马铃薯‘新疆2号’遗传转化体系的建立

doi:10.13560/j.cnki.biotech.bull.1985.2025-1016

摘要/Abstract

摘要：

目的构建马铃薯‘新疆2号’高效再生及遗传转化体系，为抗病毒育种提供技术支撑。方法以‘新疆2号’马铃薯为试验材料，用农杆菌介导法将含有植物RNA干扰载体质粒pCAMBIA2300-CP-RNAi转化马铃薯茎段外植体，探究预培养时间、植物激素组合及抗生素的种类及浓度对愈伤组织诱导及芽分化的影响；通过RT-qPCR技术，以Actin基因作为内参，检测CP基因mRNA表达水平，运用酶联免疫吸附（ELISA）技术定量检测植株中PVY病毒的实际含量，验证RNAi沉默效果及病毒抑制作用。结果当预培养、共培养时间均为2 d时，愈伤组织诱导的最适激素和抗生素组合为2.0 mg/L 6-BA + 0.8 mg/L 2,4-D + 50 mg/L Kan + 200 mg/L TMT，诱导率为85%，愈伤组织形态紧密，为鲜绿色，茎段两端膨大呈哑铃状；茎段分化的最适激素、抗生素配比为2 mg/L 6-BA + 2 mg/L ZT + 0.5 mg/L GA₃ + 50 mg/L Kan + 200 mg/L TMT时，出苗率及转化频率均达到最高，幼苗长势良好。转化苗在1/2 MS+0.6 mg/L IAA + 75 mg/L Kan + 200 mg/L TMT培养基中可形成再生植株，且根系发达。与未转化的对照植株相比，转基因植株中CP基因的相对表达量显著下调，PVY病毒的含量也明显降低，说明所构建的RNA干扰载体成功激发了特异性基因沉默效应，有效抑制了病毒在植株体内的积累。结论成功建立了稳定、高效的‘新疆2号’马铃薯遗传转化体系。构建的RNAi载体有效抑制了Y病毒在植株体内的积累。

关键词: 马铃薯, 植物激素配比, 遗传转化体系, 转基因植株

Abstract:

Objective To establish an efficient regeneration and genetic transformation system for potatoes (Solanum tuberosum L.) variety ‘Xinjiang No. 2’, and to provide technical support for virus-resistant breeding. Method Using ‘Xinjiang No. 2’ potato as the experimental material, Agrobacterium-mediated transformation was employed to introduce the plant interference vector plasmid pCAMBIA2300-CP-RNAi into potato stem explants. The effects of pre-culture duration, plant hormone combinations, and types and concentrations of antibiotics on callus induction and shoot differentiation were investigated. By real-time quantitative PCR (RT-qPCR) technology, with the Actin gene as an internal reference, the mRNA expression of the CP gene was detected. The actual content of PVY virus in plants was quantitatively measured using enzyme-linked immunosorbent assay (ELISA) to validate the RNAi silencing effect and virus suppression. Result The optimal hormone and antibiotic combination for callus induction was 2.0 mg/L 6-BA + 0.8 mg/L 2,4-D + 50 mg/L Kan + 200 mg/L TMT when pre-culture and co-culture durations were both 2 days, achieving an induction rate of 85%. The callus showed a compact morphology, fresh green color, and a dumbbell-shaped swelling at both ends of the stem segments. For shoot differentiation, the optimal hormone and antibiotic ratio was 2 mg/L 6-BA + 2 mg/L ZT + 0.5 mg/L GA₃ + 50 mg/L Kan + 200 mg/L TMT, resulting in the highest germination and transformation rates, with well-developed seedlings. Transgenic seedlings regenerated into plants on 1/2 MS medium supplemented with 0.6 mg/L IAA + 75 mg/L Kan + 200 mg/L TMT, demonstrating robust root systems. Compared to non-transformed control plants, the relative expression of the CP gene in transgenic plants was significantly downregulated, and PVY virus content was markedly reduced, confirming that the constructed RNAi vector successfully triggered specific gene silencing and effectively inhibited virus accumulation in plants. Conclusion This study successfully establishes a stable and efficient genetic transformation system for the potato variety ‘Xinjiang No. 2’. The constructed RNAi interference vector effectively suppresses Y virus accumulation in plants.

Key words: Solanum tuberosum L., plant hormone ratio, genetic transformation system, transgenic plants

赵文娟, 李辉, 杨雪莹, 王朝露, 祝建波. 马铃薯‘新疆2号’遗传转化体系的建立[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1016.

ZHAO Wen-juan, LI Hui, YANG Xue-ying, WANG Zhao-lu, ZHU Jian-bo. Establishment of a Genetic Transformation System for Potato ‘Xinjiang No. 2’[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1016.

图/表 17

表1 不同激素组合对愈伤组织诱导的影响

Table 1 Effects of different hormone combinations on callus induction (mg/L)

试验编号 Test number	6-苄氨基嘌呤 6-Benzylaminopurine（6-BA）	2，4-二氯苯氧乙酸 2，4-Dichlorophenoxyacetic acid （2，4-D）	萘乙酸 1-Naphthaleneacetic acid （NAA）	3-吲哚乙酸 Indole-3-acetic acid （IAA）	6-糠氨基嘌呤 Kinetin （KT）	玉米素核苷 Zeatin Riboside （ZR）
C1	2.0	0	0.6	0	0	0
C2	2.0	0.8	0	0	0	0
C3	0	0	0	0.2	0	2.0
C4	2.0	0.6	0	0	0.4	0

表2 不同激素组合对愈伤组织分化的影响

Table 2 Effects of different hormone combinations on callus differentiation (mg/L)

试验编号

Test number

6-苄氨基嘌呤

6-Benzylaminopurine （6-BA）

6-糠氨基嘌呤

Kinetin （KT）

玉米素

Zeatin （ZT）

3-吲哚乙酸

Indole-3-acetic acid （IAA）

赤霉素

Gibberellin A₃ （GA₃）

2，4-二氯苯氧乙酸

2，4-Dichlorophenoxyacetic acid （2，4-D）

表3 预培养时长对外植体愈伤组织诱导与分化的影响

Table 3 Effects of pre culture duration on the induction and differentiation of callus tissue in explants

预培养时间 Pre cultivation time （d）	外植体数 Number of explants	愈伤数 Number of callus	愈伤形成率 Callus formation rate （%）	再生芽数 Number of regenerated buds	出苗率 Seedling emergence rate （%）	转化频率 Conversion frequency （%）
0	60	2	3.3	0	0	0
1	60	21	35	4	19.0	0
2	60	49	81.7	23	46.9	21.73
3	60	39	65	11	28.2	9.1
4	60	11	18.3	1	9.1	0

图1 pCAMBIA2300-CP-RNAi载体

Fig. 1 pCAMBIA2300-CP-RNAi vector

表4 外植体茎段愈伤组织诱导培养基的筛选

Table 4 Selection of induction medium for stem segment callus of explants

处理 Treatment	外植体数 Number of explants	愈伤数 Number of callus	愈伤形成率 Callus formation rate （%）	愈伤组织形态 Morphology of callus tissue
C1	60	27	45	白色须状物
C2	60	51	85	鲜绿色、结构紧密
C3	60	40	66.7	黄绿色，结构松散
C4	60	30	50	白色须状物、结构松散

图2 外植体茎段愈伤组织诱导培养基的筛选

Fig. 2 Selection of induction medium for stem segment callus tissue from explants

图3 茎段外植体愈伤组织分化培养基的影响

Fig. 3 Selection of differentiation medium for stem segment explant callus tissue

表5 不同卡那霉素浓度对植株再生的影响

Table 5 Effects of different kanamycin concentrations on plant regeneration

卡那霉素浓度 Kanamycin concentration （mg/L）	外植体数 Number of explants	再生芽数 Number of regenerated buds	出苗率 Seedling emergence rate （%）	转化频率 Conversion frequency（%）
0	60	54	90	3.7
25	60	48	80	6.3
50	60	31	51.7	23.53
75	60	17	28.3	17.7
100	60	0	0	0

图4 不同卡那霉素浓度对再生植株的影响A-E表示卡那霉素浓度为0、25、50、75、100 mg/L

Fig. 4 Effects of different concentrations of kanamycin on regenerated plantsA-E refers to kanamycin concentrations of 0, 25, 50, 75, 100 mg/L, respectively

表6 不同卡那霉素浓度对植株生根的影响

Table 6 Effects of different kanamycin concentrations on plant rooting

卡那霉素浓度 Kanamycin concentration （mg/L）	外植体数 Number of explants	生根外植体数 Number of rooting explants	生根率 Rooting rate （%）
0	40	40	100
50	40	40	100
75	40	0	0
100	40	0	0

图5 不同卡那霉素浓度对植株生根的影响A-D表示卡那霉素浓度为0、50、75、100 mg/L，E表示转基因植株在75 mg/L卡那霉素浓度下的生根状态

Fig. 5 Effects of different concentrations of kanamycin on plant rootingA-D indicate kanamycin concentrations of 0, 50, 75, and 100 mg/L, respectively, while E refers to the rooting status of transgenic plants under a 75 mg/L kanamycin concentration

表7 不同抑菌剂的种类及浓度对转化的影响

Table 7 Effects of different types and concentrations of antibacterial agents on conversion

处理 Treatment	羧苄青霉素浓度 Carbenicillin concentration （mg/L）	特美汀浓度 Timentin concentration （mg/L）	诱导愈伤培养 Induced callus culture	诱导出芽培养 Induced budding culture	生根培养 Rooting culture
1	100	0	染菌	染菌	染菌
2	200	0	染菌	染菌	染菌
3	300	0	染菌	染菌	染菌
4	0	100	染菌	染菌	染菌
5	0	200	未染菌	未染菌	未染菌
6	0	300	未染菌	未染菌	未染菌

图6 不同抑菌剂的种类及浓度对转化的影响A-C表示羧苄青霉素浓度为100、200、300 mg/L，D-F表示特美汀浓度为100、200、300 mg/L

Fig. 6 Effects of different types and concentrations of antibacterial agents on conversionA-C refer to concentrations of carbenicillin at 100, 200, and 300 mg/L, while D-F refer to concentrations of timentin at 100, 200, and 300 mg/L

图7 不同浓度 IAA对转化植株根诱导及再生的影响A-C表示IAA浓度为0.2、0.6、1.2 mg/L

Fig. 7 Effects of different concentrations of IAA on the root induction and regeneration of transformed plantsA-C indicate IAA concentrations of 0.2, 0.6, and 1.2 mg/L

图8 再生植株的PCR阳性检测A：用引物CP-F，CP-R扩增的PCR产物，B：用引物CP-f，CP-r扩增的PCR产物。M：Marker；1-21：转化植株；22：阴性对照；23：空白对照；24：阳性对照

Fig. 8 PCR positive detection of regenerated plantsA: PCR products corresponding to primers CP-F and CP-R. B: PCR products corresponding to primers CP-f and CP-R. M: Marker; 1-21: transforming plants; 22: negative control; 23: blank control; 24: positive control

图9 野生型和转基因植株中CP基因相对表达量***P<0.001， ****P<0.000 1， the same below

Fig. 9 CP gene relative expression in wild-type and transgenic plants

图10 野生型和转基因植株中马铃薯PVY病毒含量测定

Fig. 10 Determination of potato PVY virus content in wild-type and transgenic plants

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