Functional Analysis Of RgMT From Rice And Its Genetic Transformation In Alfalfa

Salination soil are the key factor of environmental crisis. So the research on the gene engineering will play extreme important function for the alteration to the salination soil.We have got the rgMT gene from the rice cDNA library,Research gene induced expression under several abiotic stresses and research its function in E.coli and Yeast. Regeneration and transformation system of Medicago sativa (alfalfa) was establishment. We introduced rgMT gene into Medicago sativa by gene engineering.It can enlarge the growth range of Medicago sativa which play important role on improvement environment,prevention the loss water and soil and enchencement the nutrition of soil.We previously isolated a gene from a rice root cDNA library whose expression imprved the carbonate tolerance in E.coli;The gene open read frame is 222bp.The deduced amino acid sequence of the gene is the same as that of an MT-like protein from rice (GenBank accession no. S57768).The rgMT gene expression was induced under several abiotic stresses from salts (NaCl and NaHCO₃), drought (10% PEG 6000) and heavy metal iron (CdCl₂, CuCl₂ and ZnCl₂) in rice leaves and roots. The results suggested that the rgMT gene was in response to environmental stresses. The rgMT gene can express and obtain purified protein in E. coli, The final yield of the purified rgMT protein was about 4.8 mg g^-1 dry cells. Concentrations of various metal ions that were suitable for comparing metal-ion tolerance of transformed and non-transformed E. coli were 1.6 mM CdCl₂, 4.7 raM CuCl₂ and 1.7 mM ZnCl₂. The metal-ion (Cd²⁺, Cu²⁺,and Zn²⁺) tolerance of E. coli cells expressing rgMT fusion protein increased. These results suggest that rgMT functions in E. coli cells.The tolerance of yeasts expression rgMT was analyzed to know relation between rgMT and stress. As yeast is eukaryote organism,it has the similar metabolizing route, rgMT was constructed yeast express vector, and induced for expressing protein, Analysis of the tolerance,transformant yeast expression rgMT protein increased compared with the control. The result indicated that the increased tolerance of yeast highly expressiong rgMT also enhanced plant expressing rgMT.The explant was cotylender of alfalfa (medicago sativa),and MS was regarded as the basic medium,the different combination of 2,4-D and 6-BA were added into the medium to induce the callus. The rate of inducement ranged from 55% to 90.6%, among of them 2mg/L 2,4-D +1 mg/L 6-BA medium is the best (90.6%).The highest differentiation medium was MS+2mg/L KT+0.3mg/L 6-BA+0.15mg/L NAA using callus whose color was yellow and green. The regenerated plants were obtained in the rooting medium (1/2 MS+ 2 mg/L Yeast extract). Theestablishment of plant regeneration system has made the foundamentation for the studies on genetic transformation in plant breeding.Establishment of genetic transformation system of Medicago sativa callus mediated by Agrobacterium tumefaciens was studied by GUS colinration analysis and a effective protocol for genetic transformation of alfalfa was developed on the basis of high frequency tissue culture regeneration system and the GUS transgenic plants were achieved. Several factors affected genetic transformation system were study. 100 transgenic plant were achieved,the concentration of acetosyringone was 100Mmol/L,bacterial concentration was OD6oo=0.3-0.5, the time of co-cultivation was 4 days,concentration of kanamycin was 50mg/L. The highest transformation frequency was 80%. Then Medicago sativa callus transformed by rgMT,Northern blot analysis using the Kana resisted plant showed that the gene was integrated into genome of the plants,36 transgenic plants were achieved.