Effects Of The Over-expression BoFLC3and AtFT Genes On Flowering-Time And Cold Tolerance In Brassica Juncea

Plant flowering is the transition from vegetative to reproductive development,it is one of the most important phase in the plant life cycle, the transition determine the flowering time and quality. So it is essential to study the molecular mechanism of plant flowering time, and it is also useful to regulate plant growth and development, increase crop yield and quality.Mustard (Brassica juncea Coss.) is a typical seed-vernalization plant, bloting is affected by low temperature and long-short daylight, the premature bolting occurs frequently in production due to unsuitable sowing time, environmental condition change or inappropriate varieties, that reducethe yield and quality of vegetative organs.In order to ensure flowering time of the male and female parent meet in breeding, avoid or reduce the chance of false cross, it is necessary to research and explore plant bloting and flowering time regulation by engineering method.FLC encodes a MADS box transcription factor that is expressed mainly in vegetative shoot apices and roots, and the FLC expression is affected by several genes; Moreover, the FLC is down-regulated by vernalization (a long period of near-freezing temperatures) and autonomous pathway genes. It had been proved that FLC inhibit flowering by suppressing a group of floral promotion genes termed’floral pathway integrators’. FT gene expression promotes plant early flowering, the FT protein interacted with a bZIP transcription factor FD, the FT/FD heterodimer induce the floral meristem identity gene APETALA1(API) and FRUITFULL (FUL) expression.. Before plant bolting and flowering, the expression of endogenous FLC and FT genes inhibit each other. In B. oleracea, during the process of vernalization, the expression level of FLC increase in the apical meristem but FT expression level down. The overexpression of exogenous FLC and FT has been widely proved to delay flowering or accelerate flowering respectively. However, there is no report that overexpress the exogenous FLC and FT genes simultaneously affect plant flowering time.In mustard breeding, it was valuble to regulate the flowering time meet of parentsby controlling the consistency of parents bloting, so it is necessary to study the mustard flowering and bloting. Therefore, this thesis studied the effect of long-day or short-day on flowering time,the effect of vernalization on flowering time and the relationship between flowering time and cold tolerance by overexpressing the BoFLC3and AtFT gene in mustard (Brassica Juncea Cross.). This study provide a reference for further research to prevent immature bolting, ensure flowering time meet, and mustardbreeding. The main results were as follows:1. The generation of transgenic mustard plants PCR idetification and morphological investigation of the transgenic plants.Plant expression vectors pCABarFLC, pCABarFT, pCABarRbcSFT were transformed into mustard respectively by agrobacterium-mediated transformation method. After several rounds selection on the medium with glufosinate PPT, transgenic plants of pCABarFLC, pCABarFT, pCABarRbcSFT were obtained. The transgenic plants DNA were extracted by CTAB method. The PCR results showed that the corresponding genes had been integrated into the mustard.Morphological investigation showed that the leaf have not obvious difference from wild-type plant. But pCABarFLC#2, pCABarFLC#5, pCABarFLC#6plants produced budding abortion phenomenon, the RT-PCR analysis revealed the BoFLC expression level of pCABarFLC#2,pCABarFLC#5,pCABarFLC#6plants were obviously higher than that of pCABarFLC#1, pCABarFLC#3, pCABarFLC#4plants.2. The herbicide resistance analysis of pCABarFLC, pCABarFT, pCABarRbcSFT transgenic mustard T1and T2generationThe T1and T2generation of pCABarFLC, pCABarFT, pCABarRbcSFT transgenic mustards were investigated the herbicide resistance by a treatment of 10mg·L-1PPT at germination and15mg·L-1PPT spraying of seedling respectively.The results showed that plants with herbicide resistance growth well, while the plants without herbicide resistance soon died.3. The effect of long or short days on flowering time of T1generation of pCABarFLC, pC ABarFT, pCABarRbcSFT transgenic mustard plants(1) The lowering time of T1generation of pCABarFT transgenic mustards was30.7days a in long-days(23℃,16h light\8h night), and the pCABarRbcSFT was32.1days on average,all of them were earlier than the WT. The flowering time of pCABarFLC×pCABarFT transgenic mustards was44.8days on average, the flowering time was near with WT plants. But the flowering time of pCABarFLC×pCABarRbcSFT transgenic mustards was52.1days,which were later than the WTplants and pCABarFLC x pC ABarFT plants.(2) In short-days (23℃,8h light\16h night), the flowering time of T1generation of pC ABarFT transgenic mustards was33.1days, earlier than the pCABarRbcSFT transgenic mustards and WT(wild type) with8days and15.1days respectively, and earlier than the pCABarFLC transgenic mustards with36.5days.4. The effect of vernalization on flowering time of T1generation of pCABarFLC, pC ABarFT transgenic mustardsVernalization treatment accelerated transgenic mustards flowering, the flowering time of pCABarFLC vernalizated T2generation was17.0days earlier than the non-vernalizated of transgenic mustards.And flowering time of vernalizated pC ABarFT T2generation was3.2days earlier than the non-vernalizated plants.5. The cold tolerance analysis of pCABarFLC, pC ABarFT transgenic mustardsThere was no obvious difference of T2generation seeds germination between pCABarFLC and pC ABarFT transgenic mustards under23℃. But the cold tolerance of pCABarFLC transgenic plants were higher obviously than that of pCABarFT transgenic plants and WT plants under lower temperature.