水稻低亲和性阳离子转运蛋白基因OsLCT3的克隆与功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2023-1025

摘要/Abstract

摘要：

【目的】部分稻米镉超标严重影响我国粮食质量安全，本研究旨在鉴定对稻米镉积累有调控作用的基因，为阻控稻米镉积累提供新的基因资源。【方法】通过逆转录PCR和RACE技术，克隆了水稻低亲和性阳离子转运蛋白基因家族的一个新成员OsLCT3。通过生物信息学方法对OsLCT3的自然变异进行分析，对OsLCT3蛋白的理化性质进行预测；利用实时荧光定量 PCR分析其全生育期表达模式及对镉、锰、铁胁迫的响应；采用融合报告基因定位法探究OsLCT3的亚细胞定位。通过苗期镉胁迫水培以及镉污染土壤的成熟期植株各部位的镉及二价矿质金属元素测定，分析敲除OsLCT3对水稻二价阳离子运输的影响。此外，通过酵母细胞的异源功能互补验证OsLCT3 对酵母镉耐受性的影响。【结果】OsLCT3仅存在于部分水稻品种中，编码区全长1 263 bp，根据编码区变异其氨基酸序列可划分为5个单倍型，编码的蛋白具有12个跨膜结构域，与小麦、节节麦的LCT亲缘关系较近，与水稻OsLCT2的同源性仅52%，与籼稻、粳稻两个亚种OsLCT1的同源性分别为49%、47%。OsLCT3在生长发育的各时期均在根部高表达，在扬花期的表达最高，且在根部的表达受镉胁迫及过量铁、锰的抑制，蛋白定位于质膜。与野生型相比，oslct3敲除系苗期株高降低，地上部的镉、铁、锌含量降低，根部铁含量升高，其他元素含量不变；根部镉、铁、锌向地上部的转运率降低。大田条件下，oslct3敲除系成熟期的茎叶和糙米镉含量较野生型均显著下降，稻草和糙米锰、铜、铁、锌含量较对照无显著差异。表达OsLCT3导致酵母对镉胁迫更敏感。【结论】OsLCT3参与根部的镉、铁、锌向地上部的运输，正调控稻米镉积累。

关键词: 水稻, OsLCT3, 镉转运, 铁转运, 锌转运, 金属转运蛋白

Abstract:

【Objective】Excessive cadmium（Cd）in some rice grains seriously affect food safety in China. The aim of this study is to identify the novel gene that regulate accumulation of Cd in rice grains and provide the genetic resource for reducing Cd accumulation in rice grains. 【Method】OsLCT3, a member of the low- affinity cation transporter family in rice, was cloned by reverse transcription PCR and RACE techniques. Natural variations of OsLCT3 and physicochemical properties of its coding protein were analyzed by bioinformatics methods. Its expression profiles throughout the growth period and under Cd, Mn, Fe stress were analyzed by quantitative real-time PCR（RT-qPCR）. Its subcellular localization was explored by investigating localization of OsLCT3-GFP fusion protein in in rice protoplasts. The effect of OsLCT3 knockout on divalent cation transport in rice was analyzed by measuring cadmium and other divalent mineral metal elements in various parts of plants at seedling stage in cadmium-stressed hydroponics and at maturity stage in cadmium-polluted soil. In addition, the effect of OsLCT3 on the yeast tolerance to Cd was verified by heterologous functional complementarity in yeast cells. 【Result】OsLCT3 only existed in some indica and japonica rice varieties, with a total coding region of 1 263 bp. The amino acid sequences were classified into five haplotypes based on coding region variation, and the encoded proteins had 12 sub-transmembrane structural domains. OsLCT3 was similar to the LCT-like proteins in Triticum aestivum and Aegilops tauschii, whereas it shared only 52% sequence identity with OsLCT2, 49% and 47% sequence identity with OsLCT1 in indica and japonica rice subspecies, respectively. OsLCT3 was highly expressed in the roots at all stages of growth and development. Its expression levels in the roots were suppressed by Cd, excessive iron（Fe）and manganese（Mn）stress. OsLCT3 was localized to the plasma membrane. Compared with the wild type plants, the oslct3 knockout lines showed the reduced plant height, the lower concentrations of Cd, Fe, and Zn in the shoots, higher concentration of Fe in the roots, and the same concentrations of other bivalent mineral elements. Moreover, oslct3 knockout lines demonstrated the decreased root-to-shoot translocation rates of cadmium, iron and zinc. Under field conditions, Cd concentrations in straw and brown rice of oslct3 knockout lines at maturity were significantly lower than those of the wild type plants, and there were no significant differences in manganese, copper, iron and zinc concentrations in straw and brown rice compared with the wild type plants. Expression of OsLCT3 in yeast resulted in increased sensitivity of yeast to cadmium stress.【Conclusion】OsLCT3 is involved in the translocation of Cd, Fe, and Zn from roots to shoots, and positively regulated the accumulation of Cd in rice grains.

Key words: rice, OsLCT3, cadmium transport, iron transport, zinc transport, metal transporter

李兴容, 谭志兵, 赵燕, 李曜魁, 赵炳然, 唐丽. 水稻低亲和性阳离子转运蛋白基因OsLCT3的克隆与功能研究[J]. 生物技术通报, 2024, 40(4): 97-109.

LI Xing-rong, TAN Zhi-bing, ZHAO Yan, LI Yao-kui, ZHAO Bing-ran, TANG Li. Cloning and Functional Analysis of OsLCT3, a Low-affinity Cation Transporter Gene of Rice[J]. Biotechnology Bulletin, 2024, 40(4): 97-109.

图/表 11

表1 本研究所用的引物及序列

Table 1 Primers and sequences used in this study

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	引物用途 Primer usage
T3-CDS-F	TATCTTATGGACTGGTCTGTAGC	OsLCT3 CDS扩增、测序
T3-CDS-R	TCTGTTGTCTGATGATGATTTTG	OsLCT3 CDS amplification and sequencing
T3-C-F	ATGGTGGCCCTTTCTATCCA	OsLCT3 RACE核心序列扩增
T3-C-R	TCACTGTATCTCCAAAACCA	OsLCT3 RACE core sequence amplification
T3-3-F2	TTGCCTCTGCAATAGTTGCC	OsLCT3 3' RACE 扩增
T3-3-F3	GTACTGTAATGGTTACACTTAG	OsLCT3 3' RACE amplification
T3-5-R2	GAGGAAATACCAAGCCACCATC	OsLCT3 5' RACE 扩增
T3-5-R3	CGAAGTGATACATCCCTCAACCTG	OsLCT3 5' RACE amplification
OsLCT3-F2	ATGGTGGCTTGGTATTTCCTC	OsLCT3 DNA片段扩增
OsLCT3-R2	CACCTCATTGGCATTACTGTCC	Amplification of OsLCT3 DNA fragments
OsLCT3-F3	GAGATCCGTGCGAAAGTGCT	OsLCT3 DNA片段扩增 Amplification of OsLCT3 DNA fragments
OsLCT3-R3	AGAGTGAGTGAAAAGCCCGA	OsLCT3 DNA片段扩增 Amplification of OsLCT3 DNA fragments
OsActin1-F	TGCTATGTACGTCGCCATCCAG	OsActin1 DNA片段扩增
OsActin1-R	AATGAGTAACCACGCTCCGTCA	Amplification of OsActin1 DNA fragments
T3-qP-F	GGTGGTTCTGGGAAGACTAAA	OsLCT3 RT-qPCR分析
T3-qP-R	ATGGAGAGCAGACCTGATATTG	OsLCT3 RT-qPCR analysis
OsActin1-qP-F	CAACACCCCTGCTATGTACG	OsActin1 RT-qPCR分析
OsActin1-qP-R	CATCACCAGAGTCCAACACAA	OsActin1 RT-qPCR analysis
T3P1-F	GGCAGTACATTGGAAGCTTGTCTG	OsLCT3 敲除载体靶位点1 接头引物
T3P1-R	AAACCAGACAAGCTTCCAATGTAC	OsLCT3 knockout vector target site 1 adaptor primer
T3P2-F	GCCGTCAATTACAACGCCACCGG	OsLCT3 敲除载体靶位点2 接头引物
T3P2-R	AAACCCGGTGGCGTTGTAATTGA	OsLCT3 knockout vector target site 2 adapter primers
T3-p1132-F	cgcggtggcggccgctctagaATGGTGGCCCTTTCTATCCAT	OsLCT3亚细胞定位
T3-p1132-R	gataagcttgatatcgaattcCTGTATCTCCAAAACCACCGCG	OsLCT3 subcellular localization
pYES2-T3-F	gggaatattaagcttggtaccATGGTGGCCCTTTCTATCCAT	OsLCT3酵母载体构建
YES2-T3-R	gatggatatctgcagaattcTCACTGTATCTCCAAAACCACCG	OsLCT3 yeast vector construction
HPT-F	GCTCCATACAAGCCAACCACG	Hpt片段扩增
HPT-R	CCTGCCTGAAACCGAACTGC	Hpt fragment amplification
CAS9-F	CGAGACGAACGGTGAGACTGGTG	Cas9片段扩增
CAS9-R	GGTGCTTGTTGTAGGCGGAGAGG	Cas9 fragment amplification
OsLCT3-CAS-F	CTGCGGTATATCATCCCAATGCT	OsLCT3的靶点PCR扩增
OsLCT3-CAS-R	CAACTATGGTGAAAACTTCGGCG	PCR amplification of OsLCT3 targets
pYES2-T3-eGFP-F	gggaatattaagcttggtaccATGGTGGCCCTTTCTATCCAT	OsLCT3-eGFP酵母载体构建
pYES2-T3-eGFP-R	tgatggatatctgcagaattcTTACTTGTACAGCTCGTCCATGCC	OsLCT3-eGFP yeast vector construction

表1 本研究所用的引物及序列

Table 1 Primers and sequences used in this study

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	引物用途 Primer usage
T3-CDS-F	TATCTTATGGACTGGTCTGTAGC	OsLCT3 CDS扩增、测序
T3-CDS-R	TCTGTTGTCTGATGATGATTTTG	OsLCT3 CDS amplification and sequencing
T3-C-F	ATGGTGGCCCTTTCTATCCA	OsLCT3 RACE核心序列扩增
T3-C-R	TCACTGTATCTCCAAAACCA	OsLCT3 RACE core sequence amplification
T3-3-F2	TTGCCTCTGCAATAGTTGCC	OsLCT3 3' RACE 扩增
T3-3-F3	GTACTGTAATGGTTACACTTAG	OsLCT3 3' RACE amplification
T3-5-R2	GAGGAAATACCAAGCCACCATC	OsLCT3 5' RACE 扩增
T3-5-R3	CGAAGTGATACATCCCTCAACCTG	OsLCT3 5' RACE amplification
OsLCT3-F2	ATGGTGGCTTGGTATTTCCTC	OsLCT3 DNA片段扩增
OsLCT3-R2	CACCTCATTGGCATTACTGTCC	Amplification of OsLCT3 DNA fragments
OsLCT3-F3	GAGATCCGTGCGAAAGTGCT	OsLCT3 DNA片段扩增 Amplification of OsLCT3 DNA fragments
OsLCT3-R3	AGAGTGAGTGAAAAGCCCGA	OsLCT3 DNA片段扩增 Amplification of OsLCT3 DNA fragments
OsActin1-F	TGCTATGTACGTCGCCATCCAG	OsActin1 DNA片段扩增
OsActin1-R	AATGAGTAACCACGCTCCGTCA	Amplification of OsActin1 DNA fragments
T3-qP-F	GGTGGTTCTGGGAAGACTAAA	OsLCT3 RT-qPCR分析
T3-qP-R	ATGGAGAGCAGACCTGATATTG	OsLCT3 RT-qPCR analysis
OsActin1-qP-F	CAACACCCCTGCTATGTACG	OsActin1 RT-qPCR分析
OsActin1-qP-R	CATCACCAGAGTCCAACACAA	OsActin1 RT-qPCR analysis
T3P1-F	GGCAGTACATTGGAAGCTTGTCTG	OsLCT3 敲除载体靶位点1 接头引物
T3P1-R	AAACCAGACAAGCTTCCAATGTAC	OsLCT3 knockout vector target site 1 adaptor primer
T3P2-F	GCCGTCAATTACAACGCCACCGG	OsLCT3 敲除载体靶位点2 接头引物
T3P2-R	AAACCCGGTGGCGTTGTAATTGA	OsLCT3 knockout vector target site 2 adapter primers
T3-p1132-F	cgcggtggcggccgctctagaATGGTGGCCCTTTCTATCCAT	OsLCT3亚细胞定位
T3-p1132-R	gataagcttgatatcgaattcCTGTATCTCCAAAACCACCGCG	OsLCT3 subcellular localization
pYES2-T3-F	gggaatattaagcttggtaccATGGTGGCCCTTTCTATCCAT	OsLCT3酵母载体构建
YES2-T3-R	gatggatatctgcagaattcTCACTGTATCTCCAAAACCACCG	OsLCT3 yeast vector construction
HPT-F	GCTCCATACAAGCCAACCACG	Hpt片段扩增
HPT-R	CCTGCCTGAAACCGAACTGC	Hpt fragment amplification
CAS9-F	CGAGACGAACGGTGAGACTGGTG	Cas9片段扩增
CAS9-R	GGTGCTTGTTGTAGGCGGAGAGG	Cas9 fragment amplification
OsLCT3-CAS-F	CTGCGGTATATCATCCCAATGCT	OsLCT3的靶点PCR扩增
OsLCT3-CAS-R	CAACTATGGTGAAAACTTCGGCG	PCR amplification of OsLCT3 targets
pYES2-T3-eGFP-F	gggaatattaagcttggtaccATGGTGGCCCTTTCTATCCAT	OsLCT3-eGFP酵母载体构建
pYES2-T3-eGFP-R	tgatggatatctgcagaattcTTACTTGTACAGCTCGTCCATGCC	OsLCT3-eGFP yeast vector construction

图1 OsLCT3的 PCR 扩增 A： OsLCT3 基因克隆；B：OsLCT3多态性分析

Fig. 1 PCR amplification of OsLCT3 A: OsLCT3 gene cloning; B: OsLCT3 polymorphism analysis

图2 OsLCT3 的生物信息学分析 A：OsLCT3蛋白的疏水性分析；B：LCT家族成员的系统进化分析；C：OsLCT3单倍型的分布频率；D：OsLCT3蛋白的信号肽预测；E：OsLCT3跨膜结构域分析；F：OsLCT3单倍型分析；Ta: 小麦；Sbi: 高粱；Os: 水稻；Si: 小米；Ata: 节节麦；Hv: 大麦；Obr: 短花野生稻

Fig. 2 Bioinformatics analysis for OsLCT3 A: Hydrophobicity of OsLCT3 protein. B: Phylogenetic analysis of LCT family members. C: Distribution frequency of the OsLCT3 haplotype. D: Signaling peptide prediction of OsLCT3 protein. E: OsLCT3 transmembrane domain analysis. F: OsLCT3 haplotype analysis. Ta: Triticum aestivum; Sbi: Sorghum bicolor; Os: Oryza sativa; Si: Setaria italica; Ata: Aegilops tauschii; Hv: Hordeum vulgare; Obr: Oryza brachyantha

表2 OsLCT3 蛋白的理化性质

Table 2 Physicochemical properties of OsLCT3 protein

蛋白质 Protein	氨基酸数 Amino acid No./aa	分子式 Formula	分子量 Molecular weight/kD	等电点 pI	不稳定指数 Instability index	平均疏水性 Hydropathy index	正 / 负电残基数 Acid-base amino acid	脂溶指数 Aliphatic index
OsLCT3	420	C₂₀₈₈H₃₃₁₅N₅₁₁O₅₆₇S₂₂	45.355	6.51	25.72	0.777	28/30	121.95

图3 OsLCT3的表达模式 A：OsLCT3在不同生育期的表达模式分析；孕穗期幼穗的 OsLCT3表达水平被设置为 1，采用 2-△△Ct 法估算各个时期各个组织部位中 OsLCT3的相对表达水平（R：根；LB：叶片；LS：叶鞘；BS：茎基部；YP：幼穗；FLB：旗叶叶片；FLS：旗叶叶鞘；LLB：低位叶片；LLS：低位叶鞘；N I：倒一茎节；N II：倒二茎节；N III：倒三茎节；P：穗梗；S：小穗；H：颖壳）；B： OsLCT3对不同金属元素胁迫7 d的响应。标准差基于3个生物学重复。数据分析采用单因素方差分析，t- test 检验，*表示相同条件下与对照相比，在0.05水平上差异显著

Fig. 3 Expression patterns of OsLCT3 A: Expression analysis of OsLCT3 at different growth stages. The expression level of OsLCT3 in young panicle of booting stage was set to 1, and the 2-△△ Ct method was used to estimate the relative expression of OsLCT3 in each tissue at each stage（R: Root. LB: Leaf blade. LS: Leaf sheath. BS: Basal stem. YP: Young panicle. FLB: Flag leaf blade. FLS: Flag leaf sheath. LLB: Lower leaf blade. LLS: Lower leaf sheath. N I: Node I. N II: Node II. N III: Node III. P: Peduncle. S: Spikelet. H: Husk）. B: Response of OsLCT3 to stress of different metal elements for 7 d. Standard deviation is based on three biological replicates. One-way ANOVA was used for data analysis，* indicating a significant difference at the level of 0.05 compared with the control by student’s t- test

图4 OsLCT3的亚细胞定位 eGFP：增强绿色荧光蛋白；mCherry：红色荧光蛋白；Bright field：明场；Merge：荧光整合；比例尺=10 μm

Fig. 4 Subcellular localization of OsLCT3 eGFP: Enhanced green fluorescence. mCherry: Red fluorescent protein. Bright field: Bright field. Merge: Fluorescence integration. Scale bar=10 μm

图5 oslct3和WT水培条件下的生长情况 A-B：oslct3纯合突变体的鉴定；C-D：WT和敲除系的表型；E：株高；F：根长；*表示相同条件下与野生型相比，0.05水平上差异显著，**表示相同条件与下野生型相比，0.01水平上差异显著（t-test 检验），下同。比例尺=5 cm

Fig. 5 Growth of oslct3 and WT in hydroponic growth A-B: Identification of oslct3 homozygous mutants. C-D: Phenotypes of WT and knockout lines. E: Plant height. F: Root length. * or ** indicate statistically significant difference in comparison to WT at P< 0.05 or P< 0.01 by student’s t test, the same applies hereinafter. Scale bar=5 cm

图6 oslct3的二价矿质营养及Cd含量分析

Fig. 6 Analysis of divalent mineral nutrition and Cd content of oslct3 conc: Concentration. The same below

图7 Cd污染大田条件下oslct3 产量、稻草和糙米的金属含量分析 A：成熟期植株表型；B：株高；C：单株产量；D-E：糙米、稻草Cd含量；F-G：糙米、稻草金属元素含量。比例尺=10 cm

Fig. 7 Grain yield of oslct3 and metal contents in straw and brown rice under Cd-contaminated fields A: Plant phenotype at maturity. B: Plant height. C: Yield per plant. D-E: Cd content in brown rice and straw. F-G: Brown rice and straw metal element content. Scale bar=10 cm

图8 OsLCT3 对酵母 Cd 耐受性的影响鉴定 A：OsLCT3 在酵母中Cd耐受性表型鉴定；B：OsLCT3在酵母中的耐Cd性生长曲线分析

Fig. 8 Identification of effects of OsLCT3 on the tolerance of yeast to Cd stress A: OsLCT3 Cd tolerance phenotypic identification in yeast. B: Cd tolerance growth curve analysis of OsLCT3 in yeast

图9 OsLCT3 在酵母中的亚细胞定位比例尺=2 μm

Fig. 9 Subcellular localization of OsLCT3 in yeast Scale bar=2 μm

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