Expression And Analysis Of Cold-Responsive CBF-like Genes In Citrus And Related Species Under Low Temperature

CBF (C-Repeat/Dehydration-Responsive Element Binding Factor) transcription factors play important roles in cold response network in plants, and induced expression of their coding genes can improve the tolerance to cold and drought in many species.In order to understand the CBF cold response pathway in citrus, the expression of CBF in trifoliate orange under low temperature and abscisic acid, and in navel orange under low temperature, high salinity and abscisic acid was studied in this work. For more information on citrus CBF genomic DNA and protein sequences, PtCBF, CsCBF, CgCBF and CjCBF were isolated from four citrus genomes, and analyzed by a series of bioinformatic tools. Furthermore, citrus CBF promoters, designated as pPtCBF, pCsCBF, pCgCBF and pCjCBF were also cloned and analyzed on cis-acting elements present in them. In addition, to gain more about activity of citrus CBF promoters, genetic trasformation was introduced to perform functional characterization, preliminary results were detected by normal PCR amplification. Main results show as follows: 1. Expression analysis of citrus CBF and related genes under stressesResults showed that citrus CBFs were responsive to low temperature, high salinity and abscisic acid. However, PtCBF was different from CsCBF in stress response network. PtCBF was up-regulated, while CsCBF was down-regulated by low temperature, and both were up-regulated by abscisic acid. Additionally, PtCBF might be a target gene of MYB15, and not directly related to CORc410 in trifoliate orange; CsCBF might be modulated by MYB15 only under high salinity, and control the expression of CORc410 under abscisic acid. Finally, MYB15 could interact with CORc410 in navel orange under low temperature.2. Isolation and bioinformatic analysis of citrus CBF genomic DNA fragmentsThere were no introns in citrus CBF genomic DNA; AP2 DNA binding domain and CBF signature sequences were highly conserved in citrus CBF amino acid sequences; Three antiparallel P-sheets and one a-helix were predicted in citrus CBF protein. Citrus CBF proteins were more likely to be localized in nucleus, and regulated the expression of downstream genes. According to comparison analysis at nucleotide and amino acid level, there were no obvious deffereces among four citrus CBFs.3. Isolation and analysis of citrus CBF promotersCitrus CBF promoters shared 90% of nucleotides with each other. Transcription start site was possibly located at 185 bp away from start codon. Promoter element analysis by PLACE and PlantCARE indicated that there were several common stress-related cis-acting elements present in citrus CBF promoters, such as ABRE, MYBR, MYCR, G-Box, W-Box, circadian clock-responsive elements, and so on. However, one LTRE were detected in pPtCBF, while CBFHV in other three citrus. Two promoter sequences were amplified from rough lemon; two conseved regions and one variable region with many special motifs were observed in citrus promoters.4. Genetic transformation and functional characterization of citrus CBF promotersDuring genetic transformation, contamination was very serious, especially in the course of examination with GFP. Preliminary results showed that 2 tobacco plants with pPtCBF, only 1 with pCsCBF,3 with pCgCBF, and 7 with pCjCBF_s were positive, examined by PCR amplification.