| Maize?Zea mays L.?is the largest food crop which can be grown in a wide spectrum of soil and climatic conditions.In China,about 10%of 67 million hectares of arable land is salinized,while maize is moderately sensitive to salt stress:when salinity is more than 17 mmol·L-1,50%of the plant growth will be inhibited,salt stress can also lead to severe yield reduction.Therefore,It is of great importance to elucidate maize response to salt stress and resistance mechanisms for maize salinity resistance improvement.However,maize is a glycophyte characterized by very complicated mechanisms in salt tolerance,and salt tolerance in maize usually exhibits a quantitative genetic trait controlled by polygenes.Little progress has been made in maize salt tolerance improvement by using conventional breeding or molecular breeding methods,therefore,utilization of salt-tolerant gene resources in wild plant by foreign gene introgression or genetic modification are becoming an essential approach.At present,there have been some reports on improvement of salt tolerance of maize by transgenic method.The common way to create salt-tolerant transgenic plants is to select a few genes which are probably related to salt tolerance based on our knowledge of the salt tolerance mechanism of plant for gene manipulation.It usually takes years to obtain the transgenic plants,however,the target genes of transgenic plants often do no produce obvious phenotypes,or indeed sometimes unexpected results.There are a lot of disadvantages such as long period,low efficiency and that can not break through the frame of the existing knowledge.In order to screen and utilize salt-tolerant gene resources in wild plants rapidly and efficiently,the glycophyte Lophopyrum elongatum L.and a C4 plant Spartina anglica Hubb were employed as plant materials in this experiment,the total RNAs of each species were extracted from the seedlings treated with different concentrations of NaCl or PEG-6000 for different days and mixed stoichiometrically,and the double-strand cDNAs were synthesized by using modified SMART?switching mechanism at 5' end of RNA transcript?technology,and then,the cDNA libraries were inserted into a transgenic dual vector of pMDC83 by using the Gateway???method,and finally,the cDNA libraries of Lophopyrum elongatum and Spartina anglica Hubb were introduced into maize or tobacco BY-2 cells.A rapid screening and mining technology platform for salt-tolerance genes from cDNA libraries was preliminarily established.The main results are as follows:Lophopyrum elongatum seedlings were treated with 100 mmol·L-1,200 mmol·L-1,300 mmol·L-1,and 400 mmol·L-1 of NaCl and 20%?w/v?,30%?w/v?,50%?w/v?of PEG-6000 for 1,3,5,7 days respectively.Spartina anglica Hubb seedlings were treated with 5%?w/v?NaCl for 1,3,5,7 days respectively.RNAs were extracted separately and mixed equally for each species.The full-length cDNAs were synthesized by using a modified SMART?switching mechanism at 5,end of RNA transcript?method,and the mixed full-length entry cDNA libraries of Lophopyrum elongatum and Spartina anglica Hubb were constructed by BP reaction in Gateway technology,the titers were 4.0×106 cfu·mL-1 and 4.3×106cfu·mL-1 respectively,with an average of 0.75 kb insert fragments and 98.9%of recombination rates.The entry cDNA libraries were subsequently shuttled into the plant transgenic binary vector of pMDC83 by LR reaction to obtain the destination libraries.The original titers of the destination libraries were 6×106 cfu·mL-1 and 5.7×106 cfu·mL-1 respectively,the average length of insert fragments was 0.45 kb and the recombination rates were 100%.Five positive clones were randomly picked from each of two destination libraries for insert DNA sequencing,the resulted sequences were searched against NCBI non-redundant protein sequence database?nr?using BLASTx?http://blast.ncbi.nlm.nih.gov/?.Sequence alignment results showed that three cDNAs from Lophopyrum elongatum are highly similar to bZIP transcription factors,cell wall-associated hydrolysis enzymes and senescence-associated proteins,but no significant matches were found for the rest 7 sequences.The transgenic T0 generation maize seeds were obtained by pollinating the germinated pollens infected by Agrobacterium carrying Lophopyrum elongatum and Spartina anglica Hubb cDNA libraries.The foreign genes were proved to be successfully introduced into maize by observation of seed embryo GFP fluorescence signal coupled with PCR amplification of the marker gene of hygromycin and target genes.The Lophopyrum elongatum library was also transformed into tobacco BY-2 cells through co-incubation of Agrobacterium and BY-2 cells.The transgenic salt-tolerant BY-2 cells were screened by 25 mg·L-1 homomycin coupled with 50,100 or 200 mmol·L-1 NaCl respectively.Total 169 salt-tolerant cell lines were obtained in 50 and 100 mmol·L-1,but none was found in 200 mmol·L-1 NaCl.Our investigation paved the way for rapid and large-scaled screening of the stress resistant genes at both plant level and cell level. |
PDF Full Text Request