| Longan(Dimocarpus longan Lour.)is a subtropical fruit tree,belonging to the family Sapindaceae which has significant economic value in China.Transcriptomic sequencing using grafted new shoots of special cultivar ’Sijimi’ which can flower continuously and common cultivar ’Lidongben’ was finished before,and different expression genes between these two cultivars,including EARLY FLOWERING 4(ELF4)and TERMINAL FLOWER 1(TFL1)were found.The full length of cDNA sequences of two ELF4 homolog genes and two TFL1 homolog genes were cloned,named DIELF4-1,DIELF4-2,DITFL1-1,DlTFL1-2.Based on the research,to further study the function of DlELF4 and DITFL1,these four genes were overexpressed in Arabidopsis thaliana and tobacco in this study.Also,the technology of VIGS(virus induced gene silencing)in longan was explored and DlTFL1-1 and DITFL1-2 were introduced into recombinant plasmid with virus vector Tobacco Rattle Virus(TRV)to investigate the gene’s function.The main results of this study were as follows:1.The recombinant plasmids of DIELF4-1 and DlELF4-2 genes were transformed into wild type Arabidopsis by Agrobacterium tumefaciens floral dip method.Transformed plants were selected to get the third generation homozygous plants.The wild type Arabidopsis was used as negative control and the phenotypic changes of transgenic plants under LD and SD conditions was observed.The results showed that in the LD conditions,on average,the flowering time of wildtype plants was 27 days,and the flowering time of 3 lines of DIELF4-1 transgenic plants were 38.6±1 days,35.3±2 days and 40.3± 1 days respectively.The flowering time of 3 lines of DlELF4-2 transgenic plants were 34.01± days,36.6±1 days and 34.3±2 days.In the SD conditions,on average,the flowering time of 3 lines of DlELF4-1 transgenic plants were 48.6±1 days,47.3±1 days and 56.0±3 days respectively.The flowering time of 3 lines of DIELF4-2 transgenic plants were 44.0±2 days,46.3±1 days and 51.6±2 days.At the same time,DlELF4-1 and DIELF4-2 transgenic plants showed adventitious roots formation and increase of the number of rosette leaves.In addition,the edge of the leaves was serrated in SD condition.It is speculated that DIELF4-1 and DIELF4-2 genes may have the function of delaying flowering and regulating auxin synthesis.Expression of AtGI,AtFKFl genes in transgenic plants were analyzed by real-time quantitative PCR.The results showed that expression of AtGI and AtFKF1 were up-regulated in the roots and leaves of DIELF4-1 and DIELF4-2 transgenic plants.Combined with the analysis of the gene expression in the flower bud differentiation of longan,it is speculated that DIELF4-1 may be the repressors of flowering and DIELF4-2 have function on regulating auxin synthesis.2.The recombinant plasmids of DITFL1-1 and D1TFL1-2 genes were transformed into wild type Arabidopsis by Agrobacterium tumefaciens floral dip method and the phenotypic changes of transgenic plants under LD and SD conditions was observed.The results showed that in the LD conditions,on average,the flowering time of wildtype plants was 27 days,and the flowering time of 3 lines of DITFL1-1 transgenic plants were 35.0±1 days,37.3± 1 days and 38.3±1 days respectively.The flowering time of 3 lines of DITFL1-2 transgenic plants were 32.0 ± 1 days,34.6 ± 1 days and 33.0±2 days.In the SD conditions,on average,the flowering time of 3 lines of DlTFL1-1 transgenic plants were 42.6 ± 2 days,45.3 ± 1 days and 46.0 ± 3 days respectively.The flowering time of 3 lines of DlTFL1-2 transgenic plants were 42.0±1 days,41.0±1 days and 43.2±1 days.In addition,DITFL1-1 and DITFL1-2 transgenic plants produced more branched inflorescences,but inflorescence structure did not change.The results of the comprehensive experiment indicate that DITFL1 gene may play an inhibitory role in flower bud differentiation by maintaining the vegetative growth of plants.3.DIELF4-1,DIELF4-2,DITFL1-1,DITFL1-2 genes were transformed into tobacco by Agrobacterium tumefaciens.DIELF4-1 and D1ELF4-2 plants showed the same delay-flowering phenotypes as ectopic overexpression in Arabidopsis.The result further demonstrated that DlELF4 genes may be the repressors of flowering.DlTFL1-1 and DITFL1-2 transgenic plants also showed phenotypes of delaying flowering,and the height of plants was taller obviously.Combined with the analysis of the gene expression in the flower bud differentiation of longan,the results suggested that DITFL1-1 may play a role in regulating juvenile period,while DITFL1-2 gene may inhibit flowering by maintaining vegetative growth of plants.4.The preliminary research on technique of virus induced gene silencing on longan was carried out.The TRV virus vector with green fluorescent protein(GFP)was used to infect N.benthamian tobacco leaves and roots of longan seeds.The OD600 value of 0.5,1.0,1.5 and the time under vacuum condition of 5 min,10 min and 20 min were selected by vacuum immersion method and injection method in longan injection.The results showed that after 10 days infection,green fluorescent on tobacco leaves and longan roots were observed,and the infection was verified by PCR analysis.It showed better result when the OD600 value was 1.5 and the time under vaccum condition was between 10 and 20 min.Also,the TRV(tobacco rattle virus)virus vector of longan DITFL1-1 and DITFL1-2 gene was constructed,which lay the foundation for further research of gene function. |
