Isolation And Expression Of Genes In Tea(Camellia Sinensis (L.) O.Kuntze) Pollen Tube Under Cold Stress

Tea plant (Camellia sinensis (L.)O.Kuntze) is an economic woody crop grown in different agro-climatic zones of the world with warm and wettish climates. However, cold stress always results in low yield, poor quality and delay to market of tea, which seriously influences the production and economic benefits of tea. According to previous reports, tea pollen could geminate and elongate under low temperature while most angiosperm pollens (such as lily pollen) could not germinate. In this study, differential gene expression in tea plant pollen tube under cold stress was analyzed using cDNA-AFLP technology. Some differentially expressed transcript fragments were isolated. We analyzed the derived fragments and cloned the full length of the genes related to pollen tube growth during cold stress. A preliminary analysis of these genes sequence on bioinformatics and functions are carried out. The results are as follows:(1) Differential gene expression between normal growth and cold-stressed tea pollen tube was investigated using cDNA-AFLP technology. Thirteen differentially expressed transcript-derived fragments were isolated. Blastx comparisons showed10of them were homology to known genes, and3were unknown. TDF1had high similarity (91%) to Brassica rapa pyruvate dehydrogenase complex E1alpha subunit dehydrogenase; TDF2was similar to Vitis vinifera calcium-binding protein CML21-like(87%); TDF4was congenerous to14-3-3protein; TDF5showed74%identity with Ricinus communis Phospholipase D delta; TDF7and TDF9showed74%and71%similarity to Vitis vinifera Thioredoxin H9-lik and Petunia x hybrida beta-galactosidase2precursor, respectively。(2) Complete cDNA sequence of TDF1was cloned by RACE, and named as CsEla, and its GenBank accession is JQ999982. The expression analysis by qRT-PCR showed that CsEla expressed highest in pollen, and the transcripts of CsEla started to accumulate at the first30min during tea plant pollen tube growth, and then maintained at1h、2h、3h with a slight decline.(3) A cDNA fragment, TDF2, encoding calcium-binding protein CML21was isolated and identified. Its complete cDNA sequence was cloned by RACE, named as CsCML21, and its GenBank accession is JQ999983. Quantitative RT-PCR analysis revealed that CsCML21mRNA was strongly detected in pollen, and the abundance of CsCML21mRNA decreased during tea pollen tube growth.