Construction Of A Novel Mustard Engineered Male Sterile Line

The utilization of male sterile line is one of the most important ways for heterosis breeding,As the most promising way,the research,development and utilization of engineered male sterile line has became the focus,all over the world.At the same time,the generation,maintenance and fertility restoration of new male sterile germplasm are the core problems for the application.To maintain the engineered male sterility,there are currently two ways:one is through tissue culture method,but which is highly technical,and difficult to apply in large-scale seed production;the other is to cross with the nontransgenic maintainer line,which will produce about50%of the fertile plants in progenies.Therefore,we have to spray herbicide in field to get rid of the fertile plants and need more parent seeds for the production of hybrid seeds.In addition, those crops that fruits or seeds harvest are the aim need to restore fertility in hybrid from engineered male sterility.Therefore,it is necessary to explore the new method and the approach for maintenance and fertility restoration.of engineered male sterile line.Barnase,a RNA enzyme gene from Bacillus amyloliquefaciens,is the most frequently used male sterile gene in plant engineered male sterility.At present, commercial application of engineered male sterile line from the Barnase gene has been found in rape,chicory,and corn.The extensive study of Barnase toxic protein structure and enzyme activity showed that the protein could be split into less toxic N and C terminus polypeptide.Ssp DnaE intein,as a trans-splicing function split intein found in Synechocystis sp.PCC6803(Synechocystis, a kind of cyanobacteria),is consists of the123-amino acids N terminal and the36-amino acids C terminal.Its structure and splicing characteristics are very suitable for the structure and function recover of split protein.Therefore, based on the principle of plant nuclear gene male sterility, this study took mustard as testing materials,splitted the Barnase gene sequence into the N terminus of36amino acids and the C terminus of75amino acids, and then fused with N and C terminus of Ssp DnaE intein respectively to get fusion genes which were under the control of the tapetum specific promoter A9.The fertile transgenic plants can be obtained when each fusion gene was individually transformed into mustard,but the male sterile plants can be obtained by the co-transformation of N and C terminal fusion genes or the cross between N and C terminal fusion genes transgenic plants due to the co-expression of two fusion peptides in the tapetum cells,which resulted from the recover of Barnase toxic protein structure and function in the tapetal cells mediated by Ssp DnaE intein.And the hybrid progeny can be fertility when cross this male sterile lines with other nontransgenic plants as the result of the separation of N and C terminal fusion genes in the pollen development process.The main results are as follows:1.A series of expression vectors for mustard single transformation or co-transformation were constructed,and the transgenic mustard plants were obtained.(1) pCABarBN:A non-split Barnase toxic protein gene is expressed by Chinese cabbage tapetum-specific promoter A9,and the selection marker gene is Bar gene;(2) pCABarBN-N/Int-N:To express the fusion gene of N terminal (1-36amino acids) of Barnase and Ssp DnaE N terminual (123amino acids) using A9promoter,with the same Bar selection marker gene;(3) pCANPTInt-C/BN-C:To express the fusion gene of Ssp DnaE C terminal (36amino acids) and Barnase C terminal (37-111amino acids) using A9promoter,the selection marker gene is NPTⅡ gene;(4) pCABarBN-N:To express the N terminal polypeptide (1-36amino acids) of Barnase directly by A9promoter, and the selection marker gene is Bar gene;(5) pCANPTBN-C:To express the C terminal polypeptide (37-111amino acids) of Barnase directly by A9promoter,and the selection marker gene is NPTⅡ gene.pCABarBN,pCABarBN-N/Int-N, pCANPTInt-C/BN-C, pCABarBN-N, pCANPTBN-C were transformed into mustard respectively by agrobacterium-mediated genetic transformation method;and the pCABarBN-N/Int-N with pCANPTInt-C/BN-C,pCABarBN-N with pCANPTBN-C, pCABarBN-N/Int-N with pCANPTBN-C were co-transformed into mustard.After several rounds selection,transgenic plants of pCABarBN,pCABarBN-N/Int-N, pCABarBN-N were obtained under the glufosinate PPT selection medium;transgenic plants of pCANPTInt-C/BN-C,pCANPTBN-C were obtained using kanamycin selection;Co-transgenic plants of pCABarBN-N/Int-N and pCANPTInt-C/BN-C, pCABarBN-N and pCANPTBN-C,pCABarBN-N/Int-N and pCANPTBN-C were obtained with glufosinate and kanamycin in the screen medium.2.Molecular detection of transgenic mustard plantsThe transgenic plants seedlings DNA was extracted by CTAB method.The PCR results showed that the corresponding genes had been integrated into the mustard in all of the single transformed plants. Meanwhile,the N and C terminual gene were also integrated into the mustard genome for the pCABarBN-N/Int-N and pCANPTInt-C/BN-C, pCABarBN-N and pCANPTBN-C, pCABarBN-N/Int-N and pCANPTBN-C co-transformed plants.3.Fertility comparison of transgenic mustard plantsAfter blossoming,the fertility was analyzed all the transgenic mustard plants.The results showed all the pCABarBN-N/Int-N, pCANPTInt-C/BN-C, pCABarBN-N, pCANPTBN-C transgenic plants as well as the co-transgenic plants of pCABarBN-N and pCANPTBN-C,pCABarBN-N/Int-N and pCANPTBN-C are fertile,and all transgenic plants have bright yellow petals,long filaments,full anthers and massive pollen after anthers cracking.However,co-transgenic plants of pCABarBN-N/Int-N and pCANPTInt-C/BN-C appear typical male sterile like the pCABarBN transgenic plants.These transgenic plants have pale yellow petals,short filaments,shrunken anther,and no pollen in pollen sac.4.Pollen cell development analysis of transgenic mustard plantsUsing paraffin section and microscopic technique,the anthers development process of transgenic mustard fertile plants and sterile plants were analyzed.The results showed that the anthers of fertile plants (transgenic plants pCABarBN-N/Int-N, pCANPTInt-C/BN-C,pCABarBN-N,pCANPTBN-C,co-transgenic plants pCABarBN-N and pCANPTBN-C,pCABarBN-N/Int-N and pCANPTBN-C)developed normally, and can develop into mature pollen grains;however,the anthers development process of co-transformed plants pCABarBN-N/Int-N and pCANPTInt-C/BN-C were basically like the pCABarBN plants,the the tapetum cells in anther suffered early degradation,no normal pollen grains formed after anther mature, and anther abortion.5.The seed of transgenic mustard plantsAll the obtained transgenic plants were self-crossed.The results showed that most siliques were full and the seeds are morphology normal of pCABarBN-N/Int-N, pCANPTInt-C/BN-C, pCABarBN-N, pCANPTBN-C transgenic plants as well as pCABarBN-N and pCANPTBN-C, pCABarBN-N/Int-N and pCANPTBN-C co-transgenic plants.But the pCABarBN transgenic plants suffered early shedding flower, no silique and seed growing. The silique of the PCABarBN-N/Int-N and pCANPTInt-C/BN-C co-transformed plants after self-crossed can development well,but the siliques were shorter and empty,and no seeds in them.Pollinated with pollens fromnon transgenic plants,the pCABarBN transgenic plants as well as pCABarBN-N/Int-N and pCANPTInt-C/BN-C co-transformed plants podded normally.6.The fertility of hybrid between pCABarBN-N/Int-N and pCANPTInt-C/BN-C transgenic plantsThe hybrid offspring are obtained by the crossing between pCABarBN-N/Int-N transgenic plants and pCANPTInt-C/BN-C transgenic plants,The double resistant plants that contained simultaneously both the pCABarBN-N/Int-N and pCANPTInt-C/BN-C genes were screened out by spraying the kanamycin and glufosinate,then the PCR was performed to confirmed the plants.After flowering,it was found that these double resistant plants hybrid progeny plants also were typical male sterile,like the pCABarBN-N/nt-N and pCANPTInt-C/BN-C co-transgenic plants.