CrylAb gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and ...CrylAb gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and transformation efficiency. Different mixtures of Agrobacterium strains （EHA105 and EHA101） contained Hpt and CrylAb genes resulted in different frequencies of resistant calli. There was no correlation between the frequency of transformants with the ratio of the Agrobacterium strain mixture contained Hpt and CrylAb genes. A total of 509 transgenic plants were obtained from the four rice varieties, and 272 T2 progenies were analyzed for CrylAb and Hpt genes. PCR analysis revealed that 412 regenerated plants were Hpt positive （80.94%）, 62 plants were also CrylAb co-transformants （15.05% in total frequency）, and 42 plants among the 272 T2 progenies were CrylAb positive but Hpt negative. This suggests that marker-free transgenic plants could be produced by co-transformation mediated by mixed Agrobacterium strains with the selectable marker gene and target gene Southern blot analysis of five independent marker-free T2 transgenic lines co-transformed from Zhejing 22 showed that CrylAb gene had been inserted into rice genome with a single copy. The transgenic plants showed significantly stronger resistance to lepidopteron than the non-transgenic plants under no application of insecticides against lepidopteron.展开更多
为了比较携带mcr基因的bla_(CTX-M)阳性大肠杆菌(bla_(CTX-M)^(-)positive and mcr-positive Escherichia coli,MCRPEC::bla_(CTX-M)^(+))和携带mcr基因的bla_(CTX-M)阴性大肠杆菌(bla_(CTX-M)^(-)negative and mcr-positive Escherichi...为了比较携带mcr基因的bla_(CTX-M)阳性大肠杆菌(bla_(CTX-M)^(-)positive and mcr-positive Escherichia coli,MCRPEC::bla_(CTX-M)^(+))和携带mcr基因的bla_(CTX-M)阴性大肠杆菌(bla_(CTX-M)^(-)negative and mcr-positive Escherichia coli,MCRPEC::bla_(CTX-M)^(-))的基因组特征,并评估bla_(CTX-M)基因和mcr基因在大肠杆菌之间的共转移能力,试验以猪源MCRPEC::bla_(CTX-M)^(+)和MCRPEC::bla_(CTX-M)^(-)菌株为研究对象,通过全基因组测序利用软件或在线工具对二者进行耐药基因和毒力基因分析、bla_(CTX-M)和mcr基因定位及系统发育分析,通过接合转移试验测定mcr基因和bla_(CTX-M)基因在大肠杆菌间的共转移率,明确共转移mcr基因与bla_(CTX-M)基因的质粒组合。结果表明:共检测了57种耐药基因,MCRPEC::bla_(CTX-M)^(+)菌株和MCRPEC::bla_(CTX-M)^(-)菌株携带耐药基因种类的中位数均为11种,MCRPEC::bla_(CTX-M)^(+)菌株均匀分布在中位数两侧,而MCRPEC::bla_(CTX-M)^(-)菌株较多分布在中位数水平以下。仅MCRPEC::bla_(CTX-M)^(+)菌株携带磷霉素类耐药基因fos(A3)(35.71%)和喹诺酮类耐药基因qnrS11(32.14%),所有MCRPEC::bla_(CTX-M)^(+)菌株均携带黏菌素类耐药基因mcr-1,有94.23%的MCRPEC::bla_(CTX-M)^(-)菌株携带mcr-1基因,MCRPEC::bla_(CTX-M)^(+)菌株携带的bla_(CTX-M)基因亚型以bla_(CTX-M)^(-)55基因(42.86%)和bla_(CTX-M)^(-)65基因(41.07%)为主。共检测了8种毒力基因,MCRPEC::bla_(CTX-M)^(+)菌株和MCRPEC::bla_(CTX-M)^(-)菌株携带毒力基因种类的中位数分别为1种和1.5种,MCRPEC::bla_(CTX-M)^(+)菌株共携带3种,而MCRPEC::bla_(CTX-M)^(-)菌株共携带8种,二者均携带产溶血素基因hlyE(51.92%和51.79%)、产热稳定肠毒素基因astA(21.15%和26.79%)、产溶血素基因hlyF(5.36%和1.92%),仅MCRPEC::bla_(CTX-M)^(-)菌株携带产热不稳定肠毒素基因eltIAB-4(10.71%)、产志贺毒素基因stx2(8.93%)、产热稳定肠毒素基因estap-STa1(17.86%)和estb-STb1(10.71%)、产溶血素hlyA基因(10.71%)。有96.43%(54/56)的MCRPEC::bla_(CTX-M)^(+)菌株的bla_(CTX-M)基因由质粒携带,但不能确定质粒类型;有14.29%(8/56)的MCRPEC::bla_(CTX-M)^(+)菌株的mcr基因由染色体携带;有85.71%(48/56)的菌株的mcr基因由质粒携带,确定了其中26株菌株的质粒类型,包括IncX4型(38.46%)、IncI2型(26.92%)、IncHI2A型(11.54%)、p0111型(23.08%)。有11.54%(6/52)的MCRPEC::bla_(CTX-M)^(-)菌株mcr基因由染色体携带;有88.46%(46/52)的菌株mcr基因由质粒携带,确定了其中30株菌株的质粒类型,包括IncX4型(30.00%)、IncI2型(60.00%)、IncHI2A型(3.33%)、p0111型(3.33%)、IncY型(3.33%)。MCRPEC::bla_(CTX-M)^(+)菌株和MCRPEC::bla_(CTX-M)^(-)菌株均以A群(51.79%、34.62%)和B1群(46.43%、53.85%)为主,少量菌株属于C群(1.79%、9.62%)和D群(0,1.92%),二者均未发现B2群、E群、F群、G群。在多序列位点分型分析中,ST475、ST2、ST88、ST809、ST999在MCRPEC::bla_(CTX-M)^(+)菌株和MCRPEC::bla_(CTX-M)^(-)菌株中均有分布,MCRPEC::bla_(CTX-M)^(+)菌株主要的序列型号为ST475(12.50%)、ST2(7.14%)、ST638(5.36%)和ST86(5.36%),MCRPEC::bla_(CTX-M)^(-)菌株主要的序列型号为ST481(11.54%)、ST66(9.61%)和ST88(9.61%)。在血清型分型中,O51:H49、O-:H10、O81:H-在MCRPEC::bla_(CTX-M)^(+)菌株和MCRPEC::bla_(CTX-M)^(-)菌株中都有分布,MCRPEC::bla_(CTX-M)^(+)菌株主要的血清型为O51:H49(30.36%),MCRPEC::bla_(CTX-M)^(-)菌株主要的血清型为O116:H-和O127:H-(均为9.62%)。在接合转移试验中,有60.87%(14/23)的MCRPEC::bla_(CTX-M)^(+)菌株的bla_(CTX-M)基因水平转移至大肠杆菌EC600,bla_(CTX-M)基因的接合转移率为1×10^(-5)~1×10^(-1),其中有64.29%(9/14)的MCRPEC::bla_(CTX-M)^(+)菌株的bla_(CTX-M)基因和mcr基因共转移至大肠杆菌EC600,共转移率为4%~100%。共确定了4种共转移mcr与bla_(CTX-M)基因的质粒组合,分别为IncX4型(mcr)+IncFⅡ型(bla_(CTX-M))、IncI2型(mcr)+IncFⅡ型(bla_(CTX-M))、IncI2型(mcr)+IncI1型(bla_(CTX-M))、p0111型(mcr)+IncFⅡ型(bla_(CTX-M))。说明MCRPEC::bla_(CTX-M)^(+)菌株和MCRPEC::bla_(CTX-M)^(-)菌株携带的耐药基因、毒力基因种类及mcr基因定位表现出明显差异,bla_(CTX-M)和mcr基因可以在大肠杆菌间共转移。展开更多
基金supported by the Zhejiang Provincial Natural Science Foundation of China (Grant No.Z305650 and No.Y3080361)the Science and Technology Department of Zhejiang Province,China (Grant No.2004C12020)+1 种基金the National Key Technology Research & Development Program,China (Grant No.2006BAD01A01-5 and No.2008ZX08001-001)the 151 Foundation for the Talents of Zhejiang Province,China
文摘CrylAb gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and transformation efficiency. Different mixtures of Agrobacterium strains （EHA105 and EHA101） contained Hpt and CrylAb genes resulted in different frequencies of resistant calli. There was no correlation between the frequency of transformants with the ratio of the Agrobacterium strain mixture contained Hpt and CrylAb genes. A total of 509 transgenic plants were obtained from the four rice varieties, and 272 T2 progenies were analyzed for CrylAb and Hpt genes. PCR analysis revealed that 412 regenerated plants were Hpt positive （80.94%）, 62 plants were also CrylAb co-transformants （15.05% in total frequency）, and 42 plants among the 272 T2 progenies were CrylAb positive but Hpt negative. This suggests that marker-free transgenic plants could be produced by co-transformation mediated by mixed Agrobacterium strains with the selectable marker gene and target gene Southern blot analysis of five independent marker-free T2 transgenic lines co-transformed from Zhejing 22 showed that CrylAb gene had been inserted into rice genome with a single copy. The transgenic plants showed significantly stronger resistance to lepidopteron than the non-transgenic plants under no application of insecticides against lepidopteron.
文摘为了比较携带mcr基因的bla_(CTX-M)阳性大肠杆菌(bla_(CTX-M)^(-)positive and mcr-positive Escherichia coli,MCRPEC::bla_(CTX-M)^(+))和携带mcr基因的bla_(CTX-M)阴性大肠杆菌(bla_(CTX-M)^(-)negative and mcr-positive Escherichia coli,MCRPEC::bla_(CTX-M)^(-))的基因组特征,并评估bla_(CTX-M)基因和mcr基因在大肠杆菌之间的共转移能力,试验以猪源MCRPEC::bla_(CTX-M)^(+)和MCRPEC::bla_(CTX-M)^(-)菌株为研究对象,通过全基因组测序利用软件或在线工具对二者进行耐药基因和毒力基因分析、bla_(CTX-M)和mcr基因定位及系统发育分析,通过接合转移试验测定mcr基因和bla_(CTX-M)基因在大肠杆菌间的共转移率,明确共转移mcr基因与bla_(CTX-M)基因的质粒组合。结果表明:共检测了57种耐药基因,MCRPEC::bla_(CTX-M)^(+)菌株和MCRPEC::bla_(CTX-M)^(-)菌株携带耐药基因种类的中位数均为11种,MCRPEC::bla_(CTX-M)^(+)菌株均匀分布在中位数两侧,而MCRPEC::bla_(CTX-M)^(-)菌株较多分布在中位数水平以下。仅MCRPEC::bla_(CTX-M)^(+)菌株携带磷霉素类耐药基因fos(A3)(35.71%)和喹诺酮类耐药基因qnrS11(32.14%),所有MCRPEC::bla_(CTX-M)^(+)菌株均携带黏菌素类耐药基因mcr-1,有94.23%的MCRPEC::bla_(CTX-M)^(-)菌株携带mcr-1基因,MCRPEC::bla_(CTX-M)^(+)菌株携带的bla_(CTX-M)基因亚型以bla_(CTX-M)^(-)55基因(42.86%)和bla_(CTX-M)^(-)65基因(41.07%)为主。共检测了8种毒力基因,MCRPEC::bla_(CTX-M)^(+)菌株和MCRPEC::bla_(CTX-M)^(-)菌株携带毒力基因种类的中位数分别为1种和1.5种,MCRPEC::bla_(CTX-M)^(+)菌株共携带3种,而MCRPEC::bla_(CTX-M)^(-)菌株共携带8种,二者均携带产溶血素基因hlyE(51.92%和51.79%)、产热稳定肠毒素基因astA(21.15%和26.79%)、产溶血素基因hlyF(5.36%和1.92%),仅MCRPEC::bla_(CTX-M)^(-)菌株携带产热不稳定肠毒素基因eltIAB-4(10.71%)、产志贺毒素基因stx2(8.93%)、产热稳定肠毒素基因estap-STa1(17.86%)和estb-STb1(10.71%)、产溶血素hlyA基因(10.71%)。有96.43%(54/56)的MCRPEC::bla_(CTX-M)^(+)菌株的bla_(CTX-M)基因由质粒携带,但不能确定质粒类型;有14.29%(8/56)的MCRPEC::bla_(CTX-M)^(+)菌株的mcr基因由染色体携带;有85.71%(48/56)的菌株的mcr基因由质粒携带,确定了其中26株菌株的质粒类型,包括IncX4型(38.46%)、IncI2型(26.92%)、IncHI2A型(11.54%)、p0111型(23.08%)。有11.54%(6/52)的MCRPEC::bla_(CTX-M)^(-)菌株mcr基因由染色体携带;有88.46%(46/52)的菌株mcr基因由质粒携带,确定了其中30株菌株的质粒类型,包括IncX4型(30.00%)、IncI2型(60.00%)、IncHI2A型(3.33%)、p0111型(3.33%)、IncY型(3.33%)。MCRPEC::bla_(CTX-M)^(+)菌株和MCRPEC::bla_(CTX-M)^(-)菌株均以A群(51.79%、34.62%)和B1群(46.43%、53.85%)为主,少量菌株属于C群(1.79%、9.62%)和D群(0,1.92%),二者均未发现B2群、E群、F群、G群。在多序列位点分型分析中,ST475、ST2、ST88、ST809、ST999在MCRPEC::bla_(CTX-M)^(+)菌株和MCRPEC::bla_(CTX-M)^(-)菌株中均有分布,MCRPEC::bla_(CTX-M)^(+)菌株主要的序列型号为ST475(12.50%)、ST2(7.14%)、ST638(5.36%)和ST86(5.36%),MCRPEC::bla_(CTX-M)^(-)菌株主要的序列型号为ST481(11.54%)、ST66(9.61%)和ST88(9.61%)。在血清型分型中,O51:H49、O-:H10、O81:H-在MCRPEC::bla_(CTX-M)^(+)菌株和MCRPEC::bla_(CTX-M)^(-)菌株中都有分布,MCRPEC::bla_(CTX-M)^(+)菌株主要的血清型为O51:H49(30.36%),MCRPEC::bla_(CTX-M)^(-)菌株主要的血清型为O116:H-和O127:H-(均为9.62%)。在接合转移试验中,有60.87%(14/23)的MCRPEC::bla_(CTX-M)^(+)菌株的bla_(CTX-M)基因水平转移至大肠杆菌EC600,bla_(CTX-M)基因的接合转移率为1×10^(-5)~1×10^(-1),其中有64.29%(9/14)的MCRPEC::bla_(CTX-M)^(+)菌株的bla_(CTX-M)基因和mcr基因共转移至大肠杆菌EC600,共转移率为4%~100%。共确定了4种共转移mcr与bla_(CTX-M)基因的质粒组合,分别为IncX4型(mcr)+IncFⅡ型(bla_(CTX-M))、IncI2型(mcr)+IncFⅡ型(bla_(CTX-M))、IncI2型(mcr)+IncI1型(bla_(CTX-M))、p0111型(mcr)+IncFⅡ型(bla_(CTX-M))。说明MCRPEC::bla_(CTX-M)^(+)菌株和MCRPEC::bla_(CTX-M)^(-)菌株携带的耐药基因、毒力基因种类及mcr基因定位表现出明显差异,bla_(CTX-M)和mcr基因可以在大肠杆菌间共转移。
