| Citrus is one of the most important fruit trees in the world. Rootstock is the basis of fruit trees.The root system is a key function of organs in absorbing nutrition, water, inorganic salts and so on. Meanwhile, it is also the direct defense system against pathogen, drought, and heavy metals. Therefore, root has important biological significance in plant growth and yield. In the model plant, Arabidopsis thaliana, many of the root-specific genes and regulatory sequences have been cloned and analyzed. But in citrus, the research about root-specific genes and promoters was few. Therefore, It has important research value to clone and analyze the root-specific genes and promoters in order to obtain directionaly modified plant. This is also one of the hotspots in the citrus genetic engineering.Trifoliate orange(Poncirus trifoliate) is the most important citrus rootstock in China. This study screened singleton 297 and contig 22, two root-specific expression unigenes from a suppression subtractive hybridization (SSH) library of citrus root which was successfully constructed using roots (Tester) and leaves (Driver) of the citrus trifoliate. The two unigenes showed a high identity with the contig2 from the trifoliate orange root full-length cDNA library. Using bioinformatics, molecular biology and transgenic technology, we cloned and analyzed the contig2 gene and its promoter. The main results are as follows: 1. contig2 organization expression analysisContig2 had a high identity with the 23 ESTs of the Citrus Functional Genomics Project (CFGP). And these 23 ESTs sequences mostly came from the citrus root cDNA library.Using the root and leaves of 1-month-old seedlings of trifoliate orange, 6-month-old seedling and 20-years adult trees, we knowed that the contig2 was a root-specific expression gene for the expression levels of contig2 in root higher than in leaves (46.34,74.82,110.25 times respectively) by real-time PCR.2. contig2 genetic characteristicsThe full-length contig2 fragment has 801bp, Containing an intron (104bp) and two exons. Its open reading frame has 471bp and 3’untranslated region containing polyadenylation signal AATAAA.3. contig2 encoded protein characteristicscontig2 encode major latex-like protein (MLP-like protein), with bet_v_I functional domains. Its contains 156 amino acids, with the molecular weight 17.63 kDa and isoelectric point 5.49. The secondary structure of contig2 protein had threeα-helices and sixβ-sheet. Tertiary structure prediction revealed that it had a hydrophobic binding sites and a glycine-rich loop structure.MLP protein had the function of the absorption of water and ions, playing an important role in drought, salt tolerance in plant.4. contig2 promoter cloning and analysisThough a homologous cloning and twice YADE-PCR, the contig2 the 5’upstream 1685bp regulatory fragments were cloned, which had the core components of promoter structure(TATA-box and CAAT-box).5. contig2 promoter specific expression analysisThe expression vector pBI121-2P was constructed using contig2 promoter and pBI121. it was Transformed into Agrobacterium tumefacien. The dragon trifoliate stem segment was transferred through agrobacterium method, and a total of 21 transgenic seedlings were obtained. GUS histochemical results showed the transgenic plants expressed blue color in roots, and no blue color in leaves. Analysing five transgenic seedlings by Real-Time PCR, the results showed that the expression levels of gus gene in the trifoliate orange transgenic plants root were higher 11.53,7.76,6.02,124.78, 5.56 times than in leaves respectively. This indicated the contig2 promoter had a root tissue specificity. |
