摘要
【目的】建立发根农杆菌介导的西瓜转基因不定根过表达体系。【方法】探索利用野生型发根农杆菌K599侵染西瓜材料‘Sugar Baby’,诱导西瓜产生不定根的实验方法。借助该方法,将含有GUS基因和EGFP基因的过表达载体pCAMBIA3301和pCAMBIA1300-ProSuper导入发根农杆菌K599,侵染西瓜材料‘Sugar Baby’,通过常规PCR、qRTPCR、GUS染色和激光共聚焦显微镜检测,评价该过表达体系的效果。【结果】利用野生型和导入外源质粒的发根农杆菌均能成功诱导西瓜‘Sugar Baby’产生不定根。PCR检测结果显示,携带pCAMBIA3301和pCAMBIA1300-ProSuper载体的发根农杆菌K599诱导产生的不定根阳性率达到100%。qRT-PCR、GUS染色和激光共聚焦显微镜检测均能成功检测到GUS基因和EGFP基因的过量表达。【结论】利用发根农杆菌K599诱导西瓜产生不定根介导基因过表达的方法,具有周期短,操作方便,转化率高的特点,可实现基因功能的快速验证,为西瓜根系相关基因的功能研究提供技术支持。
【Objective】Watermelon(Citrullus lanatus)is a worldwide horticultural crop with a long history and extensive planting areas.China is the country with the largest watermelon planting area and yield in the world and also at the forefront of the world in the study on watermelon genomics and functional gene mining.Agrobacterium tumefaciens meditated transformation of watermelon has been successfully conquered by Beijing Academy of Agricultural and Forestry Sciences.However,the watermelon transgenic technology is still difficult for most researchers,which is an important factor that restricts the studying progress on watermelon genes.In this study,a rapid method of inducing adventitious roots in watermelon was established.Through the mediation of Agrobacterium rhizogenes,the exogenous genes could overexpress in adventitious roots.【Methods】The wild type A.rhizogenes strain K599 and watermelon variety‘Sugar Baby’were chosen to explore the operation method.Seedlings with two just unfolded cotyledons were inoculated.A microsyringe needle was used to pick up a piece of A.rhizogenes K599 colony and pierce through the cotyledon node.After inoculation,the seedlings were covered with plastic cups to maintain high air humidity and then cultured at 22℃in dark for 8^^-12 h.After dark incubation,the bottoms of the cups were cut off,and then the cups were placed upward on the seedlings.Vermiculite was used to fill the cups until the seedling inoculation sites were covered.Pour the vermiculite through with a spray pot.Seedlings were cultured in the light incubator with a 12 h light/12 h darkness photoperiod,and the day and night temperatures were set to 28/25℃.Vermiculite was added and seedlings were watered daily to keep the inoculation site moist and in darkness.About 20 days after inoculation,the adventitious roots appeared at the inoculation sites.The main roots and hypocotyls were cut off.Then,the plants were buried in the nutrient soil and continued to grow.With this method,the overexpression vectors pCAMBIA3301 and pCAMBIA1300-ProSuper containing GUS and EGFP genes were introduced into A.rhizogenes K599 to infect‘Sugar Baby’.The effect of the overexpression system was evaluated by common PCR.The reaction system of PCR was as follows:1μL DNA template,0.5μL of each upstream and downstream primers,5μL 2×PCR Master Mix,and 3μL ddH2O.The PCR reaction procedure for pCAMBIA3301 vector detection was pre-denaturation at 95℃for 3 min,denaturation at 95℃for 30 s,annealing at 50℃for 30 s,and extension at 72℃for 50 s for 35 cycles,and finally extension at 72℃for 8 min and preservation at 4℃.The PCR reaction procedure for pCAMBIA1300-ProSuper vector detection was pre-denaturation at 95℃for 3 min,denaturation at 95℃for 30 s,annealing at 50℃for 50 s,and extension at 72℃for 1 min for 35 cycles,and finally extension at 72℃for 10 min and preservation at 4℃.PCR products were detected by 1%agarose gel electrophoresis.To analyze the expression of GUS and EGFP in adventitious roots,qRT-PCR was conducted.First strand cDNA was synthesized using a PrimeScript^TM RT reagent Kit with gDNA Eraser(Takara,RR047A)following the manufacturer’s protocol.PCR reactions were performed on a Roche LightCycler■480 RT-PCR System using the SYBR Premix Ex Taq^TM Kit(Takara,RR420A).Expression levels were analyzed following the 2-ΔΔt method.The expression of GUS and EGFP was also detected by GUS staining and confocal laser microscopy.【Results】After seedlings were inoculated by the wild type A.rhizogenes strain K599,adventitious roots were successfully induced at the inoculation site in‘Sugar Baby’.This indicated the operation method of inducing adventitious roots in watermelon by A.rhizogenes K599 was feasible.Following this method,A.rhizogenes K599 carrying pCAMBIA3301 and pCAMBIA1300-ProSuper vectors successfully induced the adventitious roots in‘Sugar Baby’.The induction rate of adventitious roots was 70%-90%.PCR verification results showed that the positive rate of adventitious roots was 100%.GUS and EGFP genes were detected to be highly expressed by qRTPCR and verified by GUS staining and confocal laser microscopy detection.【Conclusion】Genetic transformation is an important means to analyze gene function,but the research progress on cucurbita genetic transformation is relatively slow.Although the transgenic technology of watermelon has been conquered by Beijing Academy of Agricultural and Forestry Sciences,it is still an important factor restricting many researchers to study the gene function of watermelon.In this study,A.rhizogenes K599-mediated gene overexpression system was established in watermelon.This system has the advantages of short cycle,easy operation,high transformation rate,and can realize the rapid verification of gene function in watermelon roots.It will provide technical support for the research on the mechanism of genes that specifically play roles in watermelon roots.
作者
王平勇
徐永阳
赵光伟
贺玉花
孔维虎
张健
刘水苗
户克云
侯冲
王兰菊
徐志红
WANG Pingyong;XU Yongyang;ZHAO Guangwei;HE Yuhua;KONG Weihu;ZHANG Jian;LIU Shuimiao;HU Keyun;HOU Chong;WANG Lanju;XU Zhihong(Zhengzhou Fruit Research Institute,Chinese Academy of Agricultural Sciences,Zhengzhou 450009,Henan,China;College of Horticulture of Henan Agricultural University,Zhengzhou 450002,Henan,China)
出处
《果树学报》
CAS
CSCD
北大核心
2019年第12期1763-1770,共8页
Journal of Fruit Science
基金
国家自然科学基金(31801888)
国家西甜瓜产业技术体系项目(CARS-25)
中国农业科学院科技创新工程专项经费项目(CAAS-ASTIP-2019-ZFRI)
河南省现代农业产业技术体系项目(S2014-11-G03)
中央级科研院所基本科研业务费专项(1610192019201)
关键词
西瓜
发根农杆菌
K599
过表达
Watermelon
Agrobacterium rhizogenes
K599
Overexpression
