烟草NtPsbS的亚细胞定位及编码基因表达模式分析

doi:10.7668/hbnxb.20195402

摘要/Abstract

摘要：

为了解PsbS蛋白在烟草中的表达特征,从烟草品种K326的cDNA中克隆了NtPsbS基因全长序列,利用DNAMAN软件对水稻、番茄、大豆等7种作物PsbS蛋白的氨基酸序列与NtPsbS蛋白的氨基酸序列进行比对,并使用MEGA 11软件采用邻接法建立系统发育树对其进行遗传进化分析;利用qRT-PCR技术检测烟草不同生育时期NtPsbS基因的组织表达水平;构建pS1300-PsbS-GFP植物表达载体研究其成熟蛋白亚细胞定位特性;对烟草品种K326进行各类非生物胁迫处理,研究该基因在非生物胁迫条件下的表达模式。结果表明,NtPsbS基因全长825 bp,编码274个氨基酸;NtPsbS蛋白与番茄SlPsbS蛋白相似性最高,达到91%;NtPsbS在旺长期和成熟期烟草品种K326的叶、根、茎、种子等部位均有表达,在叶片中表达量最高;成熟NtPsbS蛋白定位于烟草叶绿体内;NtPsbS在盐胁迫、冷胁迫及脱落酸ABA处理下表达量均显著升高。综上所述,NtPsbS基因在不同生育时期的烟草叶片中表达量最高,对盐胁迫、冷胁迫及脱落酸ABA处理均有响应,说明该基因可能参与烟草体内抗盐、抗冷胁迫及ABA代谢通路,为后续开展NtPsbS基因的功能特性解析提供了参考。

关键词: 普通烟草, PsbS基因, 亚细胞定位, 组织表达模式, 非生物胁迫

Abstract:

In order to reveal the expression patterns of PsbS protein in Nicotiana tabacum,the full-length NtPsbS gene sequence was cloned from cDNA of the tobacco cultivar K326,and the multiple protein sequences of NtPsbS gene and PsbS genes of seven crops including rice,tomato and soybean were compared by DNAMAN software.The phylogenetic analysis was carried out by MEGA 11 software,and the phylogenetic tree was established by neighbor-joining method.Tissue expression of NtPsbS gene in tobacco at different growth stages was detected by qRT-PCR.The plant expression vector pS1300-PsbS-GFP was constructed to find subcellular localization of the NtPsbS mature protein.Finally,the mRNA levels of NtPsbS under abiotic stresses were analyzed in K326.The results showed that the total length of tobacco NtPsbS gene was 825 bp,encoding 274 amino acids.The homology of tobacco NtPsbS protein and tomato SlPsbS protein was the highest up to 91%.The NtPsbS gene exhibited the highest mRNA levels in the leaves among the tissues,including leaves,roots,stems,seeds and other parts of K326.The mature NtPsbS protein was located in chloroplasts.For the abiotic stress,it was found that the expression level of NtPsbS was significantly increased under salt stress,cold stress and Abscisic acid(ABA)treatments.In summary,the expression level of NtPsbS was the highest in tobacco leaves at different growth stages,and it was higher when under the treatments of salt stress,cold stress and ABA,indicating that this gene may be involved in the salt and cold stress resistance and ABA metabolic pathway in tobacco,which could provide basis and reference for the functional analysis of the NtPsbS gene in future.

Key words: Nicotiana tabacum, PsbS gene, Subcellular localization, Tissue expression pattern, Abiotic stress

范普晴, 周厚良, 宋杉杉, 林法明, 石永春, 王潇然, 王燃, 张小全. 烟草NtPsbS的亚细胞定位及编码基因表达模式分析[J]. 华北农学报, 2025, 40(2): 49-55. doi: 10.7668/hbnxb.20195402.

FAN Puqing, ZHOU Houliang, SONG Shanshan, LIN Faming, SHI Yongchun, WANG Xiaoran, WANG Ran, ZHANG Xiaoquan. Subcellular Localization and Coding Gene Expression Pattern Analysis of NtPsbS in Tobacco[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(2): 49-55. doi: 10.7668/hbnxb.20195402.

http://www.hbnxb.net/CN/Y2025/V40/I2/49

图/表 6

表1 引物序列及用途

Tab.1 Primer sequences and uses

引物名称 Primer name	引物序列 (5'—3') Primer sequence (5'—3')	用途 Use
NtPsbS	F;ATGGCTCAAACAATGTTGCTGAC	基因克隆
	R:TCACTCCTCTTCCTCATCAG
qRT-NtPsbS	F:CTCTTCCTTTGAAATACCCTTC	检测NtPsbS表达水平
	R:TGAAACCAATTCCTCCTGAGG
qRT-NtL25	F:ATGGCTCAAACAATGTTGCTGAC	检测NtL25表达水平
	R:ATGGCTCAAACAATGTTGCTGAC
NtPsbS-GFP	F:CAGGGGCCCGGGGTCGACATGGCTCAAACAATGTTGCTGAC	亚细胞定位
	R:CCCTTGCTCACCATGGTACCTCACTCCTCTTCCTCATCAG

图1 NtPsbS蛋白与同源蛋白序列的比较(A)及系统发育树构建(B)

Fig.1 Comparison of NtPsbS protein and homologous protein sequences (A) and construction of phylogenetic tree(B) AtPsbS.NP_001319163.1;GhPsbS.XP_016708802.1;GmPsbS.XP_003523444.1;NtPsbS.NP_001312190.1;OsPsbS.XP_01521169.1;S1PsbS.NP_001296186.1;TaPsbS.XP_020167324.1;ZmPsbS.NP_001105228.3.

图2 NtPsbS PCR产物电泳图谱(A)、pS1300-PsbS-GFP表达载体示意图(B)及表达载体酶切鉴定电泳图谱(C)

Fig.2 Electrophoresis map of NtPsbS PCR product (A),schematic diagram of pS1300-PsbS-GFP expression vector (B)and expression vector enzyme digestion identification (C)

图3 NtPsbS基因的组织表达模式分析图柱上不同小写字母表示差异显著(P<0.05)。图5同。

Fig.3 Expression profile of NtPsbS gene in tissues Different lowercase letters on the column indicate significant differences (P<0.05).The same as Fig.5.

图4 NtPsbS-GFP蛋白在本氏烟草叶片细胞中的定位

Fig.4 Subcellular localization of NtPsbS-GFP in leaves of Nicotiana benthamiana

图5 NtPsbS基因在非生物胁迫下的表达水平

Fig.5 Expression level of NtPsbS gene under abiotic stresses

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