宁夏枸杞铁镉响应基因的筛选及其功能验证

doi:10.13560/j.cnki.biotech.bull.1985.2022-1304

摘要/Abstract

摘要：

为探讨铁（Fe）、镉（Cd）胁迫对宁夏枸杞LbFRO（ferric reduction oxidase）、LbZIP（zinc-regulated transporters and iron-regulated transporter-like protein）和LbNRAMP（natural resistance-associated macrophage protein）基因的表达模式的影响，为防治宁夏枸杞黄叶病、培育高效Fe利用枸杞品种和低镉累积品种提供理论基础及技术支持。以宁夏枸杞1号组培幼苗为试验材料，利用转录组测序技术分析筛选宁夏枸杞中与Fe和Cd吸收转运相关的基因；利用实时荧光定量PCR技术验证目的基因在Fe、Cd胁迫下的表达变化情况；利用常规PCR方法克隆获得全长序列并进行生物信息学分析；构建酵母表达载体转化Fe/Cd敏感型酵母菌株ΔCCC1/YK44进行转基因酵母功能验证。结果显示，LbIRT2、LbFRO以及LbNRAMP3受Fe和Cd胁迫后显著上调表达；系统进化树分析表明宁夏枸杞Fe、Cd响应基因与水稻和拟南芥亲缘关系较近；转基因酵母功能进一步验证了LbFRO、LbIRT2以及LbNRAMP3能增强酵母对Fe和Cd的敏感性。LbFRO、LbIRT2以及LbNRAMP3可能参与了枸杞对Fe和Cd的吸收和转运。

关键词: 宁夏枸杞, 铁胁迫, 镉胁迫, 转基因酵母, 实时荧光定量PCR

Abstract:

In this paper, the effects of iron（Fe）and cadmium（Cd）stress on the expression patterns of genes of LbFRO（ferric reduction oxidase）, LbZIP（zinc-regulated transporters and iron-regulated transporter-like protein）and LbNRAMP（natural resistance-associated macrophage protein）in Lycium barbarum L. were investigated for providing theoretical basis and technical support in preventing and controlling yellow leaf disease of wolfberry in Ningxia, cultivation of high-efficiency Fe-utilizing wolfberry varieties and low-cadmium accumulation varieties. The seedlings of L. barbarum L. No. 1 were used as experimental materials, and the genes related to iron（Fe）and cadmium（Cd）absorption and transport were isolated by transcriptome sequencing technology; the expression patterns of the target genes under Fe and Cd stress were verified by real-time fluorescence quantitative PCR; the full-length sequences of the target genes were cloned by conventional PCR method; and the yeast expression vector of those genes were constructed and transformed into Fe/Cd sensitive yeast strain ΔCCC1/YK44 for functional verification. The results showed that LbFRO, LbIRT1, LbIRT2 and LbNRAMP3 were significantly up-regulated under Fe and Cd stress, phylogenetic tree analysis showed that those genes of L. barbarum L. were closely related to rice and Arabidopsis thaliana, the functions of transgenic yeast further verified that LbFRO, LbIRT2 and LbNRAMP3 genes enhanced the sensitivity of yeast to iron（Fe）and cadmium（Cd）, indicating that these genes may be involved in the absorption and transport of Fe and Cd in L. barbarum L.

Key words: Lycium barbarum L., iron stress, cadmium stress, transgenic yeast, real-time fluorescence quantitative PCR

余慧, 王静, 梁昕昕, 辛亚平, 周军, 赵会君. 宁夏枸杞铁镉响应基因的筛选及其功能验证[J]. 生物技术通报, 2023, 39(7): 195-205.

YU Hui, WANG Jing, LIANG Xin-xin, XIN Ya-ping, ZHOU Jun, ZHAO Hui-jun. Isolation and Functional Verification of Genes Responding to Iron and Cadmium Stresses in Lycium barbarum[J]. Biotechnology Bulletin, 2023, 39(7): 195-205.

图/表 9

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引物Primer	序列Sequence（5'-3'）	用途Purpose
LbIRT2	F: ATGGCTGCTCAAACCCAAGT R: TCACAACTCCATGCCAGTAGTAGA	基因克隆 Gene clone
LbFRO	F: ATGGGTGAACTCTCAGGGCAA R: CTAGAGGTCAAAATTGTGGCAGTTG	基因克隆 Gene clone
LbNRAMP3	F: ATGCCTCCACACGATGAAGAACA R: TCAACTCTCTATGCTCGTGATACTCTT	基因克隆 Gene clone
LbActin	F: TCTACGAGGGTTACGCTTTG R: TCCCGTTCAGCAGTGGTT	枸杞内参引物 Internal primer of Lycium barbarum L.
qRT-LbFRO	F: TCAAGCCACCCAGGGAACT R: CATGAAAATTGGTAAACTGAACATCA	荧光定量引物 Fluorescent quantitative PCR primer
qRT-LbNRAMP3	F: GCAGAGAGCGTTAATAACAAGAAGTTG R: TTAAGCCACTCGTTGAGAACATCT	荧光定量引物 Fluorescent quantitative PCR primer
qRT-LbIRT1	F: AGGAGCAATTCATCAAGATGCA R: TGCTACATGCTCCGGCTACTAG	荧光定量引物 Fluorescent quantitative PCR primer
qRT-LbIRT2	F: TCCAAATTACCATTCCTAAAGACCAT R: CGAACCCACTGTGATCTTTGAG	荧光定量引物 Fluorescent quantitative PCR primer
qRT-LbYSL	F: TGCAGTGTTCATGAGCGGTATAA R: TCACATCCTCCGAATTCTTCTTC	荧光定量引物 Fluorescent quantitative PCR primer

引物Primer	序列Sequence（5'-3'）	用途Purpose
LbIRT2	F: ATGGCTGCTCAAACCCAAGT R: TCACAACTCCATGCCAGTAGTAGA	基因克隆 Gene clone
LbFRO	F: ATGGGTGAACTCTCAGGGCAA R: CTAGAGGTCAAAATTGTGGCAGTTG	基因克隆 Gene clone
LbNRAMP3	F: ATGCCTCCACACGATGAAGAACA R: TCAACTCTCTATGCTCGTGATACTCTT	基因克隆 Gene clone
LbActin	F: TCTACGAGGGTTACGCTTTG R: TCCCGTTCAGCAGTGGTT	枸杞内参引物 Internal primer of Lycium barbarum L.
qRT-LbFRO	F: TCAAGCCACCCAGGGAACT R: CATGAAAATTGGTAAACTGAACATCA	荧光定量引物 Fluorescent quantitative PCR primer
qRT-LbNRAMP3	F: GCAGAGAGCGTTAATAACAAGAAGTTG R: TTAAGCCACTCGTTGAGAACATCT	荧光定量引物 Fluorescent quantitative PCR primer
qRT-LbIRT1	F: AGGAGCAATTCATCAAGATGCA R: TGCTACATGCTCCGGCTACTAG	荧光定量引物 Fluorescent quantitative PCR primer
qRT-LbIRT2	F: TCCAAATTACCATTCCTAAAGACCAT R: CGAACCCACTGTGATCTTTGAG	荧光定量引物 Fluorescent quantitative PCR primer
qRT-LbYSL	F: TGCAGTGTTCATGAGCGGTATAA R: TCACATCCTCCGAATTCTTCTTC	荧光定量引物 Fluorescent quantitative PCR primer

基因ID Gene ID	基因名称 Gene name	log₂ （T12hR/CKR）	log₂ （T48hR/CKR）	长度 Length/bp	相似度 Similarity/%	筛选部位 Isolation location
CL7788.Contig3	离子转运蛋白基因 Ion transport protein	6.67	6.82	1 948	89.94	根系Root
Unigene39380	OPT寡肽转运蛋白基因 OPT oligopeptide transporter	8.372 403	10.0 693	1 739	99.47	根系Root
Unigene61286	锌转运体5 Zinc transporter 5-like	4.88	5.86	1 431	82.17	根系Root
CL11868.Contig2	ZIP家族锌转运蛋白基因 ZIP Zinc transporter	7.28	7.23	1 167	98.77	根系Root
CL4044	Nramp3金属离子转运蛋白基因 Metal transporter Nramp3-like	5.3	4.23 493	3 830	92.62	根系Root
CL1976.Contig4_	机械敏感通道蛋白基因8 Mechanosensitive ion channel protein 8-like	7.852 761	7.92	3 088	72.81	根系Root
Unigene60599	黄色条纹蛋白基因YSL2 Metal-nicotianamine transporter YSL2-like	2.178 219	2.525 766	2 161	86.78	根系Root
CL1155.Contig	黄色条纹蛋白基因YSL3 Metal-nicotianamine transporter YSL3-like	9.070 304	6.348 448	3 045	91.95	根系Root
CL3488.Contig3	铁氧还原酶基因6 Ferric reduction oxidase 6-like	1.875 425	2.782 025	3 193	89.01	叶片Leaf
Unigene44038	铁氧还原酶基因8 Ferric reduction oxidase 8	2.515 402	2.019 701	1 935	89.08	叶片Leaf
CL9188.Contig6	液泡膜铁离子转运蛋白基因4 Vacuolar iron transporter homolog 4-like	3.63 367	2.881 373	588	84.66	叶片Leaf
CL12735.Contig1	铁氧还原酶基因2 Ferric reduction oxidase 2-like	3.527 493	4.06 572	2 158	91.53	叶片Leaf
Unigene21912	寡肽转运蛋白基因4 Oligopeptide transporter 4	2.264 557	0.763 512	1 713	98.03	叶片Leaf
CL1352.Contig23	多向耐药性PDR跨膜转运蛋白基因1 Pleiotropic drug resistance protein 1-like	9.955 166	10.28 298	5 054	90.33	叶片Leaf
CL17567.Contig5	Nramp6金属离子转运蛋白基因 Metal transporter Nramp6	6.758 793	6.640 834	1 159	95.06	叶片Leaf
Unigene36942	Nramp5金属离子转运蛋白基因 Metal transporter Nramp5	8.152 608	7.285 907	415	95.48	叶片Leaf

基因ID Gene ID	基因名称 Gene name	log₂ （T12hR/CKR）	log₂ （T48hR/CKR）	长度 Length/bp	相似度 Similarity/%	筛选部位 Isolation location
CL7788.Contig3	离子转运蛋白基因 Ion transport protein	6.67	6.82	1 948	89.94	根系Root
Unigene39380	OPT寡肽转运蛋白基因 OPT oligopeptide transporter	8.372 403	10.0 693	1 739	99.47	根系Root
Unigene61286	锌转运体5 Zinc transporter 5-like	4.88	5.86	1 431	82.17	根系Root
CL11868.Contig2	ZIP家族锌转运蛋白基因 ZIP Zinc transporter	7.28	7.23	1 167	98.77	根系Root
CL4044	Nramp3金属离子转运蛋白基因 Metal transporter Nramp3-like	5.3	4.23 493	3 830	92.62	根系Root
CL1976.Contig4_	机械敏感通道蛋白基因8 Mechanosensitive ion channel protein 8-like	7.852 761	7.92	3 088	72.81	根系Root
Unigene60599	黄色条纹蛋白基因YSL2 Metal-nicotianamine transporter YSL2-like	2.178 219	2.525 766	2 161	86.78	根系Root
CL1155.Contig	黄色条纹蛋白基因YSL3 Metal-nicotianamine transporter YSL3-like	9.070 304	6.348 448	3 045	91.95	根系Root
CL3488.Contig3	铁氧还原酶基因6 Ferric reduction oxidase 6-like	1.875 425	2.782 025	3 193	89.01	叶片Leaf
Unigene44038	铁氧还原酶基因8 Ferric reduction oxidase 8	2.515 402	2.019 701	1 935	89.08	叶片Leaf
CL9188.Contig6	液泡膜铁离子转运蛋白基因4 Vacuolar iron transporter homolog 4-like	3.63 367	2.881 373	588	84.66	叶片Leaf
CL12735.Contig1	铁氧还原酶基因2 Ferric reduction oxidase 2-like	3.527 493	4.06 572	2 158	91.53	叶片Leaf
Unigene21912	寡肽转运蛋白基因4 Oligopeptide transporter 4	2.264 557	0.763 512	1 713	98.03	叶片Leaf
CL1352.Contig23	多向耐药性PDR跨膜转运蛋白基因1 Pleiotropic drug resistance protein 1-like	9.955 166	10.28 298	5 054	90.33	叶片Leaf
CL17567.Contig5	Nramp6金属离子转运蛋白基因 Metal transporter Nramp6	6.758 793	6.640 834	1 159	95.06	叶片Leaf
Unigene36942	Nramp5金属离子转运蛋白基因 Metal transporter Nramp5	8.152 608	7.285 907	415	95.48	叶片Leaf

基因名称 Gene name	氨基酸长度 Amino acid length/aa	分子量 Molecular weight/kD	等电点 pI	脂肪指数 Aliphatic index	不稳定指数 Instability index	亲疏水性 GRAVY
LbIRT2	215	22 607.41	8.81	97.26	40.50	0.340
LbFRO	740	83 469.16	8.48	112.19	36.60	0.371
LbNRAMP3	511	56 347.21	5.83	119.10	37.17	0.556