马铃薯GARP转录因子家族分析及StARR14-X1基因功能验证

doi:10.13560/j.cnki.biotech.bull.1985.2025-0701

摘要/Abstract

摘要：

目的挖掘彩色马铃薯块茎中花青素积累调控的关键基因，为分子育种技术提供可行的基因资源和技术参考。方法基于紫色马铃薯不同发育时期块茎转录组测序数据，筛选GARP转录因子，对其进行生物信息学分析，并对花青素调控相关的StARR14-X1基因进行功能验证。结果通过转录组数据共筛选得到12个GARP转录因子，这些蛋白质的氨基酸序列长度集中在148-686 aa之间，分子量在16.66-75.44 kD之间，pI值在5.13-8.05之间，脂肪族指数在71.36-97.36之间；不稳定系数在35.59-75.94之间，均属于亲水性蛋白，亚细胞定位预测其在细胞核、细胞质、叶绿体中均有分布。利用农杆菌介导法的叶盘法成功将StARR14-X1基因转化到四倍体烟草中，获得了StARR14-X1过表达转基因烟草植株。StARR14-X1过表达转基因株系茎和花瓣的花青素含量显著升高。结论 StARR14-X1基因在花青素积累中具有正调控功能，促进花青素的积累。

关键词: 马铃薯, 花青素, GARP转录因子, 正调控

Abstract:

Objective Identifying key genes regulating anthocyanin accumulation in the tubers of colored potatoes (Solanum tuberosum L.) provides feasible genetic resources and technical references for molecular breeding technology. Method GARP transcription factors were screened based on transcriptome sequencing data of purple potato tubers at different developmental stages. Bioinformatics analysis was conducted, and functional verification of StARR14-X1 gene related to anthocyanin regulation was performed. Result The 12 GARP transcription factors were screened through on transcriptome data. The amino acid sequence lengths of 12 GARP transcription factors were concentrated between 146-686 aa, the molecular weight ranged from 16.66 to 75.44 KD, the pI value ranged from 5.13 to 8.05, the fatty index ranged from 71.36 to 97.36, and the instability coefficient ranged from 35.59 to 75.94, all of which belonged to hydrophilic proteins. Subcellular localization prediction showed that 12 GARP transcription factors were distributed in the nucleus, cytoplasm and chloroplasts. The StARR14-X1 gene was successfully transformed into tetraploid tobacco using the Agrobacterium-mediated leaf disc method, and transgenic tobacco plants overexpressing StARR14-X1 were obtained. The anthocyanin contents in the stems and petals of StARR14-X1-overexpressing transgenic lines increased significantly. Conclusion This study clarifies that the StARR14-X1 gene has a positive regulatory function in anthocyanin accumulation and promotes anthocyanin accumulation.

Key words: potato, anthocyanin, GARP transcription factor, positive regulation

武小娟, 赵雪雯, 王沛捷, 聂虎帅, 李楠, 马宇, 杨恩泽, 马杰茹, 马艳红. 马铃薯GARP转录因子家族分析及StARR14-X1基因功能验证[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0701.

WU Xiao-juan, ZHAO Xue-wen, WANG Pei-jie, NIE Hu-shuai, LI Nan, MA Yu, YANG En-ze, MA Jie-ru, MA Yan-hong. Analysis of the PotatoGARPTranscription Factor Family and Functional Validation of StARR14-X1[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0701.

图/表 12

图1 StARR14-X1转基因烟草遗传转化流程图A：侵染叶片；B：愈伤培养；C：诱导芽分化；D：生根培养

Fig. 1 Genetic transformation process diagram of StARR14-X1 transgenic tobaccoA: Infected leaves. B: Callus culture. C: Induction of shoot differentiation. D: Rooting culture

表1 马铃薯GARP转录因子理化性质及亚细胞定位

Table 1 Physicochemical properties and subcellular localization of potato GARP transcription factors

基因 Gene name	氨基酸数Number of amino acids （aa）	分子量 Molecular weight （Da）	理论等电点 PI	不稳定系数 Instability index	总平均亲水性 Gravy	脂肪族指数 Aliphat ic index	亚细胞定位 Subcellular localization
StORR24-like-1	686	75 439.3	6.36	43.01	-0.627	71.36	细胞核
StORR9	226	25 635.06	5.38	63.55	-0.611	85.35	细胞核
StORR9-like	226	25 628.02	5.13	67.28	-0.62	84.91	细胞核
StARR5-like	248	27 532.26	5.29	75.94	-0.458	80.52	细胞核
StARR1-like	424	46 469.1	5.92	54.21	-0.501	74.29	细胞核
StORR10-like	163	18 701.66	6.74	40.77	-0.29	97.36	细胞核
StARR2-like	675	74 652.52	5.93	45.56	-0.564	74.64	细胞核
StARR5	202	22 405.65	5.38	58.08	-0.373	93.07	细胞核
StARR17-like	148	16 656.27	6.08	35.59	-0.345	86.89	细胞质
StORR24-like-2	669	74 026.52	6.78	48.79	-0.693	71.69	细胞核
StARR14-X1	394	42 295.94	8.05	62.75	-0.298	83.15	叶绿体、细胞核
StARR11	481	54 100.85	6.39	44.27	-0.592	72.43	细胞核

图2 马铃薯GARP转录因子的基序相对位置

Fig. 2 Relative positions of motifs in potato (Nicotiana tabacum) GARP transcription factors

图3 马铃薯GARP转录因子的保守基序

Fig. 3 Conserved motifs in potato GARP transcription factors

表2 12个GARP转录因子的功能预测

Table 2 Prediction of the functions of 12 GARP transcription factors

基因名称 Gene name	GO 注释的基因名称 Gene name of GO annotation	GO 编号 GO ID	GO 信息 GO term
StORR24-like-1	PGSC0003DMG400007823	GO：0031537	花青素代谢过程的调节
StORR9	PGSC0003DMG400029839	GO：0000156	磷酸响应调节因子活性
StORR9-like	PGSC0003DMG400029852	GO：0000156	磷酸响应调节因子活性
StARR5-like	PGSC0003DMG402022640	GO：0006351	DNA模板的转录
StARR1-like	PGSC0003DMG400023534	GO：0031537	花青素代谢过程的调节
StORR10-like	PGSC0003DMG400003084	GO：0005634	细胞核
StARR2-like	PGSC0003DMG401025624	GO：0016049	细胞生长
StARR5	PGSC0003DMG400024578	GO：0005515	蛋白质结合
StARR17-like	PGSC0003DMG400016643	GO：0006351	DNA模板的转录
StORR24-like-2	PGSC0003DMG400003196	GO：0005634	细胞核
StARR14-X1	PGSC0003DMG400029514	GO：0031537	花青素代谢过程的调节
		GO：0048364	根的发育
StARR11	PGSC0003DMG400011980	GO：0003677	DNA结合

图4 转录组样品相关性系数图相关系数r在±1到±0.8之间为高度正负相关；颜色越红相关性越强，颜色越白，相关性越弱

Fig. 4 Correlation coefficient graph of transcriptome samplesThe correlation coefficient r is highly positive and negative between ± 1 and ± 0.8. The redder the color, the stronger the correlation; the whiter the color, the weaker the correlation

图5 12个GARP基因在马铃薯华颂66号三个时期中的表达模式块茎形成期为S11、S12、S13三次重复均值，块茎膨大期为S21、S22、S23三次重复均值，块茎成熟期为S31、S32、S33三次重复均值。使用迈维云平台对基因在样本水平进行归一化处理［13］，归一化后数值在-2-2

Fig. 5 Expression patterns of 12 GARP genes in three development stages of potato Huasong 66The tuber formation stage is the mean value of three replicates (S11, S12, S13); the tuber expansion stage is the mean value of three replicates (S21, S22, S23); and the tuber maturation stage is the mean value of three replicates (S31, S32, S33). The Maiwei Cloud Platform was used for normalization of genes at the sample level, and the normalized values range from -2 to 2

图6 8个GARP转录因子在不同品种马铃薯的3个时期的表达分析S1：块茎形成期；S2：块茎膨大期；S3：块茎成熟期。不同小写字母表示在0.05水平差异显著（邓肯测验），下同

Fig. 6 Analysis of the expressions of 8 GARP transcription factors in three periods of different potato varietiesS1: Tuber formation stage. S2: Tuber expansion stage. S3: Tuber maturity stage. Different lowercase letters indicate significant differences at 0.05 level (Duncan test). The same below

图7 StARR14-X1转基因烟草阳性植株PCR检测电泳图M：D2 000 DNA Maker；WT为阴性对照；CK：空白对照（ddH2O）；P：含有pCAMBIA1302-StARR14-X1质粒的菌液；1-8：转基因烟草株系

Fig. 7 PCR electropherogram of positive plants of StARR14-X1 transgenic tobaccoM: D2 000 DNA Marker; WT: the negative control; CK: the blank control (ddH2O); P: the bacterial solution containing the pCAMBIA1302-StARR14-X1 plasmid; 1-8: the transgenic tobacco lines

图8 野生型植株与转基因植株根、茎、叶、花的相对表达水平A、B、C、D依次为根、茎、叶、花的相对表达水平；WT为野生型植株；OE1、OE4、OE5、OE8为转StARR14-X1基因烟草的4个不同株系，下同

Fig. 8 Relative expressions of the roots, stems, leaves and flowers in wild-type and transgenic plantsA, B, C, and D are the relative expressions in the roots, stems, leaves, and flowers, respectively. WT refers to the wild-type plant; OE1, OE4, OE5, OE8 are four different lines of transgenic tobacco with StARR14-X1 gene, the same below

图9 野生型烟草及转StARR14-X1基因烟草的植株形态

Fig. 9 Plant morphology of wild-type tobacco and transgenic tobacco overexpressing StARR14-X1

图10 野生型植株与转基因植株花青素含量

Fig. 10 Anthocyanin contents in wild-type versus transgenic plants

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