Cloning And Analysis Of CBF-like Genes From Citrus

As a subtropical fruit, citrus is restrained by low temperature on its cultural area. Natural recurrent low temperature often induces decrease of fruit productivity, decline of fruit quality, and even causes death of tree, all of which may decrease economic and ecological profit greatly. So, avoiding or protecting citrus from low-temperature damage is an important problem on enlarging the cultural area and increasing the profit of citrus production. Main measures on solving this problem are improving field management, selecting or cultivating novel varieties with high tolerance to low temperature. At present, genetic engineering becomes the main trend of plant breeding. Cold tolerance is a complex quantitative trait of plant which is a result of cooperation of many related genes. As a result, single gene transformation of plant can only enhance cold tolerance in a very limited degree. Being a key factor in regulating the transcription of a series of genes related to cold acclimation, CBF (CRT/DRE binding factor) is induced by low temperature rapidly and improve transcriptional level of these genes by binding to CRT/DRE (C-repeat/dehydration responsive element) specially, resulting in the synthesis of saccharide, proline, proteins contributed to the stabilization of biomembrane, and some other substances related to cold resistance, thus enhance the cold tolerance of plant. Further studies on gene transformation testified that the cold tolerance of plant containing high expressed foreign CBF gene is improved greatly. This suggests a new way of improving cold tolerance of plant by genetic engineering technique.In this study, core AP2 fragments of CBF-like genes from trifoliate orange (Poncirus trifoliata (L.) Raf), navel orange (Citrus sinensis (L.) Osbeck.) and lemon (Citrus Limon (Linn.) Burm. f.) were isolated to testify whether there are also CBF existing in citrus on controlling of cold acclimation. Further cloning the full-length cDNA of CBF-like genes from trifoliate orange, which is the most cold-tolerant species of citrus, and corresponding bioinformatical analysis were also carried out to pave the way of improving cold tolerance of citrus by using these CBF-like genes. The major results are as following:1. Guanidinum thiocyanate, Trizol reagent and hot phenol extracting methods were tried to extract total RNA from leaves of citrus. Method using hot phenol extraction and LiCl precipitation was identified as the best one. Total RNA extracted by this method was most intact with least contaminant of DNA, protein and polysaccharide. 2. Four degenerate primers were designed according to conserved domain of CBF from Arabidopsis thaliana and some other plants, the best pair of which was picked out for further RT-PCR amplification. AP2 fragments of a CBF-like gene (CBFa gene from citrus) were cloned from trifoliate orange, navel orange and lemon respectively, and another AP2 fragment of a different CBF-like gene (CBF1 gene from trifoliate orange) was also cloned from trifoliate orange. Sequence analysis by BLASTN algorithm showed that the four fragments had high homology with CBF from many other plants. Both AP2 fragments of CBFa genes from navel orange and lemon were the same in nucleotide sequence, while had little difference from AP2 fragment of CBFa gene from trifoliate orange at several loci.3. The 5' and 3' cDNA ends of CBFa and CBF1 genes from trifoliate orange were amplified respectively using nested PCR according to SMART RACE technique, with pairs of nested primers which were designed based on the two AP2 fragments from trifoliate orange. After confirmation by sequencing, the full-length cDNA of CBFa and CBF1 genes from trifoliate orange were obtained by PCR method with a pair of primers located in 5'-UTR and 3'-UTR or enzyme cutting and connecting method successfully.4. GenBank, ExPASy, EMBOSS, Jalview and some other databases and softwares were used to analyze the two CBF-like genes from trifoliate orange. The result showed that both of them possessed signature sequences of CBF, nuclear targeting sequence, conserved AP2 DNA binding domain and potential acid domain, which was the typical structure of all CBF genes. Besides, some other loci such as N-myristoylation site, Tyrosine sulfation site, Amidation site, and phosphorylation sites of protein kinase C and casein kinaseâ…¡were also conserved in both CBF-like genes. It predicted that these loci might be involved in process after translation and regulation of activity, thus concerned with its normal function.