Analysis Of Tissue Specific Expression Genes And Identification Of Leaf-specific Promoter From Litchi

Transgenic breeding, which has the advantage of target-improved properties and short period, is an important technology and a potential way to genetic improvement of litchi. The promoter is the core component of vector that mediates the expression of foreign genes in certain tissues. The vector with the tissue-specific promoter can regulate the expression of exogenous genes in a specific tissue and has more particular uses. In this study, the tissue-specific expression genes were identified by comparing the transcriptome of root, leaf, peel, and seed from litchi. The promoters of two leaf-specific expression genes were cloned and integrated with LcTLP gene. After the construction of plant expression vectors confirmed by sequence, the expression profile of two promoters were investigated using transient expression with particle bombardment and further studied in transgenic seedlings obtained by the method of pollen tube pathway. The main results of this work were described as follows:1. A comparative analysis of transcriptome data has been carried out, and 2072 tissue-specific expression genes of litchi were identified. There are 1123 leaf-specific expression genes,556 root-specific expression genes,98 peel-specific expression genes, 128 pulp-specific expression genes and 167 seed-specific expression genes, respectively. The expression of several genes were confirmed by real-time quantitative PCR.2. Two promoters of leaf-specific expression genes, LcFKBP16-2 and LcGRX, were cloned by the method of PCR. There were a lot of structure similarities between the two promoters, such as both of the two promoters contained a large number of CAAT-box,TATA-box and elements relate to light response, revealed by the bioinformatic analysis on the sequences of the two promoters. Both the two promoter sequences contain many other cis-regulatory elements.3. Two plant expression vectors, named pCAMBIA1304-FKBP16-2-TLP and pCAMBIA1304-GRX-TLP, were constructed by replacing the CAMV 35S promoter of the basic vector pCAMBIA1304 with the promoter of LcFKBP16-2 and LcGRX, respectively. A thaumatin like protein gene(LcTLP), which was regarded as a disease-resistant related gene, was also integrated in the two plant expression vectors. The expression characteristics of these two expression vectors were investigated with particle bombardment. The results showed that these two promoters could regulate the expression of GUS gene in leaf, root, peel and seed of litchi, but not in pulp.4. Several seedlings of 'Ziniangxi'and'Baitangying' litchi were obtained by transfering the two vectors via pollen tube pathway. Only five positive plants of 'Baitangying'containing the pCAMBIA1304-FKBP16-2-TLP were identified.