| Cucumber (Cucumis sativus i.)i originated in tropics and sub-tropics ,is a kind of vegetable which grows well only under temperature at about 25-32℃, their cold temperature is at the range of 10-12℃, they will stop growth when the temperature is under 10℃,and will be injuried by the temperature at about 5℃, seedlings can endure 1-2℃ cold temperature for steak after cold acclimation. There is no cold resistance material in the species, so, prominently improving the cold endurance can not be generated through general genetic hybrid breeding, the only method can be used is the gene engineering method through which other species' cold-regulated genes and cold-induced transcriptional factors(genes) can be transferred into cucumber, promote abundant of genes expression at the level of transcription and break down the constraint of plasmgerm deficientin the genetic group, provide, a new plasmgerm for cucumber chilling-tolerance breeding.P- RD29A is a kind of promoter which can be induced into expression by dehydration, plant hormone ABA, and low-temperature. Cor15a gene takes an important role in cold resistance of Arabidopsis thaliana, cold-induced transcriptional factors genes CBF3 can promote the expression of cold-regulated genes. In this paper, two plant expression vectors concerning chilling tolerance were constructed. pVCT2016 consists of RD29A-corl5a-Tnos expression box,pRD29A-CBF3 consists of RD29A-corl5a-Tnos and RD29A-CBF3-Tnos tandem expression boxes. Through Agrobacterium-mediated transformation, two expression boxes were transformed into cucumber genomics, and a new chilling tolerance cucumber was created.1. The construction of plant expression vector 1.1 The construction of expression vector pVCT2016:After digestion of clone vector pSAU1001 with restriction endonuclease Xba I, filled in the cohesive end with DNA polymerase I Klenow fragment, then, digested with Sad, after electrophoresis, the band of 426bps in 1% argose was collected, and the fragment was purified; the expression vector pRD29A-GUS-T was digestion with SmaI and SacI, and 13550bps fragment was collected and purified, and the two fragment was ligated with T4 DNA ligase, the ligated product was transformed into E coli strain XL1-BLUE, select the white colony in plate of LB including Kanamycin 50mg/L,and the positive colony was detected, and further identification of restriction endonuclease digestion performed. Exact the pVCT2016 from XL1-Blue, and the plasmid was transformed into LBA4404(pAL4404),and the plant expression vector was constructed.1.2 The construction of expression vector pRD29A-CBF3:In order to construct plant expression vector pRD29A-CBF3, two middle vectors was constructed. After double digestion of clone vector pVCT2012 with restriction endonucleases HindIII and EcoRI, two fragments of 2635bps and 1949bps produced and the band of 1949bps was cut from argarose and purified , pBS II KS+ digested with Hindlll and EcoRl, precipitation with ethanol, 12bp fragment was discarded, and 2849bp fragment was purified, ligated the two fragments with T4 DNA ligase, transformed into XL1-Blue, the middle vector pBS-RD29A-CBF3 was constructed. After pBS-RD29A-CBF3 was digested with BamHI/Cla I, purified the 1974bp fragment, pGEM-7Zf digested with BamHI/Cla I, purified the 2988bp fragment, ligated the two fragments with T4 DNA ligase, transformed into XL1-Blue, the middle vector pGEM-RD29A-CBF3 was constructed. pGEM-RD29A-CBF3 was digested into 2983bp and 1978bp two fragments with EcoRl, and the 1978bp fragment was purified, pVCT2016 was digested into linear DNA with EcoRI, and subsequent ligation was performed, the two fragments was ligated and transformed into Escherichia coli(XL1-BIue) and Agrobacterium tumefaciens (LBA4404/pAL4404) , and the plant expression vector pRD29A-CBF3 was constructed.2.Study on transformation of plant expression vectors into cucumber cotyledons2.1 Optimization of regeneration system on cucumber cotyledons genetic transformationOn the base of other people's anterior work, the cucum...
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