The Primary Study Of New Type Male Sterility Lines Of Rice

Male sterile lines play a decisive role in the use of heterosis in rice.A large number of male nuclei sterility can't be used in production due to lack of maintainer lines.This study designed a new male sterility system according to the SPT which invited by Pioneer Hi-Bred International,Inc.The system consists of two parts,male sterile line and the maintainer line respectively.The maintainer line contains wild-type fertility gene which corresponding with sterile lines,pollen lethal genes and herbicide selectable marker genes.Because of carrying the lethal gene,the maintainer line does not produce transgenic pollen.Offspring which hybridize between maintainer line and sterile line are sterile lines which do not contain transgenic genes.Offspring of selfing between maintainer lines are half with transgenic genes and half without transgenic genes.In order to create this system,this study carried out the following work:1.We identified one male sterile line named W908 from MingHui86 transgenic offspring.Based on the early gene location results,we found that the key gene PTC1 which controlling the fertility changed by analyzing the sequencing results of genome and cDNA and the mutant whose right border of the first intro changing resulted in the cut mode changed.2.We obtained site-directed mutation of male sterile genes PTC1 and TDR by TALENs and also created male sterility lines of PTC1 and TDR.We constructed TALEN expression vector with designing one targeted site of PTC1,and transformed it into minghui 86,Oryza sativa L.japonica.cv.Nipponbare and xiushuil34 mediated by Agrobacterium strains EHA105.We have obtained 40,30 and 33 seedlings respectively,and we have found 20 transgenic seedlings by vector gene PCR.Which no mutation was found by sequence alignment with wild-typed plant.We constructed TALEN expression vector with designing two targeted sites of TDR.And transformed it into XiuShui 134 mediated by Agrobacterium strains EHA105.At last,one of the two sites TALEN-TDR-1,we have obtained 59 seedlings including 15 positive seedlings and 0 mutants.For the other site TALEN-TDR-2,we have obtained 101 seedlings including 43 positive seedlings and 2 mutants.3.Site-directed knocked out TDR and PTC1 by CRISPR/Cas9 to create male sterility lines.We have designed three targeted sites of TDR and two targeted sites of PTC1.And constructed CRISPR/Cas9 plant expressed vectors,which were transformed into Oryza sativa L.japonica.cv.Nipponbare mediated by Agrobacterium strains LBA4404.The deletion or insertion mutants have been found in both targeted sites of PTC1.No mutants have been detected in transgenic seedlings of TDR gene targeted site4.Vector construction of Maintainer line.We have constructed plant expression vector containing wide-type PTC1 male fertility gene and pollen lethal gene.We still need to add a selection marker gene in subsequent experiments.The vector we constructed will be the foundation for the completion of the artificial male sterility system.