Modification And Application Of Xa27 Resistance Gene To Bacterial Blight

Bacterial blight（BB）and bacterial leaf streak（BLS）of rice,caused by Xanthomonas oryzae pv.oryzae（Xoo）and Xanthomonas oryzae pv.oryzicola（Xoc）,respetively,are the representative diseases resulted in the most severe reduction of rice output.The cultivation of resistant varieties was considered as the most effective and economical option to control diseases.Few rice high resistance（R）genes were characterized for bacterial blight,but no R genes have been identified for rice leaf streak,therefore modificated and utilization of the cloned genes is an important supplementary and fast way to cultivate both-against BB and BLS.People always pay more attention to the safety of genetically modified crops.They believe that the marker genes in the transgenic process are potential safety hazards.Therefore,safety of genetically modified crops will be greatly improved and the basic requirements for approval of safety application certificates when transgenic crops without marker genes and stable expression of foreign genes.However,maker-free foreign genes often have unintended expression due to the lack of flanking sequence protection.In order to solve the above problems,we basis on the vector system of marker-free double right border vector,take the bacterial blight resistance gene Xa27 as remodel object.Then inset multiple transcription activator-like effectors（TALE）identified by bacterial blight and bacterial leaf streak recognize TALE binding-elements（EBE）to Xa27’s promoter.For avoid the influence of the reverse endogenous promoter,we retain the 1149bpupstream of the EBE element of the Xa27 gene promoter,and add a reverse rice seed 16KD prolamin gene terminator（16KD）after the terminator.The main results are as follows:1.By comparing the field resistance performance and expression analysis of the marker-free Xa21 transgenic rice and the marker-free Xa23 transgenic rice obtained earlier in the research group,the results revealed that the presence of the endogenous promoter at the integration site of the exogenous gene may cause the exogenous gene The main factor of expected expression.2.Using the Agrobacterium transformation method,the plant expression vectors pDRB-Xa27 and pDRB-Xa27-KD were transferred into the rice variety TP309 respectively.After molecular identification and resistance identification the two maker-free transgenic plants showed strong resistance to bacterial blight and bacterial leaf streak disease;The expression results showed that two transgenic plants with 16KD terminator added the exogenous Xa27’s expression was expected that was induced by bacterial blight and bacterial leaf streak disease,adversely the transgenic plants without the 16KD terminator3.were constitutively expressed.So the addition of the 16KD terminator can effectively reduce the unexpected expression of the foreign gene.4.By testing transgenic rice of T₁generation pDRB-Xa27-KD-1 and pDRB-Xa27-KD-2,we got two plants without marker genes and carrying Xa27 gene.