Key words: Nitraria tangutorum, CBL gene, salt stress, drought stress, gene cloning, gene expression

中图分类号:

S795

黎梦娟,朱礼明,霍俊男,等. 唐古特白刺NtCBL1、NtCBL2基因克隆及表达分析[J]. 南京林业大学学报（自然科学版）, 2021, 45(3): 93-99.

LI Mengjuan, ZHU Liming, HUO Junnan, ZHANG Jingbo, SHI Jisen, CHENG Tielong. Cloning and expression analyses of NtCBL1,NtCBL2 gene of Nitraria tangutorum[J].Journal of Nanjing Forestry University (Natural Science Edition), 2021, 45(3): 93-99.DOI: 10.12302/j.issn.1000-2006.202009025.

图/表 7

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引物名称 primer	序列(5'-3') sequence(5'-3')
NtCBL1-F	5'-ATGGGGTGTTTTCAGTCAAA-3'
NtCBL1-R	5'-TTATGTGGCAAGCTCATCCA-3'
NtCBL2-F	5'-ATGGTGCAGTGCATAGAGGGA-3'
NtCBL2-R	5'-TCAGGTATCTTCAACTCGTGAATG-3'
qCBL1-F	5'-AGCGTGAAGAGGTCAAGCAA 3
qCBL1-R	5'-ACCTGGTTTGCATCGGCTT-3'
qCBL2-F	5'-AGCTACACTTGCTGAGTCGG-3'
qCBL2-R	5'-ATGGATGTCGCAGGACAAGG
β-Actin-F	5'-CTTATAACGCTATCATAGGGCGTCTGAGC-3'
β-Actin-R	5'-TCGGTCAATATCTAGCCATGCGGG-3'
gNtCBL1-F	5'-ttccaccatggcgtgcaggtcgactctagaATGGGGTGGTT TTCAGTCAAAGGT-3'
gNtCBL1-R	5'-cagctcctcgcccttgctcaccatggatccTGTGGCAAGCTC ATCCACTTCT-3'
gNtCBL2-F	5'-ttccaccatggcgtgcaggtcgactctagaATGGTGCAGT GCATAGAGGGA-3'
gNtCBL2-R	5'-cagctcctcgcccttgctcaccatggatccGGTATCTTCAA CTCGTGAATGGAATACAAAAC-3'

唐古特白刺NtCBL1、NtCBL2基因克隆及表达分析

Cloning and expression analyses of NtCBL1,NtCBL2 gene of Nitraria tangutorum

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