| Cabbage(Brassica oleracea L.var capitata)is a variety of Brassica species in the cruciferous family.Cabbage is a typical cross-pollinated plant,the heterosis breeding mainly depend on its self-incompatibility.But it is difficult to guarantee 100% purity using self-incompatibility lines for seeds production,and the propagation of self-incompatibility lines depends on artificial bud pollination,which is inefficient and costly.In present,cabbage heterosis breeding has gradually shifted to the utilization of male sterile lines.Expanding the male sterile source and breeding male sterility mutant lines have become an important work for cabbage breeding.The pollen development regulatory network based AG(AGAMOUS)gene pathway in model plant Arabidopsis has been understanding clealy.MALE STERILITY1(MS1)is a key gene in the network,which encodes PHD-finger structural transcription factors involving the male sterility in Arabidopsis,napus,rice and barley.Except for participating in defense system,jasmonic acid plays an important role in the anthers dehiscence.At present,the heterosis breeding of cabbage by male sterility approach is mainly depending on ogura-CMS Cytoplasmic Male Sterility from radish,it is a potential risk for breeding.Therefore,it is necessary to enlarge the male sterile resources and breed male sterility lines of cabbage,and to overcome the self-incompatibility that hinder the reproduction of male sterility line.In this study,RNA interference technique and CRISPR/Cas9 technique were used to regulate and edit BoMS1 gene,BoAOS gene and self-incompatibility BoSRK gene,the aim is to develop a male sterility lines but compatibile with its maintainer.The results of this study are as follows:1.The vector pCA13BarPMS1MS1 for Arabidopsis thaliana ms1ms1 mutant complementary and a BoMS1 gene RNAi vector pCA13BarPMS1iMS1 were constructed.Total twelve T1 generation Arabidopsis thaliana plants of pCA13BarPMS1MS1 were obtained by inflorescence infection,three transgenicArabidopsis plants with ms1/ms1 background were genotyped,the transgenic plants showed normal pollens after flowering,the other phenotypes were not different from those of Arabidopsis thaliana with genotype MS1/ms1 and the pods were normally elongated and expanded,same as the phenotype of MS1/ms1.Seven transgenic PCA13BarPMS1iMS1 cabbage plants were obtained,one of them showed completely sterile,protruding stigma,short filament,hypertrophy anther and no pollen.Pollen was found in the other plants with decreased pollen vitality after 0.5% TTC staining.All these results indicated that the BoMS1 gene involves in the fertility regulation of cabbage.2.The plant expression vector pCA13BariMS1/iSRK for double interference of BoSRK gene and BoMS1 gene and the vector pCas-tBoMS1-ABCD/ tBoSRK-ABCD for edting of BoSRK gene and BoMS1 gene were constructed.Twenty-one pCA13BariMS1/iSRK transgenic plants were obtained,most of them showed degenerated anther and no pollen after flowering.Among of them,one transgenic cabbage had whitening pollen producing,but showed inactive after 0.5% TTC staining.Eighteen pCas-tBoMS1-ABCD/ tBoSRK-ABCD transgenic plants were obtained and 6individuals produced BoMS1 and BoSRK genes deletion simultaneously.The mutation frequency of the BoMS1 gene was 33.33%,BoSRK gene mutation frequency reached72.22%,and double mutation frequency was 33.33%.Except for one plants died because of disease infection,other 5 plants bloomed normally.At the early stage of low temperature(< 20?),the anther stopped growing before the petals opened,and the anther was aborted without pollen.When the temperature rose to 25 ?,anthers developed normally in 4 plants but accompanied decreased pollen vitality with varying degrees.However,there was one plant showed abortive anther and without pollen.The self-compatibility of pCA13BariMS1/iSRK and pCas-tBoMS1-ABCD/tBoSRK-ABCD transgenic plants(investigated by crossing with wild type at Blooming and bud stage)were examined by fluorescence microscopy observation.The results showed that the germination of pollen tube during bud stage was the same as that of wild type,but the germination of pollen tube during flowering stage was better than that of wild type.The results showed a promise way to produce a male sterility line but compatibile with its maintainer by the BoMS1 gene and BoSRK gene regulation.3.The plant expression vector pCA13BariAOS/iSRK for double interference of BoAOS gene and BoSRK gene and the vector pCas-BoAOS-AB/tBoSRK-ABCD for edting BoAOS gene and BoSRK gene were constructed to transform the hypocotyls ofcabbage.Nine pCA13BariAOS/iSRK transgenic plants were obtained.In order to investigate the interference effect of BoAOS gene,the BoAOS and BoOPR3 genes of the synthesis and metabolic pathways of jasmonic acid in two transgenic plants were analyzed by RT-PCR before and after soft rot disease.The results showed that both the expression of BoAOS gene and BoOPR3 gene in wild-type plants increased after inoculation,but the expression of the two genes decrease after inoculation in transgenic plants.,indicated an effect interference for BoAOS gene.But normal anther development was observed in all plants with varied degree pollen vitality decreased after flowering.In adition,increased compatibility at flowering stage in some transgenic plants were found by fluorescence microscopy.Total 50pCas-BoAOS-AB/tBoSRK-ABCD transgenic plants were obtained,and 7 of them produced BoMS1 and BoSRK genes deletion.The mutation frequency of the BoAOS gene was 14.00%,BoSRK gene mutation frequency reached 86.00%,and double mutation frequency was 14.00%.In the early stage of flowering,pollen dispersal was abnormal,and the pollen viability was lower than that of wild type,the pollen dispersal was normal at the later stage,but there were still differences in pollen viability among different plants.The self-compatibility pCas-BoAOS-AB/tBoSRK-ABCD transgenic plants was examined by fluorescence microscopy observation.The results showed that pollen tube germination at flowering stage was stronger than that in wild type plants. |
PDF Full Text Request