| Our previous work has proved that the expression of TaSI1 and TaSC are induced by salt respectively. And both of them can improve the salt stress tolerance in transgenic Arabidopsis . Otherwise, through two-dimensional gel electrophoresis, TaSI1 was isolated from the salt-tolerant materials RH8706-49 . In this study ,consi- dering the close evolution relationship between rice and wheat , we over-expressed TaSI1 and TaSC respecyively in order to study their salt tolerance .At the same time , choosing pTCK303 as the vector ,we constructed pTCK303-TaSI1, and pTCK303- TaSC expression vector.We all know that homologous genes play the similar role in biological function. So we blasted by the NCBI website in order to search the homologous genes of TaSI1 and TaSC in rice. As a conseconce ,we found two homologous gene named OsSI and OsSC respectively. Furtherly, in the experiment we studied the function of OsSI and OsSC by RNA interference in rice .We used pTCK303 as the expression vector for RNAi experiment and selected the specific section of OsSI and OsSC gene as a cDNA fragment, which were named OsSIi and OsSCi respectively. Through twice connection, first being connected forwardly and then is reversely ,we finished the consruction of pTCK303-OsSIi and pTCK303-OsSCi expression vector.Through several process including the co-culture, select-culture and so on, we have got many transgenic plants of T0 generation by Agrobacterium-mediated transformation method. These transgenic lines as follows: pTCK303-OsSIi summed up to 27, pTCK303-TaS11 to 20, pCAMBIA 1300 ProTaSI1::GUS to 11, pTCK303- OsSCi to 14, pTCK303-TaSC to19 and pTCK303 to 4.Real time-PCR method was used to detect the transcripts of OsSI and OsSC mRNA interference lines respectively in the mRNA level. The result was that the two lines of OsSI gene, line i-3, i-4, fell 50%, 70% respectively in the mRNA level; three transgenic lines of OsSC gene, line i-1, i-2, i-20 decreased by 20%, 25%, 50% respectively. All of these showed that mRNA interference experiment was very successful. At the same time, we detected the transcription of TaSI1 and TaSC gene over-expression transplant by semi-quantitative RT-PCR, in which four transgenic lines of TaSI1 gene, OX-1, OX-2, OX-5, OX-9, increased significantly in mRNA level; three transgenic lines of TaSC gene , OX-10 increased significantly in mRNA level, but the OX-1and OX-2 rose less than OX-10 transgenic lines.Finally , We detected the functions of the transplants mentioned above in stress responses .The results of salt stress tolerance analysis showed that of three lines of TaSI1 over-expression transgenic plants, OX-2, OX-5, OX-9, growed well compared with the control, and the survival rate was higher about 20 percent than empty vector transplants; three lines of TaSC over-expression transgenic plants did not have obvious difference between OX-1, OX-2 and control transplant, but survival rate of OX-10 line was higher less than 20 percent than the control. At the same time, T1 generation RNAi transgenic plants of OsSI and OsSC gene showed that the two lines of OsSI, i-3 and i-4 growed significantly better than control, and the survival rate was lower in salt stress tolerance analysis ; three RNAi lines of OsSC, i-1,i-2,i-20, was significantly lower than that of control in survival rate .At the same time, histochemical localization analysis of TaSI1 gene promoter-GUS activities showed that the gene may express in the callus, stems, internode, leaf tongue, stamen, and the buds, but did not express in the roots and leaves. |
PDF Full Text Request