Separation Of Differentially Expressed Genes And Full Length CDNA Cloning Of Selective Genes During Periodic Albinism In Anjibaicha(Camellia Sinensis)

Anjibaicha is an albino mutant tea variety which can inherit stably. The leaf color is sensitive to temperature at the bud-emerging stage in early spring. The leaves gradually change from light green to pale green, then milky white, and finally return to green thoroughly after some time at the albinism stage. The contents of different amino acids are also changing along with the leaf color change. Anjibaicha, at the albinism stage, whose total free amino acid content is two times more than common green tea cultivars, amounting to more than6%, is especially suitable for the production of high quality green tea. However, the periodic albinism of Anjibaicha is influenced by environmental conditions, leading to instability of the contents of amino acids, which restricts its development and utilization to some extent. Therefore, studying the molecular mechanisms of periodic albinism and amino acid enrichment not only plays an important role in controlling the quality of Anjibaicha, but also helps to promote the development and utilization of Anjibaicha and its functional components. In this paper, cDNA-AFLP was used to separate genes which are differentially expressed in Anjibaicha’s periodic albinism process. And SMART RACE was used to clone the full length of some genes which perhaps were related to periodic albinism and amino acid enrichment of Anjibaicha. The main results of the study are as follows:1. CDNA-AFLP approach was used to identify the differentially expressed genes during the periodic albinism. The results showed that the differentially expressed genes were in four different expression patterns:(1) Compared to the pre-albinism stage and the re-greening stage, some genes showed up-regulation at the albinism stage;(2) Compared to the pre-albinism stage and the re-greening stage, some genes showed down-regulation at the albinism stage;(3) Some genes showed up-regulation from the pre-albinism stage to the re-greening stage;(4) Some genes showed down-regulation from the pre-albinism stage to the re-greening stage. And one hundred and twenty seven differentially expressed fragments (DEFs) were successfully sequenced. After the127DEFs were compared against databases of NCBI by BlastX,60of the127DEFs showed high homology to sequences with known functions and represented transcripts.67of the127DEFs were nonhomologous to sequences with known functions or did not show any significant homology to known genes. Based on the putative physiological functions, the identified transcripts could be classified into some groups, including transcription factors, ubiquitination related genes, chloroplast biogenesis related genes, signal transduction genes, stress-related genes, cell cycles related genes, and material and energy metabolism related genes.2. Real-time fluorescent quantitative PCR was used to detect the expression of seven genes which may be related to albino mutation and amino acid enrichment of Anjibaicha during its periodic albinism process. The study found that PPR gene, HD-Zip transcription factor, WRKY transcription factor, ubiquitin ligase gene, ubiquitin protein gene, ubiquitin carbon terminal hydrolase gene and retrotranspo son showed up-regulation from the pre-albinism stage to the albinism stage but down-regulation from the albinism stage to the re-greening stage. These results were consistent with the results of cDNA-AFLP analysis.3. SMART-RACE approach was used to obtain the full length cDNA of PPR gene, ULE gene, SUMO gene and UCH gene of Anjibaicha. Sequence analysis showed that PPR gene full-length cDNA is2063bp, encoding605amino acids. Its amino acid sequence similarity with Grape, Alfalfa, Soybean and Arabidopsis thaliana was73%,68%,66%and57%, respectively. ULE gene full-length cDNA is1839bp, encoding384amino acids. Its amino acid sequence similarity with Grape, Soybean, Arabidopsis thaliana, and Glandulifera genus was72%,63%,63%and60%, respectively. SUMO gene full-length cDNA is803bp, encoding98amino acids. Its amino acid sequence showed high homology with Pea, Tobacco, Grape, Soybean, and Arabidopsis thaliana. The similarities were95%,93%,92%,91%and89%, respectively. UCH gene full-length cDNA is1878bp, encoding461amino acids. Its amino acid sequence similarity with Arabidopsis thaliana was59%.