Effects Of Heterotopic Expression Of Populus Euphratica DWF4(PeDWF4) On The Growth,callus Formation And Salt Tolerance Of Populus Bolleana

Brassinosteroids(BRs) are phytohormones found in 1970 s playing important roles in many plant developmental events including cell elongation and division, photomorphogenesis, leaf and root development, fertility, fruit maturation, seed dormancy and germination, vascular tissues differentiation, senescence, biotic and abiotic stess tolerance, and so on. DWF4 is the gene of a key enzyme functioning in the speed-limiting steps of BR biosynthesis. For studying the mechanism of plant tolerance to abiotic stress and improving the plant character in growth and development and in abiotic stress tolerance, in this study, 1) Populus euphratica(P. euphratica)DWF4 gene(Pe DWF4) was introduced into Populus bolleana(P. bolleana) genome, and Pe DWF4 transgenic lines were constructed, and their phenotypes were analyzed; 2) Leaf of Pe DWF4 transgenic line, P. bolleana and P. euphratica were used as explant to generate calluses,and the reactions of three kinds of calluses to the hormones containing in medium were analyzed;3) the changes of expression level of BR biosynthesis enzyme gene DWF4, BR signaling transduction gene BZR1, Na+/H+ transporter gene NHX1 and Ca2+ binding protein gene CBL10 of three kinds of calluses under salt stress were analyzed. Results as follows:1) The leaf of P. bolleana was used as meterial, and PeDWF4 was introduced into the genome of P. bolleana via agrobacterium mediated method. Subsequently 4 transgenic line, TL3,TL4, TL6 and TL10 were obtained by Hyg selection and PCR identification. The transgenic lines were transfered from MS medium to soil. Compared with the wild type, the transgenic lines showed, in phenotypes, increased leaf size, decreased leaf angle, enlarged stem, accelerated growth of the internode. Meanwhile, the number of lateral root was reduced and the length of primary root was shortened compared with the wild type, among them, the phenotype of TL6 was the most significant.2) TL6, P. bolleana(WT) and P. euphratica leaves were set on the MS medium containing2,4-D(TL6 and P. bolleana) or 6-BA+NAA(P. euphratica) to induce calluses. These three kinds of calluses grew with similar texture and color, but compared with the WT, TL6 had the advancedcallus formation and increased growth rate.3) Culturing those three kinds of calluses on the mediums containing 0, 50, 100 and 150mmol/L NaCl respectively, when compared with contol(0 mmol/L), the decline of growth rate of P. euphratica/WT/TL6 calluses under 50, 100, 150 mmol/L NaCl were 1.7%/81.5%/45.6%,16.4%/91.1%/59.9%, and 58.2%/93.7%/85.2% respectively. This result showed that the TL6 was more tolerant to salt stress than WT, but less tolerant than P. euphratica.4) Compared with control(0 mmol/L Na Cl), RT-PCR analysis suggested that under the same salt stress(100 mmol/L Na Cl), the expresssion of DWF4 and NHX1 in P. euphratica, WT and TL6 were increased, and the expresssion of BZR1 was decreased, however the amplitude of upgrade or downgrade of those genes in these three kinds of calluses were varied but have a similar trend: P.euphratica>TL6>WT. Furthermore, the expresssion of CBL10 was increased in P. euphratica and TL6 calluses, and decreased in WT callus, while the expression level of P. euphratica callus was increased more than that of TL6.All the results above implied that heterotopic expression of Pe DWF4 could promote plant growth and development and increase the tolerance to salt stress through modulating BR biosynthesis, therefore it has potentially big application value to improve the characters of trees and crops.