Cloning And Functional Analysis Of The Promoter Of PtCBF1, A CBF1-Like Genes From Poncirus Trifoliata(L.) Raf

Citrus ranks the number one in the world fruit production.Many citrus cultivars are susceptible to cold damage.Thus,creating a cold tolerance variety is one of main objections for citrus breeding researchers.Modest improvement of the freezing tolerance of commercial citrus cultivars will potentially save growers millions of dollars in tree losses.However,the process of conventional genetic breeding is hampered because of pollen/ovule sterility,nucellar polyembryony,genetic heterozygosity,wide sexual incompatibility and long juvenility in most citrus cultivars.Fortunately,with the development of genetic engineering,a new strategy is provided to improve citrus cold resistance.Many attempts of genetic modified citrus for cold tolerance have been done in the past few years,yet it has met with limited success.The main reason to the fact is that the mechanism for cold resistance in plant,especially in citrus,has not been understood deeply.In addition,in the earlier stage,the single foreign gene expression driven by constitutive promoters had also affected the availability of transgenic varieties.Thus,the utilization of promoter in response to low temperature and profound understanding of the mechanism of cold resistance in citrus is a guarantee of success in citrus cold engineering.The main purpose of the present research is to isolate the promoter of PtCBF1,which is an important gene in cold-response pathway in Poncirus trifoliata,an extremely cold-tolerant interfertile Citrus relative,investigate their performance in transgenic Poncirus trifoliata and evaluate their potential applications in citrus and plant gene engineering.Moreover,based on the research,the special cis-acting elements in PtCBF1 promoter are studied,to find the foremost functional element which decides the promoter activity,and to obtain further knowledge about the molecular mechanism involved in citrus cold tolerance.The main results achieved are as following:1.The PtCBF1 DNA sequence of coding region was amplified from Poncirus trifoliate genomic DNA with utilization of a pair of primers designed based on PtCBF1 cDNA sequence.It was observed that the gene didn't contain intron,which was the same as its homologue in other species,and it did not retain the general characteristics of eukaryotic genes.2.1115 bp upstream sequence was isolated via two step chromosome walking based on TAIL-PCR and SON-PCR.Then a PCR analysis was performed to verify the exact upstream sequence of PtCBF1.3.Bioinformatics analysis of the upstream sequence was performed and the essential cis-acting elements for the promoter were found,furthermore,various potential environmental stress response CAREs were identified,including 7 ABREs,3 HSEs,7 MYB recognition sites,16 MYC recognition sites,8 G-boxes,5 Ca²⁺ responsive elements,2 auxin responsive elements,5 GA responsive elements,several light responsive and tissue-specific elements and so on.In addition,three kinds of statistically significant motifs were predicted by MEME.4.The promoter was fused to GFP gene,and the construct was introduced into onion epidermal cell by gene gun transduction and into epicotyl segments of P.Trifoliata via Agrobacterium-mediated transformation.The results of fluorescence detection showed that the promoter worked under cold stress,however,further experiments were needed to confirm its cold-induced activity.5.To gain further insight into citrus gene regulation under low temperature,we performed partial deletion and gain-of-function analysis of the PtCBF1 promoter.The results revealed that a sequence between-555 to-355 nucleotides from the translation start site had a great impact in the function of the promoter,and the three MYC recognition sites in this region may played an important role.It's highly possible that an ICE1-like bHLH protein activated by cold signaling recognized the element to induce the activity of the promoter and the expression of the downstream gene in trifoliate orange. Thus,it is speculated that a similar cold-response ICE1-CBF pathway of Arabidopsis existed in citrus,at the same time some citrus-specific cold-response regulations were also existed.