一种高效花生遗传转化体系的建立与优化

doi:10.11983/CBB25010

摘要/Abstract

摘要： 为建立高效的农杆菌介导的花生(Arachis hypogaea)遗传转化体系, 选取了11个花生品种, 从中筛选出胚小叶芽丛诱导率最高的品种胚小叶为试验材料, 通过对农杆菌菌株、菌液OD值、AS (乙酰丁香酮)浓度、表面活性剂浓度、侵染方式及时间、共培养时间等影响因素进行筛选并优化, 获得花生胚小叶转基因植株。结果显示: 以花育9133胚小叶为受体, 利用含有eGFP (绿色荧光蛋白)和含GUS (β-葡萄糖苷酸酶)蛋白的重组农杆菌侵染转化, 发现侵染液为MS液体+LBA4404菌株+100 μmol∙L^-1 AS+150 mg∙L^-1表面活性剂Silwet-77+菌液OD600为0.7时, 侵染方式为抽真空15 min+浸泡20 min+共培养4 d时花生转化率最高, CaMV 35S:eGFP、AhUBQ4:GUS阳性率分别为52.67%、57.67%。经组织培养法诱导的转化再生植株, 其中含eGFP蛋白的经eGFP绿色荧光和PCR检测鉴定为转基因阳性植株, 含GUS蛋白的经GUS染色和PCR检测鉴定为转基因阳性植株。该研究成功建立并优化了花生遗传转化体系, 为花生基因功能研究、抗性品种的培育、品质改良、生物技术研究等提供参考。

关键词: 胚小叶, 遗传转化体系, eGFP, GUS

Abstract: INTRODUCTION: To establish an efficient Agrobacterium-mediated genetic transformation system for peanuts and lay a foundation for the study of peanut gene functions and variety breeding.
RATIONALE: In this study, 11 peanut varieties were selected, and the cotyledonary leaflets from the variety with the highest bud cluster induction rate were screened out as experimental materials. By screening and optimizing influencing factors such as Agrobacterium strains, the optical density (OD) value of the bacterial suspension, the concentration of acetosyringone (AS), the concentration of surfactants, the infection method and duration, and the co-culture time, transgenic plants of peanut cotyledonary leaflets were obtained.
RESULTS: The results showed that using the embryo leaflet of Huayu 9133 as the receptor, the recombinant Agrobacterium containing eGFP (green fluorescent protein) and GUS (β-glucosidase) protein was used to infect and transform. It was found that when the infection solution was MS liquid+LBA4404 strain+100 μmol∙L^-1 AS+150 mg∙L^-1surfactant Silwet-77 + bacterial solution OD600 was 0.7, the infection method was vacuuming for 15 min + soaking for 20 min + co-culture for 4 d, the peanut conversion rate was the highest. The positive rates of CaMV 35S:eGFP and AhUBQ4:GUS were 52.67% and 57.67%, respectively.The transgenic plants were induced by tissue culture method. The transgenic plants containing eGFP protein were identified as transgenic positive plants by eGFP green fluorescence and PCR detection, and the transgenic plants containing GUS protein were identified as transgenic positive plants by GUS staining and PCR detection.
CONCLUSION: This experiment successfully established and optimized the peanut genetic transformation system, which provided a reference for the study of peanut gene function, the cultivation of resistant varieties, quality improvement and biotechnology research.

CaMV 35S:eGFP侵染材料荧光示意图(a: 再生芽丛明场, b: 再生芽丛荧光, c: 再生苗明场, d: 再生苗荧光)

Key words: Embryo lobule leaflet, Genetic transformation system, eGFP, GUS

李婷婷, 闫志文, 崔媛媛, 郭昊松, 陈方军, 张倩倩, 刘晓芹. 一种高效花生遗传转化体系的建立与优化. 植物学报, DOI: 10.11983/CBB25010.

Tingting Li, Zhiwen Yan, Yuanyuan Cui, Haosong Guo, Fangjun Chen, Qianqian Zhang, Xiaoqin Liu. Establishment and Optimization of an Efficient Peanut Genetic Transformation System. Chinese Bulletin of Botany, DOI: 10.11983/CBB25010.

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