甘蔗硝酸盐转运蛋白1/肽转运蛋白家族6.4基因(ScNPF6.4)克隆及其调控分蘖功能分析

doi:10.3724/SP.J.1006.2024.44002

作物学报 ›› 2024, Vol. 50 ›› Issue (8): 2131-2142.doi: 10.3724/SP.J.1006.2024.44002

甘蔗硝酸盐转运蛋白1/肽转运蛋白家族6.4基因(ScNPF6.4)克隆及其调控分蘖功能分析

李旭娟¹^,²(), 李纯佳¹^,², 田春艳¹^,², 孔春艳¹^,², 徐超华¹^,², 刘新龙¹^,²^,^*()

¹热带作物生物育种全国重点实验室, 云南昆明 650205
²云南省农业科学院甘蔗研究所 / 云南省甘蔗遗传改良重点实验室, 云南开远 661699

收稿日期:2024-01-02 接受日期:2024-04-01 出版日期:2024-08-12 网络出版日期:2024-04-24
通讯作者: * 刘新龙, E-mail: lxlgood868@163.com
作者简介:E-mail: lixujuan2011@163.com
基金资助:
云南种子种业联合实验室(202205AR070001-09);国家自然科学基金项目(31760412);国家重点研发计划项目(2022YFD2301102-20);云南省科技人才与平台计划项目(202205AM070001)

Identification of nitrate transporter protein 1/peptide transporter protein family 6.4 gene (ScNPF6.4) and functional analysis of its regulation of tillering in sugarcane

LI Xu-Juan¹^,²(), LI Chun-Jia¹^,², TIAN Chun-Yan¹^,², KONG Chun-Yan¹^,², XU Chao-Hua¹^,², LIU Xin-Long¹^,²^,^*()

¹National Key Laboratory for Biological Breeding of Tropical Crops, Kunming 650205, Yunnan, China
²Sugarcane Research Institute, Yunnan Academy of Agricultural Sciences / Yunnan Key Laboratory of Sugarcane Genetic Improvement, Kaiyuan 661699, Yunnan, China

Received:2024-01-02 Accepted:2024-04-01 Published:2024-08-12 Published online:2024-04-24
Contact: * E-mail: lxlgood868@163.com
Supported by:
Yunnan Seed Laboratory(202205AR070001-09);National Natural Science Foundation of China(31760412);National Key Research and Development Program of China(2022YFD2301102-20);Yunnan Science and Technology Talent and Platform Program(202205AM070001)

摘要/Abstract

摘要：

甘蔗(Saccharum spp. hybrid)是重要的糖料和能源作物, 分蘖是影响其产量的重要性状之一。硝酸盐转运蛋白1/肽转运蛋白家族(NITRATE TRANSPORTER 1 (NRT1)/PEPTIDE TRANSPORTER (PTR) family, NPF)成员在植生长发育中发挥重要作用, 甘蔗中NPF基因的挖掘利用可为甘蔗品种分蘖遗传调控和产业升级奠定重要基础。本研究通过前期转录组数据结合反转录PCR (reverse transcription PCR, RT-PCR)从甘蔗品种ROC22中获得其NPF家族成员6.4基因的cDNA全长序列(命名为ScNPF6.4), 并对其进行序列结构、理化性质、系统进化等分析, 然后开展了该基因在苗期甘蔗中的组织特异性、甘蔗腋芽萌发过程中的表达情况以及响应激素处理的表达模式分析, 最后将该基因遗传转化水稻过表达进行功能验证。结果表明, ScNPF6.4的cDNA包含1个1806 bp的开放阅读框, 编码601个氨基酸, 属于易化子超家族(major facilitator superfamily, MFS)蛋白, 其蛋白分子量为63.9 kD, 理论等电点为9.23, 包含12个跨膜螺旋区。该蛋白不存在信号肽, 具疏水性, 属于一类稳定的非分泌蛋白, 系统进化分析表明其属于NPF 6.4亚族蛋白。亚细胞定位分析表明该蛋白定位于内质网。组织特异性分析发现, ScNPF6.4在苗期甘蔗根中相对表达量最高, 在叶和茎基部相对表达量较低; ScNPF6.4在不同甘蔗品种腋芽萌发阶段均上调表达; 适量浓度的植物外源激素6-BA、ABA、GA₃、IBA、乙烯利和SLs均能诱导苗期甘蔗中ScNPF6.4的上调表达; 水稻中异位过表达ScNPF6.4可增加水稻分蘖数并提前孕穗。以上结果表明, ScNPF6.4正调控甘蔗分蘖芽萌发, 其表达受外源植物激素调控, 该基因过表达可增加水稻分蘖数并促进提前孕穗, 克隆该基因可为甘蔗分蘖早生快发高产育种提供重要的基因资源。

关键词: 甘蔗, ScNPF6.4, 分蘖, 植物外源激素, 功能分析

Abstract:

Sugarcane is an important sugar and energy crop, and tillering is one of the important traits affecting its yield. Nitrate transporter 1 (NRT1) / peptide transporter (PTR) family (NPF) protein plays an important role in plant vegetative growth and development, mining and exploitation of NPF genes in sugarcane could lay an important foundation for genetic regulation of tillering in sugarcane. In this study, the full-length cDNA sequence of the NPF6.4 gene (ScNPF6.4) was obtained from sugarcane variety ROC22 by combining pre-transcriptomic data with RT-PCR. Subsequently, the sequence structure, physicochemical properties and phylogeny were analyzed. The tissue-specific expression of the gene in sugarcane at seedlings stage, the expression in axillary bud germination, and the expression pattern in responsive to hormone treatments were analyzed. Finally, the gene was genetically transformed into rice to verify the over-expression for functional verification. The results showed that the cDNA of ScNPF6.4 contained an open reading frame of 1806 bp encoding 601 amino acids, which belonged to the major facilitator superfamily (MFS) protein with a molecular weight of 63.9 kD and a theoretical isoelectric point of 9.23, and included 12 transmembrane helical regions. The protein was hydrophobic in nature and had no signal peptide, whereas it belonged to a class of stable non-secretory proteins. In addition, phylogenetic analysis illustrated that it belonged to the NPF family and 6.4 subfamily of proteins. Likewise, subcellular localization indicated that the protein was localized in the endoplasmic reticulum. The tissue-specific analysis revealed that the relative expression level of ScNPF6.4 was the highest in roots and lower in leaf and stem bases of sugarcane seedling. Moreover, ScNPF6.4 was up-regulated at the axillary bud sprouting stage of different sugarcane varieties, and appropriate concentrations of exogenous plant hormones such as 6-BA, ABA, GA₃, IBA, ethylene, and SLs could induce the up-regulated its expression in sugarcane at seedling stage, while ectopic over-expression of ScNPF6.4 increased the tillering number and induced early heading in rice. Thus, it could be inferred that ScNPF6.4 could regulate the germination of sugarcane tiller buds, and its expression was up-regulated by exogenous phytohormones, whereas the over-expression of this gene could increase the tillering numbers and induce early heading in rice. This study provides an important genetic resource for the breeding of sugarcane to improve tillering ability, which is beneficial for the early and rapid growth and high cane yield formation.

Key words: sugarcane, ScNPF6.4, tillering, exogenous plant hormone, functional analysis

李旭娟, 李纯佳, 田春艳, 孔春艳, 徐超华, 刘新龙. 甘蔗硝酸盐转运蛋白1/肽转运蛋白家族6.4基因(ScNPF6.4)克隆及其调控分蘖功能分析[J]. 作物学报, 2024, 50(8): 2131-2142.

LI Xu-Juan, LI Chun-Jia, TIAN Chun-Yan, KONG Chun-Yan, XU Chao-Hua, LIU Xin-Long. Identification of nitrate transporter protein 1/peptide transporter protein family 6.4 gene (ScNPF6.4) and functional analysis of its regulation of tillering in sugarcane[J]. Acta Agronomica Sinica, 2024, 50(8): 2131-2142.

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[45]	Wang J, Wan R, Nie H, Xue S, Fang Z. OsNPF5.16, a nitrate transporter gene with natural variation, is essential for rice growth and yield. Crop J, 2022, 10: 397-406. doi: 10.1016/j.cj.2021.08.005
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引物名称Primer name	引物序列Primer sequence (5°-3°)	引物作用 Primer function
NPF-54F	GTCGTCCTCGCTCCCAGTC	基因克隆 Gene cloning
NPF-2143R	CGCCGTATGCCTCCTATGTT
NPF-138F	ATGGTTTCCGCTGGGG
NPF-1943R	CTACACGTCCATTCCTTCG
Q-ScNPF-208F	TCCAACTCCAAGTCCGCCAAC	表达分析(甘蔗) Relative expression analysis (in sugarcane)
Q-ScNPF-358R	CAAGCTCACGCCGGTGGC	表达分析(甘蔗) Relative expression analysis (in sugarcane)
GAPDH-F	CACGGC CAC TGGAAGCA	内参基因(甘蔗) Reference gene (in sugarcane)
GAPDH-R	TCC TCAGGG TTC CTG ATG CC	内参基因(甘蔗) Reference gene (in sugarcane)
Q-ScNPF6.4-F	CGCGAACGCCCGTGGTCCGC	表达分析(水稻) Relative expression analysis (in rice)
Q-ScNPF6.4-R	CGGACACGTTCGCCTTGAGACCCCCC	表达分析(水稻) Relative expression analysis (in rice)
OsActin	TGCTATGTACGTCGCCATCCA	内参基因(水稻) Reference gene (in rice)
OsActin	AATGAGTAACCACGCTCCGTC	内参基因(水稻) Reference gene (in rice)

甘蔗硝酸盐转运蛋白1/肽转运蛋白家族6.4基因(ScNPF6.4)克隆及其调控分蘖功能分析

Identification of nitrate transporter protein 1/peptide transporter protein family 6.4 gene (ScNPF6.4) and functional analysis of its regulation of tillering in sugarcane

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