The Transformation Of BAN Genes By Agrobacterium Mediate And Its Expression Analysis In Alfalfa

Alfalfa is one of the most widely distributed perennial legume species in the world.It has been taken as the King of Forage for it’s excellent forge quality,rich nutrition and strong palatability.However,the ruminant feeding with overdose of fresh alfalfa would easily cause tympanites,even kill them,which was the main reson of limited utilize of alfalfa in grazing area.The present study found that low content of condensed tannins in alfalfa leaf is the major reason that caused ruminant tympanites.Therefore,the risk of tympanite can be reduced effectively by increasing properly condensed tannins content of alfalfa.So we think that the research on this issue has great meaning both in theory and reality.In this experiment,the gene of a key enzyme for condensed tannins biosynthesis was transformed into alfalfa genome mediated by agrobacterium.Besides,the activity of the key enzyme weas determined and the contents of condensed tannins were measured and analyzed for the T0 generation of alfalfa.It laid the foundation of the better alfalfa varieties for ruminant feeding.The concrete research content and the result brief elaboration are as follows:1.The establishment of efficient regeneration systems.Using the hypocotyls of Gannong No.3 alfalfa cultivar as explant,callus induction and differentiation and root culture were studied.Research shows that the optimal hormone ratio of callus induction was 3.0 mg·L^-12,4-D and1.5 mg · L^-1NAA.Callus induction achieved 84.67%.The optimal hormone ratio of callus differentiation was 0.5 mg·L^-1KT and 0.5 mg·L^-1NAA,the embryoid induction rate reached as high as 92.00%,and following budding rate also can reached as high as 81.52%;the optimal ratio for rooting was 1/2MS and NAA 0.5 mg·L^-1.Rooting induction can reached 86.00%.Using this system,the plant root would be longer and stronger,fibrous root also be flourish.This would be helpful to the transplant regeneration.2.Optimized the agrobacterium mediated gene transformation of BAN gene.Using the hypocotyls of Gannong No.3 alfalfa cultivar as explant and agrobacterium carryied with the CPB-BAN-GFP gvector as mediator.The optimized genetic transformation system is as follows:The suitable glufosinate-ammonium selection concentration was 2.0 mg · L^-1and the best selection time was during callus differentiation culture.The best antibiotic concentration was Cef300 mg · L^-1to obtain bacteria-free tissue.The best infection time is 15 minutes;the bestconcentration of bacteria suspension is OD600=0.6⁰.8;co-cultivation period was 3 days which was helpful for genetic transformation.3.The gene expression analysis of transgenic alfalfa.Based on the optimized gene transformation and regeneration system,8 lines of glufosinate resistant transgenic alfalfa have been screened initially.PCR detection demonstrated that 4 lines of resistant plant amplified the target gene of a 1020 bp band.Southern blot analysis confirmed the integration of BAN gene in the genome of transgenic alfalfa.RT-PCR analysis and q RT-PCR detection revealed that BAN gene is normally expressed in transgenic alfalfa at the m RNA level of transcription.4.Measurement of ANR activity in genetically modified alfalfa and the content of condensed tannins.The analysis showed that the activity of ANR was significantly increased than wild-type plants and condensed tannins content of only one plant has greatly improved.The ANR enzyme plays important role for the synthesis of anthocyanin.It has contributed to the purplish red on some of the leaves of the transgenic plant,which also demonstrated that BAN gene excessive expressed in transgenic plant.