Study On Floral Organ Protein Properties Of Catalpa Bungei C.A. Meyer And Its Self-incompatibility

Catalpa bungei C.A. Meyer is a precious timber and ornamental species. The cultivationand application of Catalpa bungei have been paid attention more and more recently due to itswidely uses in architecture, furniture and landscape. However, the seedlings shortage of Catalpabungei resulting from its self incompatibility and difficulty in vegetative reproduction severelystunted the extensive development of Catalpa bungei plantation. Therefore, studying thecrossbreeding and sexual propagation of Catalpa bungei would be very helpful in supplying theseedling resources and in developing the Catalpa bungei plantations. The Catalpa bungei C.A.Meyer from Yuantai mountain of Lianyungang （CB-1）, Catalpa bungei C.A. Meyer fromLaoshan Forest Farm of Nanjing （CB-2） and Catalpa fargesii Bur. f. duelouxii （Dode） Gilrnourfrom Nanjing Forestry University （CF） were selected to study the storage conditions of theirpollen and the factors affecting their pollen germination and pollen tube development in thisthesis. The electrophoretic analysis of specific proteins in the pollen wall and other floral organsof catalpa were also done. By the techniques of fluorescence microscopy, the dynamics of pollengermination in the stigma and pollen tube development in the style after cross-pollination werestudied and the self-incompatibility types and their traits of Catalpa bungei species wereidentified. The proteins in the style and in the ovary of Catalpa bungei after cross pollinationwere separated, purified and identified and the properties of the protein associated withself-incompatibility of Catalpa bungei were primarily explicated. The main results were showedas follow:1. The optimal conditions for pollen germination of Catalpa bungei were culture in the darkand25℃for6h, at which the pollen germination rate was over80%and the pollen tube lengthwas over300μm. The responses of the pollen to low and ultralow temperature varied with theCatalpa bungei species and the influences of temperature conditions on pollen viability variedwith the storage time. It was found that when needed to be stored for a long term, the Catalpabungei pollen was feasibly stored in the liquid nitrogen frozen at the temperature of-70℃, whenneeded to be stored for a short term, the optimal storage temperature was-20℃or4℃. Thepollen could not be sorted at the room temperature at which its viability was easily lost. Theoptimal method of thawing for the frozen pollen in the liquid nitrogen was heating the pollen for5minutes at35-38℃in the electric-heated thermostatic water bath.2. There were several factors affecting the Catalpa bungei pollen germination and the pollentube development under in vitro culture. The optimal temperature for the pollen in vitro culturewas24-28℃, both higher and lower than the optimal temperature could inhibit the pollengermination and the growth of pollen tube. The pollen germination and the growth of pollen tube were promoted when the concentration of sucrose and polyethylene glycol （PEG-4000） and pHin the medium were the lower while they were inhibited when the concentration of sucrose wasover15-25g·L^-1, the concentration of PEG-4000over20g·L^-1, and the pH value over5.0～5.6.3. The ultrasonic extraction was the suitable for the protein in the pollen wall and theoptimal parameters were ultrasonic power of400W for3seconds at an interval of6seconds, andthe ultrasonication of120times were finished in3rounds at an interval of5minutes. Thesolution of Tris-HCl with a7.8pH was suitable for the extraction of the pollen protein. By thetechnique of SDS-PAGE, it was found that the common protein86.8kD,74.2kD,70.0kD,45.0kD,43.0kD,37.1kD,35.4kD,34.2kD,33.0kD,32.3kD,30.7kD,29.7kD,28.2kD,20.7kD,19.8kD,17.0kD,15.2kD, and12.4kD were existed in all three Catalpa bungei tree speciestested. However, the specific proteins of CB-1and CB-2were53.8kD and23.4kD, the specificprotein of CB-2and CF was38.5kD, and the specific protein of CB-1and CF was26.5kD. Thespecific protein35.0kD was found only in the CB-1species. In comparison with the analysis ofSDS-PAGE, the isoelectric point （pI） of the specific protein35.0kD in the CB-1species was4.75in the analysis of IEF-SDS-PAGE, and the pI of the specific protein26.5kD in CF and CB-1species was5.55.4. The soluble protein content in the floral organs was in the order of ovary> calyx> style>petal. The analysis of SDS-PAGE indicated that the common proteins included96kD,45kD,32kD,29kD,28kD,27kD,17kD,16kD,13kD, and12kD in the style. The specific protein42kDwas found in the style of CB-1, the protein58kD and24kD was found in the CB-2and CF species,the64kD and19kD in the CB-2, and the37kD in the style of CF species. In the ovary, thecommon protein include protein45kD,32kD,29kD,28kD,27kD,25kD,23kD,21kD,20kD,19kD,17kD,15kD, and12kD, while the specific protein41kD,38kD, and23kD were found inCB-1species. In the calyx, the common proteins included protein72kD,45kD,38kD,37kD,32kD,30kD,29kD,28kD,24kD,23kD,22kD,21kD,20kD,18kD,17kD,15kD,14kD,13kD,and12kD, while the specific protein88kD and65kD were found in the CB-1, and the protein52kD,40kD, and26kD in CB-2and CF species. The protein bands was relative few in the petaland mainly distributed in12-45kD, and the specific protein63kD was found in the CB-1species.5. The time required for the pollen germination in stigma was4-8h after self-pollinationwhile it was2-3h after cross-pollination. The growth of pollen tubes in cross-pollination wasfaster and only in40h after cross-pollination the pollen tubes were entered into the ovary.However, the growth of pollen tubes in self-pollination in style was slower and some tubes wereinterrupted in the1/3of style. In the79-143h after self-pollination there were some pollen tubesentered into the ovary, and the abnormal morphology of the pollen tubes such as bent, curved,and growing downward were observed during the pollen tubes growth. This indicated thatCatalpa bungei was of the characteristics of self-incompatibility and cross-compatibility.6. The proteins in the style and in the ovary were separated and purified after self-pollinationand cross-pollination. Five main peaks were obtained from the style of self-pollination and four main peaks from the style of cross-pollination. While from the ovary, two main peaks wereobtained in self-pollination and three peaks in cross-pollination. The identification of proteinsfrom the peaks showed that the proteins form the peak S1（first peak）, S2（second peak）, and S3（third peak） in the styles of self-pollination had obvious inhibitions on the germination of selfpollen. The main protein components were52kD and38kD in the peak S1, while52kD,44kD,38kD and29kD were mainly existed in the S2and38kD and29kD in the S3. Meanwhile, theprotein52kD,44,38, and29were also identified in the ovary of self-pollination, and theseproteins had relatively strong inhibitions on the germination of self pollen either. These suggestedthat catalpa species was of the characteristic of late-acting ovarian self-incompatibility due to theproteins inhibited pollen tubes in the style and in the ovary after self-pollination.