茎尖转化法转基因小麦后代的遗传特性分析
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摘要
为了探讨农杆菌转化微创芽生长点法获得的转基因小麦后代的遗传特性,以3个世代的转基因小麦后代为材料,通过抗性筛选、PCR和PCR-Southern blot等方法分析外源基因遗传在T_2、T_3、T_4中的遗传分离情况。结果表明:T_2代33个株系中外源基因完全丢失的株系占61%,其余39%的株系能够检测到外源基因,但分离比例分散偏离了孟德尔遗传分离规律;T_3代42个株系中检测不到外源基因的株系占55%,能检测到外源基因但分离比例复杂的株系占43%,符合孟德尔分离定律占2%;T_4代8个株系中检测不到外源基因的株系占25%,能检测到外源基因但分离比例复杂的株系占50%,转基因植株与非转基因植株的分离比例符合孟德尔分离定律占25%。可见,利用农杆菌转化微创芽生长点法获得的转基因小麦后代中,外源基因丢失的比例较高,转基因植株与非转基因植株的分离多不符合孟德尔分离比例,但随着世代的增加,外源基因遗传稳定性有所提高。
In order to investigate the genetic characteristics of transgenic wheat progenies,which were obtained by shoot apical point pathway.We used three generations of transgenic wheat progeny as material,the genetic characteristics of exongenous gene in T_2,T_3 and T_4 were analyzed through the resistance screening,PCR,PCR-and Southern blot method.The results showed that in 33 strains of T_2,the exogenous gene completely missing strains accounted for 61%,and the exogenous gene accounted for 39%,it could be detected,but it deviated from Mendel's genetic law of segregation.In 42 strains of T_3,the exogenous gene completely missing strains accounted for 55%;the exogenous gene accounted for 43%,it could be detected but deviated from Mendel's genetic law of segregation;the exogenous gene separation ratio was consistent with Mendel's genetic law of segregation,accounting for 2%.In 8 strains of T_4,2 strains could not be detective exogenous gene;4 strains could be detected but the segregation ratio of exogenous gene was complex;the segregation ratio of the 2 strains of transgenic plants and non-transgenic plants in accordance with Mendel's law of segregation.In conclusion,the progenies of transgenic wheat by shoot apical point transformation,the separation of transgenic plants and non transgenic plants were dispersion,genetic stability of foreign genes increased significantly with the multiplication of selfing generation.
In order to investigate the genetic characteristics of transgenic wheat progenies,which were obtained by shoot apical point pathway.We used three generations of transgenic wheat progeny as material,the genetic characteristics of exongenous gene in T_2,T_3 and T_4 were analyzed through the resistance screening,PCR,PCR-and Southern blot method.The results showed that in 33 strains of T_2,the exogenous gene completely missing strains accounted for 61%,and the exogenous gene accounted for 39%,it could be detected,but it deviated from Mendel's genetic law of segregation.In 42 strains of T_3,the exogenous gene completely missing strains accounted for 55%;the exogenous gene accounted for 43%,it could be detected but deviated from Mendel's genetic law of segregation;the exogenous gene separation ratio was consistent with Mendel's genetic law of segregation,accounting for 2%.In 8 strains of T_4,2 strains could not be detective exogenous gene;4 strains could be detected but the segregation ratio of exogenous gene was complex;the segregation ratio of the 2 strains of transgenic plants and non-transgenic plants in accordance with Mendel's law of segregation.In conclusion,the progenies of transgenic wheat by shoot apical point transformation,the separation of transgenic plants and non transgenic plants were dispersion,genetic stability of foreign genes increased significantly with the multiplication of selfing generation.
引文
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[13]GOSAL S,GILL R,SINDHU A S A S,et al.Introducing the CryIA(c)Gene into Basmati Rice and Transmitting Transgenes to R3Progeny[M]//Advances in Rice Genetics:(In 2 Parts).Singapore:World Scientific Publishing Company,2003:558-560.
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[15]HIEI Y,KOMARI T.Stable inheritance of transgenes in rice plants transformed by Agrobacterium tumefaciens[J].Rice Genetics,2008,3:131-142.
[2]SCHRAMMEIJER B,SIJMONS P C,VANDEN DENELZEN P J M,et al.Meristem transformation of sunflower via Agrobacterium[J].Plant Cell Reports,1990,9(2):55-60.
[3]GOULD J,BANISTER S,HASEGAWA O,et al.Regeneration of Gossypium hirsuumand barbadense from shoot apex tissues for transformation[J].Plant Cell Reports,1991,10(1):12-16.
[4]WEEKS J T,YE J,ROMMENS C M.Development of an in planta method for transformation of alfalfa(Medicago sativa)[J].Transgenic Research,2008,17(4):587.
[5]GOULD J,DEVEY M,HASEGAWA O,et al.Transformation of Zea mays using agrobacterium tumefaciens and the shoot apex[J].Plant Physiology,1991,95(2):426-434.
[6]SUPARTANA P,SHIMIZU T,SHIOIR H,et al.Development of simple and efficient in planta transformation method for rice(Oryza sativa L.)using agrobacterium tumefaciens[J].Journal of Biocience and Bioengineering,2005,100(4):391-397.
[7]SUPARTANA P,SHIMIZU T,NOGAWA M,et al.Development of simple and Efficient in planta transformation method for wheat(Triticum aestivum L.)using Agrobacterium tumefaciens[J].Journal of Biocience and Bioengineering,2006,102(3):162-170.
[8]HAMADA H,LINGHU Q,NAGIRA Y,et al.An in planta biolistic method for stable wheat transformation[J].Scientific Reports,2017,7(1):11443.
[9]董福双,高义平,吕孟雨,等.利用卡那霉素“浸种法”准确筛选转基因小麦种子的方法研究[J].麦类作物学报,2013,33(6):1105-1106.DONG F SH,GAO Y P,LM Y,et al.Study on accurately kanamycin screening method of transgenic wheat seed by seed-immersion[J].Journal of Triticeae Crops,2013,33(6):1105-1106.
[10]马盾,周小云,黄乐平.花粉管通道法转基因棉花后代的遗传特性[J].西北农业学报,2008,17(3):147-149.MA D,ZHOU X Y,HUANG L P.Genetic characters on the future generations of transgenic cotton obtained by pollen tube pathway[J].Acta Agriculturea Boreali-occidentails Sinica,2008,17(3):147-149.
[11]李三和,林刚,张金锐,何光源.转基因小麦外源基因在主栽小麦种质中的遗传规律[J].麦类作物学报,2005,25(5):23-26.LI S H,LIN G,ZHANG J R,et al.Inheritance and stability of transgene in elite wheat(Triticum aestivum L.)varieties from transgenic wheat[J].Journal of Triticeae Crops,2005,25(5):23-26.
[12]VAIN P,JAMES V,WORLAND B,et al.Snape.Transgene Structure and Expression in a Large Population of Rice Plants and Their Progenies[M]//Advances in Rice Genetics:(In 2 Parts).Singapore:World Scientific Publishing Company,2003:550-551.
[13]GOSAL S,GILL R,SINDHU A S A S,et al.Introducing the CryIA(c)Gene into Basmati Rice and Transmitting Transgenes to R3Progeny[M]//Advances in Rice Genetics:(In 2 Parts).Singapore:World Scientific Publishing Company,2003:558-560.
[14]SANGTONG V,MORAN D,CHIKWAMBA R,et al.Expression and inheritance of the wheat Glu-1DX5 gene intransgenic maize received[J].Theoretical and Applied Genetics,2002,105(6-7):937-945.
[15]HIEI Y,KOMARI T.Stable inheritance of transgenes in rice plants transformed by Agrobacterium tumefaciens[J].Rice Genetics,2008,3:131-142.