番木瓜胚性细胞悬浮系高效遗传转化体系的建立  

Establishment of a high-efficiency genetic transformation system for papaya using embryogenic cell suspensions as genetic transformation receptors

作　　者：杨敏 周陈平 李庆萌 邝瑞彬[1] 吴夏明 魏岳荣[1] YANG Min;ZHOU Chenping;LI Qingmeng;KUANG Ruibin;WU Xiaming;WEI Yuerong(Institute of Fruit Tree Research,Guangdong Academy of Agricultural Sciences/Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization,Ministry of Agriculture and Rural Affairs/Guangdong Province Key Laboratory of Tropical and Subtropical Fruit Tree Research,Guangzhou 510640,Guangdong,China)

机构地区：[1]广东省农业科学院果树研究所·农业农村部南亚热带果树生物学与遗传资源利用重点实验室·广东省热带亚热带果树研究重点实验室,广州510640

出　　处：《果树学报》2023年第10期2089-2097,共9页Journal of Fruit Science

基　　金：广州市重点研发计划项目(2023B03J1369);广东省基础与应用基础研究基金项目(2022A1515010697,2021A1515010739);广东省农业科学院果树研究所培育项目(22100);广东省省级乡村振兴战略专项资金种业振兴项目(2022-NPY-00-032);国家高端外国专家引进计划项目(G2023030034L);国家自然科学基金项目(32102355)。

摘　　要：【目的】建立番木瓜高效遗传转化体系,为番木瓜基因功能研究和重要农艺性状改良提供新的技术支撑。【方法】以紫晖番木瓜胚性细胞悬浮系(embryogenic cell suspensions,ECS)为遗传转化受体,利用植物表达载体pCAMBIA1301和农杆菌介导法进行遗传转化,对抗生素浓度筛选、侵染时间、继代培养、抗性胚的诱导与萌发以及植株再生整个过程进行探索,最后获得抗性再生植株。【结果】通过设置不同浓度的头孢霉素和潮霉素处理,观察ECS细胞状态,筛选、确定头孢霉素和潮霉素最适处理质量浓度分别为200 mg·L^(-1)和5 mg·L^(-1)。工程菌和ECS共培养侵染2 d后转到含有头孢霉素和潮霉素的液体筛选培养基上进行继代培养,继代周期为14 d。经GUS染色验证,表明继代3次后的ECS几乎全部为转化细胞。将以上ECS转移到液体胚诱导培养基中进行培养,2个月后可获得大量球形体细胞胚,且GUS组织染色为蓝色。将球形体细胞胚转到半固体成熟培养基上培养,2个月后可获得成熟子叶期体细胞胚。子叶期体细胞胚在萌发培养基上光培养30 d后,可获得再生芽。任意选取再生芽进行GUS染色,均可染成蓝色。抗性再生芽经促根培养可成功获得再生植株。利用PCR检测抗性再生植株,可以确定GUS基因已经整合到番木瓜基因组中。【结论】成功建立了一种以番木瓜ECS为转化受体的农杆菌介导的高效遗传转化体系。在该技术体系中,经农杆菌侵染后的ECS继代筛选3次后,几乎全部为转化细胞,这些转化细胞经体胚诱导、成熟、萌发和生根过程可成功获得再生植株,抗性体胚得胚率为43.65%,抗性体胚萌发率为73.26%,植株再生率为80.55%,大大提高了番木瓜遗传转化效率。该体系为番木瓜基因功能研究和分子育种提供了新的途径。【Objective】Papaya(Carica papaya L.)is a wildly cultivated tropical and subtropical fruit with a high nutritional and medicinal value.However,papaya disease is very serious,which is an important problem restricting the development of papaya industry.The key to solve this problem is to use molecular breeding to breed disease-resistant varieties of papaya,and the establishment of efficient genetic transformation system is an important premise of molecular breeding.Therefore,the aim of this study was to establish an efficient genetic transformation system,and provide new technical support for the study of important gene functions and molecular genetic improvement of papaya.【Methods】In this study,immature zygote embryos of Zihui papaya were used as explants to obtain embryogenic cell suspensions(ECS)by induction,proliferation,screening and liquid oscillation culture.Using ECS as the receptors for genetic transformation,and the plant expression vector pCAMBIA1301 was transformed by Agrobacterium-mediated method.This study explored the suitable conditions of antibiotic concentration,infection time,subculture,induction,maturation and germination of resistant embryos,and finally obtained resistant regenerated plants.【Results】First,ECS were treated with different concentrations of cefotaxime sodium(0,100,200,300,400 mg·L^(-1))and hygromycin(0,3,5,7,10 mg·L^(-1))respectively,and then the growth state and cell morphology of suspended cells were observed.The results showed that the optimal concentrations of cefotaxime sodium and hygromycin were 200 mg L^(-1)and 5 mg L^(-1),respectively.After that,the engineered bacteria containing target genes were prepared,and the prepared ECS were co-cultured with the engineered bacteria for 2 days and transferred to the liquid screening medium containing cefotaxime sodiums and hygromycin for subculture.The subculture period was 14 days.GUS staining showed that after 3 subgenerations,almost all the suspended cells were transformed cells.These suspended cells were transferred t

关 键 词：番木瓜 胚性悬浮系 根癌农杆菌 遗传转化 共培养 

分 类 号：S667.9[农业科学—果树学]