Identification Of Self-incompatibility Model,Cloning And Expression Of Correlative Gene In Camellia Sinensis (L.) O. Kuntze

Tea plant (Camellia sinensis (L.) O. Kuntze), which belongs to the family Theaceae, is a perennial cross-pollinated plant with highly heterozygosity. Its long life cycle and the few application of inbreeding and hybridization in its genetic breeding limits the research on the genetic mechanism in tea, and also makes the research of tea genetics lags far behind that of other crops. In addition, a low seed set and sterility in self-pollination make it show self-incompatibility. So, it is an important issue to study the mechanism of self-incompatibility in tea plant for its genetics breeding.In this paper, the mechanism of self-incompatibity in tea was studied at physiology and molecular biology levels including the optimum condition for keeping the pollen alive in vitro and pollen germination, pollen tube growth in style and ovary after self-and cross-pollination, cDNA-AFLP analysis of gene expression in flower between self-and cross-pollination, gene clone of house-keeping gene GAPDH and quantitative PCR, gene clone and expression analysis of CsiCDPK which associated with self incompatibility in tea. Results as follows,1. How to keep the pollen alive and which condition was the best for them to germinate are very important for developmental biology of tea plant. The differences of pollen germination conditions and pollen germination capacity in different cultivars will determine the sexual propagation capacity and its value in tea breeding. H3BO4, Ca(NO3)2, PEG-4000, carbon source and pH value were chosen as the factors to determine the best germinated condition. It was showed that the best condition is with0.01%H3BO4,0.05%Ca(NO3)2,5%PEG-4000,5%maltose and at pH5.0-6.0(with30mmol·L-1MES). The germinated rate is82.24%, and the length of the pollen tube is49.2μm in this condition. The pollen can be kept alive at4℃for1～5days. But if it needs to keep much longer time, it should be kept at-70℃after frozen by liquid N2.2. It acts as an important basis in identification and mechanism of the self-incompatibility that if the pollen could germinate and where they would be terminated. C.sinensis cv. Keemenzhong, C.sinensis cv. Longjin-changye, C.sinensis cv. Fudign-dabaicha and C.sinensis cv. Yabukita were chosen to be treated by cross-and self-pollinated. Style and the ovary were taken as sample at different time after treatment. With the fluorescence staining by aniline blue, both the cross-and self-pollen were observed went into ovary after they penetrated through the style. Further analysis of paraffin section of ovary showed that the cross-pollen tube can go into ovary easily but the self-ones can not. For these, the mechanism of the self-incompatibility in tea plant should not be the gametophytic self-incompatibility (GSI) but the late-acting self-incompatibility (LSI) or ovary self-incompatibility (OSI).3. The technology of cDNA-AFLP, which combined technology of RT-PCR and AFLP, can compare the difference of gene expression in specified stage by using cDNA with different transcription level as template in special tissue. In this experiment, the difference gene expression was compared between cross-and self-pollination pistils with cDNA-AFLP, with the un-pollination as blank control. After sequencing the different gene bands and having blasted in Genbank, we got15fragments which may associated with self-incompatibility including calcium-dependent protein kinase, senescence-associated protein, beta family G-protein and cyclin-like protein.4. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the most commonly used housekeeping genes used in comparisons of gene expression data. Forward and reward primers based on the conserved sequences of GAPDH, identified by alignment the GAPDH sequences of Arabidopsis thaliana, longan and peas from Genbank, was used for PCR amplification of Camellia sinensis GAPDH from cDNA. After cloning, transformation, sequencing and blasting, a fragment of Camellia sinensis GAPDH, about880bp in length, was obtained and named as CsGAPDH. The predicted amino acid sequences of CsGAPDH revealed80%,83%, and81%identities to the published GAPDH from Arabidopsis, longan and peas, respectively. The whole sequence was then obtained by3’and5’-RACE. And the reproducibility of the gene was evaluated. The expression of GAPDH can be used to normalize quantitative gene expression of objective genes in tea plants.5. Expression of7fragmetns from cDNA-AFLP in pistil after self-pollinated24hours were detected by QT-PCR. It showed that they all expressed specially. A cDNA fragment encoding a calcium-dependent protein kinase gene was isolated and identified by cDNA-AFLP (Amplified Fragment Length Polymorphism). The complete cDNA sequence was cloned by RACE (rapid amplification of cDNA ends), named CsiCDPK (tea calcium-dependent protein kinase) gene. The whole sequence of CsiCDPK was2281bp in length and the predicted amino acid sequence was with760aa.To identify differential expression of CsiCDPK gene, QT-PCR experiments were performed in pistil after self-pollinated24hours. The results showed that the CsiCDPK gene expressed17.1times than GAPDH, while only4.6and2.9times in un-pollinated and cross-pollinated. That hinted that CsiCDPK might play a key role in self-incompatibility of tea plant.