摘要
【目的】探索适合籼稻明恢86多基因遗传转化的条件,为创制含高产、抗逆、抗虫、抗病等基因的水稻新材料奠定基础。【方法】以籼稻明恢86为受体材料,将构建好的含作物高产基因RRM2、耐旱基因HS1、抗除草剂基因EPSPS、抗虫基因Bt、细胞凋亡抑制基因iap和促细胞再生基因p35等多基因载体(载体分别命名为P5和P8)进行遗传转化。在此基础上,分别对多基因遗传转化体系的受体材料、农杆菌浓度、侵染时间、共培养方式、G418筛选浓度和草甘膦筛选浓度等主要影响因素进行试验,探讨其适宜的转化条件。【结果】受体材料的筛选结果表明,幼胚的出愈率显著高于成熟胚,且其愈伤组织状态相对较好;各转化条件的筛选结果,农杆菌侵染浓度OD600为0.4~0.6、侵染时间15~20min、共培养2~3d、培养基上添加无菌滤纸、G418筛选浓度150mg·L^-1和草甘膦筛选浓度800mg·L^-1是提高转化效率的优化条件;PCR分析结果,多基因载体P5中的GUS基因成功转入籼稻明恢86。【结论】通过对培养条件的优化,可使籼稻明恢86愈伤组织的诱导愈伤率和抗性愈伤率得到显著提高。
【Objective】Conditions for simultaneous transformation of multiple genes in Indica rice,Minghui 86,were studied to facilitate the development of high yield and resistant breeds.【Method】Two multi-gene vectors designated as P5 and P8 that harbored the high-yield RRM2,drought-tolerant HS1,herbicide-resistant EPSPS,and insect-resistant Bt as well as the apoptosis-inhibiting iap and gene that promotes cell regeneration p35 were transformed to the receptor Minghui 86 using agrobacterium-based transformation methodology.The receptor,callus culture,agrobacterium concentration,infection time,co-culture,and screening concentrations of G418 and glyphosate were evaluated for the selection.【Result】 Under same culture conditions,the recovery rate of Minghui 86 immature embryos was significantly higher with a better callus quality than that of the mature embryos.The optimal transformation conditions were determinated to be a bacterial concentration of OD 600 =0.4-0.6,an infection time of 15-20 m,co-culture for 2-3 d with the addition of sterile filter paper in medium,and the screening concentration of G418 at 150 mg·L^-1 and of glyphosate at 800 mg·L^-1.The PCR detection confirmed that GUS in the polygenic vector P5 was successfully transferred into Indica cv.Minghui 86.【Conclusion】 The optimized culture conditions afforded significantly improved callus and resistance induction rates in Minghui 86.
作者
魏林艳
连玲
魏毅东
罗曦
何炜
谢鸿光
谢华安
张建福
WEI Lin-yan;LIAN-Ling;WEI Yi-dong;LUO Xi;HE Wei;XIE Hong-guang;XIE Hua-an;ZHANG Jian-fu(Rice Research Institute,Fujian Academy of Agricultural Sciences,Fuzhou,Fujian 350019,China;Key Laboratory of Germplasm Innovation and Molecular Breeding of Hybrid Rice for South China,Ministry of Agriculture and Rual/Fuzhou Branch,National Rice Improvement Center of China/Fujian Engineering Laboratory of Crop Molecular Breeding/Incubator of National Key Laboratory of Crops Germplasm Innovation and Molecular Breeding Between Fujian and Ministry of Science and Technology/Base for South-China,National KeyLaboratory of Hybrid Rice for China,Fuzhou,Fujian 350003,China;National Engineering Laboratory of Rice of China,Fuzhou,Fujian 350003,China)
出处
《福建农业学报》
CAS
CSCD
北大核心
2019年第6期621-629,共9页
Fujian Journal of Agricultural Sciences
基金
福建省科技计划项目——省属公益类科研院所基本科研专项(2016R1020-12)
国家重点研发计划项目(2017YFD0100100)
关键词
基因
遗传转化
籼稻
明恢86
gene
genetic transformation
indica rice
Minghui 86
