| Periodic freezing injury occurred frequently in the worldwide, which brings about gigantic economic losses. Introduced cold tolerance genes into citrus through transgenic technique can shortened the breeding cycle, and obtain the cold tolerant citrus cultivars quickly. However, there were few cold tolerance genes had been cloned and identified from citrus, which limiting the development of breeding cold hardy citrus cultivars through genetic engineering.In this research, three important genes in cold responsive gene expression pathway were cloned from citrus cold hardy relative trifoliate orange (Poncirus trifoliata (L.) Raf.), which were named PtrICE1, PtrHOS1 and PtrLOS2 respectively. These genes were analyzed in detail by bioinformatics. The space-time expression patterns of these genes after different stress treatments were also detected in this research. In addtion, the function of PtrICE1 which encodes an upstream transcription factor was identified through transgenic technique. Overexpressed PtrICE1 in tabacco could improve the cold tolerance of transgenic tabacco seedings. Moreover, the upstream promoter sequence of PtrICE1 was also cloned from trifoliate orange, and the cold and wounding responsive activity of PtrICE1 promoter was proved through transgenic technique. This research provided novel genes and theoretic bases for obtaining cold hardy citrus cultivated varieties through genetic engineering and transgenic technique. The main results of this research were as follows:1. A bHLH (basic Helix-Loop-Helix) family gene PtrICE1 was cloned from trifoliate orange. The length of PtrICE1 was 1933 bp with an open reading frame of 1464 bp, encoding a protein of 487 amino acids with a molecular weight of 53.6 kDa and a theoretical isoelectric point of 5.30. PtrICE1 protein shared high identity with other plants ICE1 family proteins and had conserved SUMO (small ubiquitin-related modifier) protein conjugation motif, bHLH domain and C-terminal region. The analysis results of phylogenetic tree revealed that the plants ICE1 famliy proteins could be divided to dicotyledonous plant group and monocotyledonous plant group. PtrICE1 protein belonged to dicotyledonous plant group.2. The PtrICE1 was constitutively expressed in leaves, stems and roots of trifoliate orange under normal growth condition. After cold treatments, the PtrICE1 expression in leaves and stems was up-regulated first and down-regulated afterwards. However, the PtrICE1 expression in roots was down-regulated first and up-regulated afterwards after cold treatments. After ABA treatments, the PtrICE1 expression in leaves, stems and roots was up-regulated first and down-regulated afterwards. These results suggested that the PtrICE1 expression in different organs of trifoliate orange was influenced by cold and ABA treatments.3. The overexpression vector pMVICE1 of PtrICE1 was constructed and transformed to tabacco through Agrobacterium tumefaciens mediated leaf-disc method. The transgentic tabacco seedings overexpressed PtrICE1 were detected by PCR and 17 positive T0 generation transgentic tabacco seedings were obtained in this research. The T0 generation transgentic tabacco growed slower and flower later (delayed about 10 d) than wild type tabacco. After flowering, there were no obvious phenotype difference between T0 generation transgentic and wild type tabacco seedings.4. T0 generation transgentic tabacco seeds were collected to produce T1 generation transgentic tabacco seedings by Km resistance selection. After cold treatments, the T1 generation transgentic tabacco seedings have lower relative electrical conductivity and higher survival rate compared to the wild type. Moreover, the growth of T1 generation transgentic tabacco could recover after normal temperature treatment. These results suggested that the transgentic tabacco seedings had stronger cold tolerance than wild type. Thus, we concluded that overexpressed PtrICE1 in plants could improve plants cold tolerance.5. The PtrICE1 DNA sequence was also cloned in this research. Four exons and three introns were found in PtrICE1 DNA sequence. The PtrICE1 promoter sequence was cloned by pMD18-T mediated adapter PCR. Several potential stress tolerance related cis-elements were predicted to be involved in the PtrICE1 promoter using the PLACE Program, including cold responsive elements (CRT/DRE, MYC), light responsive elements (GT1, GATA), dehydration and ABA responsive elements (ABRE, MYB) and several auxin responsive elements (ARF, ASF, GARE, WBOX).6. The pBIPROICE1:GUS expression vector used to detected the PtrICE1 promoter activity was constructed in this research. The pBIPROICE1:GUS vector was transformed to tabacco through Agrobacterium tumefaciens mediated leaf-disc method and obtained positive T0 generation transgentic tabacco seedings by PCR. The results of GUS histochemical staining revealed that the leaves of wild type tabacco could not be blue stained. Before cold treatment, a little of blue spots were observed in the leaves of T0 generation transgentic tabacco but the cut of which were deep blue stained. After cold treatments, more blue spots were observed in the leaves of T0 generation transgentic tabacco and the cut of which was also deep blue stained. These results suggested that the PtrICE1 promoter has cold and wounding responsive activities.7. The PtrHOSl gene was cloned from trifoliate orange. The length of PtrHOSl was 3434 bp with an open reading frame of 2922 bp, encoding a protein of 974 amino acids with a molecular weight of 110.2 kDa and a theoretical isoelectric point of 5.55. The amino acid sequence of PtrHOS1 protein shared high identity with that of other plants HOS1-like proteins and had a conserved RING finger domain in its N terminal. The plants HOS1-like proteins could be divided to dicotyledonous plant group and monocotyledonous plant group. PtrHOS1 protein belonged to dicotyledonous plant group. The PtrHOS1 was constitutively expressed at high level in leaves, stems and roots of trifoliate orange under normal growth condition. After cold and ABA treatments, the PtrHOS1 expression had several declined periods in leaves, stems and roots, which suggested that the PtrHOS1 expression in different organs of trifoliate orange were down-regulated both by cold and ABA.8. The PtrLOS2 gene was cloned from trifoliate orange. The PtrLOS2 cDNA is 1662 bp in length with a 1338 bp open reading frame, encoding a deduced 445 amino acid residue protein with a predicted molecular mass of 47.79 kDa and an isoelectric point of 5.54. The amino acid sequence of the PtrLOS2 protein shared high identity (over 86%) with Arabidopsis LOS2 protein and other plants enolase, which suggested that the PtrLOS2 probably encodes an enolase. Moreover, PtrLOS2 protein had a conserved DNA-binding and a repression domain. PtrLOS2 was constitutively expressed in leaves, stems and roots of trifoliate orange under normal growth condition. In addition, PtrLOS2 expression in roots and stems was much higher than that in leaves under normal growth condition. As a whole, the expression of PtrLOS2 was up-regulated first and down-regulated afterwards in leaves and stems, but down-regulated in roots after cold treatments. After ABA treatment, the expression of PtrLOS2 was up-regulated in leaves. However, PtrLOS2 expression in stems and roots was down-regulated first and up-regulated afterwards. These results suggested that the PtrLOS2 expression in different organs of trifoliate orange was influenced by cold and ABA treatments. |
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