| Cold resistance is an important agronomy property that a lot of vegetables and field crops need. Expressions of a series of its COR(cold-regulated) genes are promoted transcriptionally by transferring CBF(C-repeat banding factors) gene into plants then 1~2 of outside efficient COR genes are transformed, which is an essential direction of genetically improving of cold resistance in plants and an important approach in enhancing cold resistance of plants effectively.In this paper, three plant expression vectors concerning chilling tolerance were constructed. LBA4404(pBIN+-RD29A-GUS + pAL4404) was used to transform explant materials of cucumber and Micro-tomato, and the transient expression of GUS gene was detected, whether chilling inducing expression trait of dehydration and low-temperature inducing promoter P-RD29A are charactered in it was primarily discussed. Plant expression vector LBA4404(pBIN+-35S-CBF3 + pAL4404) was used in the genetic transformation of cucumber and Micro-tomato. Establishment of high efficiency regeneration system and determination of parameters involved in Agrobacterium-mediated transformation, screening and characterization of transformant were systemically researched.The main results are as follows:1. The cloning, sequencing and analysis of dehydration and low-temperature inducing promoter P-RD29AAccording to the sequence of the promoter fragment of dehydration and low-temperature inducing gene RD29A in Arabidopsis thaliana, two specific primers, P1 ( 5' -ctgcaagaatctcaaacacg-3' ) and P2 (5' -tccaatagaagtaatcaaacc-3' ) were designed. Target fragment P-RD29A from Arabidopsis thaliana Columbia DNA was amplified by PCR with high fidelity pfu-DNApolymerase.The PCR products were purified and cloned into pUCm-T vector then vector pP-RD29A was constructed. By white-blue screening, colony PCR and enzyme digest analysis, the result showed the plasmid containing P-RD29A fragment was transferred into E.coli XL 1-Blue.The sequencing of the single recombinant bacterium containing the inserted P-RD29A fragment was carried out by Baiasia Bio-engineering CO.Ltd. The results showed that the fragment was 890bp, nearly consistent to the sequence of P-RD29A isolated from Arabidopsis thaliana (D13044) .2. The cloning, sequencing and analysis of transcriptional activator CBF3 geneAccording to the sequence of transcription activator CBF3 in Arabidopsis thaliana, two specific primes, P3 (5'-agcaaaccataccaacaaaaa-3') and P4 ( 5'-gtactaaaaatggaaataataatctg ag-3') were designed. The same methods as the above 1 were used for cloning, sequencing and analysis.The constructed clone vector was defined as pCBF3 and sequenced by Sangon Bio-Engineering CO. Ltd. The results showed that the fragment was 760bp, encoding 216 amino acid and consistent to the sequence and amino acid of CBF3 isolated from Arabidopsis thaliana (AF076155) .3. The construction of middle vector and plant expression vectorAfter pP-RD29Aplasmid with target fragment P-RD29Aand vector plasmid pSAU2006 were double-digested by PstI+Bam HI,the target fragments were collected and purified respectively, after being ligated, middle vector pRD29A-GUS with RD29A-GUS fragment was constructed. Plasmid pRD29A-GUS was double-digested by Smal+Ecl136, the larger fragment was collected and purified, and then self-ligated of blunt end, middle vector pVCT2011 with RD29A-Tnos fragment was constructed. The target fragment CBF3 was obtained after plasmid pCBF3 having been digested by Bgll II, which correctly inserted into linetype vector that was obtained after pVCT2011 had been digested by BamHI, then middle vector pVCT2012 containing RD29A-CBF3-Tnos gene expression box was constructed. After plasmid pSAU2006 double-digested by BamHI+HindIII and pVCT2012 double-digested by BglII +Hind III, the CaMV 35S Promoter fragment and the larger fragment were collected and purified, then were ligated, middle vector pVCT2014 containing 35S CBF3-Tnos gene expression box was constructed.Linetype vector...
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