Research On Agrobacterium Rhizogenes-mediated IRT1Gene Transformation In Cd Hyperaccumulator Brassica Campestris L

ABSTRACT:PURPOSE:It iswidespread concerned thatheavy metal Cadmium (Cd) ishigh carcinogenicity. Soil and waterwouldbe polluted by Cd through industrial waste water and waste residue. Irreversible damageto animals and human beings could be caused by Cdvia the food chain. With the continuous development of biotechnology, it shows good application potentialusing plants for Cd pollution repair.There exist Cd hyperaccumulation plants in nature, which are able to survive in the environment with the certain concentration of Cd,immobilizingand uptaking Cd using their own metabolism mechanism. As it is known to all that Cd hyperaccumulation plants has some disadvantages like low enrichment coefficient, limited enrichment and so on, which influenced the practical application.With the development of genetic engineering technology,genetical modificationof Cd hyperaccumulation plantsis an important wayto promote the development of Cd pollution environment phytoremediation technology.Cd hyperaccumulationplants Brassica campestris L.was chosenfor genetically modificationdue to itswide distribution and easily availability. Our aim is toestablish the genetically modified Brassica campestris L. hairy root systemas the research platform for future genetically modificationof Cd hyperaccumulation plants.Methods:Agrobacterium-mediated technology was used to carry out the induction of transgenic Brassica campestris L. hairy roots.Leaf explant was obtained after asepsisculture ofseed. At the same time AgrobacteriumrhizogenesATCC15834was cultured and activated. The activated bacteria liquid and leaf explants were combined for co-culture, and then were transferred to MS medium containing cefotaxime sodium to remove the bacteria. Hairy roots would appear after10days.PCR was performed to check whether rol B gene has been integrated into the hairy roots tissue. Meanwhile, different infection time and different concentration of naphthalene acetic acid (NAA) was analyzed for the influence on hairy roots induction.Based on establishment ofhairy roots system, pRI101plasmid from Takara was used to transferforeign IRT1genes associated with Cd accumulation into ATCC15834strains. Leaf explants were infected by the modified ATCC15834strains and IRT1gene was transferred into hairy rootPCR was also used for check the existence of rol B gene andforeign IRT1gene in hairy roots tissue.Finallyhairy rootswith IRT1gene were used for Cd enrichment experiments. The hairy rootswith IRT1gene and wild type hairy rootswere cultured on solid MS medium containing different concentrations of Cd in darkness.Biomass of hairy roots,residual Cd in medium and Cd absorbed by hairy rootswould be measured after7days and14days for discussing the possibility of IRT1genes forCd enrichment in hairy roots.Results:Thefragment ofrol B gene (423bp) was successfully amplified through PCR, which means that rol B gene in Ri plasmid had been integrated into the hairy roots tissue of Brassica campestris L.Till now mere is no reports about the successful induction of Brassica campestris L. hairy root.Different infection time has influence onthe efficiency of hairy roots induction.In a certain range of infection time, induction rate is Gaussian distribution; infection time of4h had the highest induction efficiency. Adding NAA to medium could promote the growth of hairy roots. In the range of0-1.0mg/L NAA, the efficiency of the hairy root inductionincreased with the higher concentration of NAA.Recombinant plasmid IRT1-pRI101was built successfully. The transgenicIRT1hairy roots wereacquired by infection of recombinant ATCC15834strains. PCR showed423bp and471bp stripes, proving that therol B gene andforeign IRT1gene had been integrated into the hairy roots tissue.Experiments of Cd enrichment showed that hairy rootswithIRT1gene grew flourishingly than wild type hairy roots under different concentrations of Cd.With the increase of Cd concentration and incubation time,the content of Cd enriched by wild type hairy rootsand hairy rootswithIRT1gene also increased. Theenrichment content of Cd reached33.161±0.026μg per100mg hairy root and the absorptivity reached7.261%±0.006%in hairy roots withIRT1gene at14th day, increasing13.73%±.01%than wild type which the absorptivity reached6.384%±0.005%.The results provided the theoretical basis of using IRT1-transgenic regeneration plant for repairing Cd pollution environment in the future and established the reliable experimental system for study the mechanism of heavy metal adsorption through hairy roots.