Studies On Screening And Building Up Of Regeneration System In Winter Wetlands Plant And Constructing Vector Of Acds Gene

At present, most of the wetland plants applied in subtropical regions will die or will be cessation of growth in winter, and the prevalence of small biomass resulting in total lack of absorption. In this paper we study some plants which are safe in winter and have a certain capacity of decontamination among 25 kinds of wetland plants, and establish cress and German iris regeneration system, and at the same time construct ACC deaminase (ACDS) gene expression vector. The main findings are as follows:(1) To establish a scientific evaluation system for screening wetland plants and their purification potential, we studied all index by cluster analysis, combined with adverse circum stance enzyme and matrix enzyme activities from 25 species of wetland plants. The results showed that the 25 plants differed in their root quantities, root length, vigor, leaf peroxide enzyme activity, biomass quantity, average concentration of nitrogen and phosphorus, and ability to accumulate nitrogen and phosphorus. Also the matrix urease activities and phosphoric acid enzyme activities from the 25 plants'root systems were different. According to the comprehensive evaluation of potential purification system we can get the 6 major categories of clustering for 25 kinds of plants, from strong to weak including Oenanthe clecumbens., Var. capitata Linn., Juncus effusus, Saxlfraga stolonlfera(L.)Meerb., Iris gevmanica,Osmanthus fragrans and other 19 plants.(2) built up and optimizied of regeneration system in Iris gevmanica and Oenanthe javanica, which had strong purification potential.1) Iris gevmanica:To acquire asepsis seeding by the embryo of seed in the Iris gevmanica explant, the best medium for inducing callus induction was MS+6-BA0.5mg·L-1+NAA0.2mg·L-1+2,4-D0.1mg·L-1, the callus induction rate reached 60.5%, and the callus were developed by 49 days every time. The best medium for callus subculture was MS+6-BA0.5mg·L-1+NAA 0.2mg·L-1,in this process, adding 100mg·L-1 active carbon, polyvinylpyrro-lidone (PVP), sodium thiosulphate and vitamin C ...etc. And also could inhibit browning phenomenon, especially active carbon.The best medium for callus differentiation was MS+6-BA 1.0mg·L-1+ NAAO.1mg·L-1, The best medium for rooting was 1/2MS+IBA0.5mg·L-1+NAA0.5mg·L-1, the average length was 4.25cm. The survive rate of the first class rooting plantlets could reach 95% when they were transplanted to the medium composed with one part field soil and one part peat and one part river sand or three parts perlite and sever parts peat.2) Oenanthe javanica:For the growing points of stem tip in the Oenanthe javanica explant, the best medium for inducing callus induction was MS+6-BA 0.5mg·L-1+NAA1.0mg·L-1+2,4-D0.5mg·L-1, the callus induction rate reached 67.5%, the callus were developed by 40 days every time.The best medium for callus subculture was MS+6-BA0.5mg·L-1+NAA 1.0mg·L-1,in this process, adding 100mg·L-1 active carbon, polyvinylpyrro-lidone (PVP), sodium thiosulphate and vitamin C ...etc. And also could inhibit browning phenomenon, especially active carbon.The best medium for callus differentiation was MS+6-BA1.0mg·L-1+NAA0.1mg·L-1, The best medium for rooting was 1/3MS+IBA0.6mg·L-1+NAA0.05mg·L-1, the average length was 4.45cm. The survive rate of the first class rooting plantlets could reach 95% when they were transplanted by the medium composed with following parts:one part stove ash slag, one part vermiculite,one part silver sand or two parts perlite and three parts vermiculite.(3) Constructed expression vector of ACDS (ACC-deaminase). Genomic DNA from Enterobacter cloacae UW4 strain was isolated and purifiedby the modified mini-scale bacterial total DNA preparation method. A PCR product of1.02Kb was amplified from the total DNA of Enterobacter cloacae UW4 strain, using degenerated oligonucleotide primers designed based on some reported sequences of ACDS from different microorganisms. The product was recovered and ligated into pCR-2.1-TOPO vector and sequenced. The sequencing data indicated that the PCR productwas an intact open reading frame (ORF) with a 1017bp coding region that encoded 338amino acid residues.only eight nucleotide acids were different from the sequencereported in GenBank (AF047710), resulted from change of four sequencereported in GenBank (AF047710), resulted from change of four amino acid residues, showing 99.1% of identity in nucleotide acids. And we constructed the expression vector containing ACDS by using CaMV35S promoter.