摘要
【目的】探寻一种由农杆菌介导的杜梨叶片瞬时转化的最佳方法。【方法】以杜梨组织培养的幼嫩叶片为材料,将含有GUS基因的pBI121植物表达载体转化农杆菌GV3101和EHA105,设计3因子3水平正交试验L9(33),采用不同农杆菌菌液浓度、不同真空渗入时间,侵染叶片组织细胞,取不同时间共培养后的叶片观察GUS染色情况。【结果】GV3101和EHA105均能高效侵染转化杜梨叶片,使GUS蛋白表达,但其转化效率不同。农杆菌GV3101在菌液OD600为0.8,真空渗入20 min,共培养4 d后,杜梨叶片的转化效率即可达到100%,且叶片坏死率仅为13.09%;EHA105在OD600为0.8,真空渗入30 min,共培养6 d,或菌液OD600为1.0,真空渗入10 min,共培养2 d的条件下转化效率最高,均为83.33%,但是,叶片坏死率分别为27.78%和15.26%,然而在菌液OD600为1.0,真空侵染时间20 min,共培养4 d后,叶片转化效率为75.79%,但叶片坏死率大大降低,仅为5.00%。【结论】菌株GV3101和EHA105均能高效侵染转化杜梨叶片,最高转化效率分别为100%和83.33%,所以菌株GV3101转化效率高于EHA105。考虑到叶片坏死率,菌株GV3101瞬时转化杜梨叶片的最适条件为:菌液OD600为0.8,真空渗入时间20 min,共培养4 d,转化效率100%,叶片坏死率13.09%;菌株EHA105瞬时转化杜梨叶片的最适条件为:菌液OD600为1.0,真空渗入20 min,共培养4 d,转化效率75.79%,叶片坏死率5.00%。
【Objective】The aim of this study was to seek for the optimal conditions for transient expression of the genes to a high level in the young leaves of Duli pear(Pyrus betulifolia)using Agrobacterium-mediated transformation strategy.【Methods】The young leaves of the tissue cultured seedlings of Duli pear(Pyrus betulifolia)were used as the experimental materials.The pBI121 expression vector containing the GUS gene under the 35S promoter was firstly transformed into two different Agrobacterium tumefaciens strains GV3101 and EHA105 through the freeze-thaw method.100 ng of pBI121 plasmid DNA was added into the competent cells DH5αunder aseptic conditions,mixed gently,and let to stand for 5 minutes in an ice water bath;The mix was quickly froozen in liquid nitrogen for 5 minutes,and the centrifuge tube was quickly placed into a 37℃water bath for 5 minutes;then 5 minutes in an icewater bath;800μL of antibiotic-free LB liquid medium was added into the tube under aseptic conditions,and the tube was shaken at 28℃for 2 hours to revive the bacteria;then it was centrifuge at 6000rpm for 1 minute to harvest the bacteria,about 100μL of supernatant was left.The obtained bacteria was gently suspended again,an appropriate amount of bacteria liquid was taken to,spread on the LB plate containing the corresponding antibiotics.The culture was inverted in a 28℃incubator until 2mm plaques had appeared.Colony PCR positive identification was performed using primers 35SPro-F:5’-CTATCCTTCGCAAGACCCTTC-3’,GUS-R:5’-ATCGCTGATGGTATCGGTGT-3’,M13F:TGTAAAACGACGGCCAGT and M13R:CAGGAAACAGCTATGAC,then agarose gel electrophoresis was used for analysis.A 3-level orthogonal experiment was designed with 3 factors,i.e.,3 bacterial concentrations at OD600=0.6,0.8 and 1.0,3 vacuum infiltration durations of 10 min,20 min and 30 min,vacuum negative pressure was set to-0.09 MPa,and co-culture time of 2 d,4 d and 6 d,respectively.The cocultivation medium was adjusted to pH 5.8 using 4.4 g·L^(-1) MS+30 g·L^(-1) sucrose+8 g·L^(-1) agar.The
作者
李刚
宋平丽
王翔
马青翠
张海霞
张玉星
许建锋
亓宝秀
LI Gang;SONG Pingli;WANG Xiang;MA Qingcui;ZHANG Haixia;ZHANG Yuxing;XU Jianfeng;QI Baoxiu(College of Horticulture,Hebei Agricultural University,Pear Engineering and Technology Center of Hebei Province,Baoding 071001,Hebei,China;School of Pharmacy and Biomolecular Sciences,Liverpool John Moores University,Liverpool L33AF,United Kingdom)
出处
《果树学报》
CAS
CSCD
北大核心
2021年第11期2006-2013,共8页
Journal of Fruit Science
基金
国家现代农业产业技术体系建设专项(CARS-28-9)
河北省研究生创新资助项目(CXZZBS2018116)。
关键词
杜梨
组织培养
叶片
农杆菌
瞬时转化
β-葡糖醛酸酶
Duli pear
Tissue culture
Leaf
Agrobacterium tumefaciens
Transient transformation
βglucuronidase
