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Construction And Application Of An Intelligent Male Sterility System Recoverable By Chemical Inducement

Posted on:2019-03-13Degree:DoctorType:Dissertation
Country:ChinaCandidate:N ChenFull Text:PDF
GTID:1360330542984652Subject:Biochemistry and Molecular Biology
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Genetic engineering male sterility(GMS),since its birth,shows advantages over natural one in expanding the production of hybrid F1 seeds to exploit heterosis of crops and reducing production cost,in particular when it is used for "two lines" system of F1 production.Currently,most GMS used in the two lines system is "inducible male sterility" or "regulatable male sterility",that is,by default,the male is fertile,and over applying chemical inducers or compensators,the male turns to sterile.In this system,continuous applying the inducers is imperative to ensure complete male sterility,which is labor-consuming and costly.In order to produce F1 seeds more easily and more economically,two intelligent male sterility systems recoverable by chemicals were established and verified in Arabidopsis Thaliana,based on the filament-specific promoter AtDAD1 or classic tobacco tapetum-specific promoter TA29,in this study.Here are the main results.The DADB fragment of AtDAD1 promoter region could direct GUS gene to express strongly and specifically in the stamen filament of Arabidopsis,but not in other organs and/or tissues of the plant.The expression started from flower stage 12 and remained until pollination was completed,and the filament-specificity was controlled by some cis-elements in the region-1938 bp to-2947 bp of DADB,according to its 5'and 3'-terminal deletion results.With the DADB driving mBarnV(an improved version of Barnase gene,can be easily cloned in prokaryotic cells and express well in eukaryotic cells)as "male sterility" gene,the transgenic Arabidopsis displayed much shorter stamen filaments which keep the anthers below the stigma,significantly decreased number of pollens and completely invalid pollens,but functional gynoecium.The plant did not produce any seeds when auto-crossed,but produced seeds when hand-pollinated with pollens from wild-type plants.When this male sterile gene was coupled with the Barstar gene driven by a modified PR-1a promoter,PR2as,and transferred into Arabidopsis,the transgenic plants(T1)were all male sterile,by default.However,when sprayed with salicylic acid(SA),the male turned to fertile,and the plant did produce seeds by self-pollination.This chemical-recoverable male sterility passed stably to the offspring,and SA(0.1 mM)spraying once in every three days was enough to recover the male fertility.These results indicated an intelligent male sterility system(IMS)was established and functional.The IMS was applied to Brassica napus cv.West-4 through Agrobacterium-mediated transformation,and a few of transgenic plants were obtained.The To plants,in the natural condition,showed also shorter stamen filaments,decreased pollen number and aborted pollens,but only in some flowers.In Ti plants,the flower number with sterile male increased,although whole plant still could produce seeds even without SA spraying.The reason for this kind of flower diversity is still to be further studied.In parallel,the tapetum-specific promoter TA29 was used in the place of DADB to drive mBarnV,and the modified PR-la promoter 8AnBx to replace PR2as to direct Barstar.This construct-transgenic Arabidopsis Ti plants showed poorly developed anthers and could not produce normal siliques or seeds under natural conditions.However,after continuous spraying SA in the specific stages of anther development,the anthers became phenotypical normal,and the plant could realize self-pollination and produce normal siliques and seeds.Comparing the DADB-based system with TA29-based one,the former has a wider time window than the latter to apply chemical inducers to recover the male sterility,which and thus can be more convenient in practice and more economical in production of F1 seeds.Taken together,this study not only established two intelligent male sterility systems recoverable by chemical inducement,but also provided new ideas and practical instruction for construction of new intelligent male sterility systems to better exploit crop heterosis.
Keywords/Search Tags:genetic engineering male sterility, filament-specific promoter, inducible promoter, intelligent male sterility, Brassica napus
PDF Full Text Request
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