The Establishment Of The Bio-safe Selective Marker Gene Transformed Into The Wheat

With the fast development of genetic engineering, the kinds, the quantity and the output of the transgenic crops increase rapidly in the world, because transgenic plants have multiple merits such as high grade, high yield, herbicide resistant, drought resisting as well as salt resistance, the kinds, the quantity and the output of the transgenic crops increase rapidly in the whole world. With the development of the transgenic crops products’commercialization, people bigin to doubt the food safty of the transgenic plants and the production aspects. The reason is that the resistance marker gene and target gene transform together to the callus to get the resistance to antibiotics or herbicides.After the choice of agent selection, inducting and forming transgenic plants at the end.Once transgenic plant regenerates success, the selectable mark genes are no longer useful, even harmful on some species degree. Therefore the problem that the security of the selectable mark genes in genetically-modified crop has caused concerns among the public, how to remove the selectable mark, how to cultivate the genetically-modified crop with non-selectable marker. The innovation of this study is considering the target gene as the selectable marker gene.That means one gene playing two function on the plants’screening. This research has good three aspects:the gene of good agronomic traits to build the safe marker carrier,to establish a stable Agrobacterium-mediated genetic transformation system and to establish the safety marker screening system t.All the study purposes to develop new Ideas and methods on the safety of genetic engineering and to gain the security marker transgenic wheat at the same time, finally to eliminate the public’ concerns.about the safety of the transgenic cropsSo my thesis is composed of three parts:building the safe marker carrier of the TaSTRG gene and Na+/H÷antiporter gene,establishing a stable Agrobacterium-mediated genetic transformation system and establishing the safety marker screening systemThe first part is related to build the safe marker carrier of the TaSTRG gene and Na+/H+ antiporter gene. My laboratory room has proved that the expression of TaSTRG and Na÷/H+ antiporter gene is induced by salt in Arabidopsis and rice. The result demonstrates that they can increase the resistance of plants, especially in terms of salt tolerance. Therefore, these two genes as a selectable marker has two advantages:firstly, they all come from the wheat which can eliminate food safety concerns; Secondly, their function is strong, and the phenotype is obvious.So it can play the good role on the screeningThe pBZ1024 used in this study remoulds through the vector pCAMBIA1300 transformation. Firstly, removed hygromycin resistance gene on the vector pCAMBIA1300, thereby eliminating concerns about the safety of the carrier,.Join the two MluI and Eco91I restriction sites for the safe marker genes to insert in this position. Secondly, the Ubi promoter and multiple cloning sites and the NOS terminator fragment of the pTCK303 vector was inserted into the remoulded pCAMBIA1300 vector to facilitate other genes such as high yield, high efficiency and quality, etc. to insert. After the TaSTRG and Na+/H+ antiporter gene sequencing are cloned,build a good pBZ1024-TaSTRG expression vector and pBZ1024-Na+/H+ antiporter gene expression vector into EHA105 through transformation, the process of digestion and a series of connections,which is to prepare the transformation of wheat callus, and finally to explore the feasibility of safe marker.The second part is the establishment of the Agrobacterium-mediated genetic transformation of wheat mature embryos. Because Agrobacterium-mediated genetic transformation in wheat has some limitations, the research for the transformation system is mainly concentrated in the mature embryo induction Optimization, Agrobacterium infection to determination of concentration and time, co-culture Temperature and time selection, differentiation, efficiency and so on. Luan 02-1 for the division of this wheat variety suitable for Agrobacterium infection. Through the exploration of Agrobacterium infection’s concentration and time of Luan02-1, bacterial concentration of 0.1, infection time for the 0.5t, are more efficient. Meanwhile, in order to eliminate the browning of the callus into the impact of glutamine in the induction of differentiation, we explore aspects of elimination of browning. Through the exploration of this study combined with the existing literature and my preliminary workroom, mature embryos rate has reached higher levels.The third part is to establish screening system of security marker.First,the division for Shi Luan 02-1 this genotype screening concentration of salt to explore, through a large number of callus reached 0.5% salt screening, screening both maximize the role, but also can improve seedling callus after chance.After the transformation of the mature wheat embryo division Luan 02-1 in the absence of salt and salt can be selected under the callus differentiation related statistics, data analysis showed that screening by salt can reduce the workload of late. Finally, The results of the PCR identification of transgenic plants, showed that.the ideal genes have transformed into the wheat. Currently there are few reports for salt selection marker gene for wheat transformation reports, in order to solve the safty o grnr better, so we try to stress resistance marker genes in the security applied to the genetic transformation of wheat and has initially established a security Marker screening system.