百合參與花粉萌發和花粉管生長之基因的特性分析

Abstract

當植物進行有性生殖時，花粉管的正常的萌發與生長，對於成功地完成雙重授受精是必需的。在進行百合花粉管萌發實驗時發現花粉經-20℃儲存一段時間後，其萌發和生長速率有顯著減緩的現象，然而一旦它們萌發之後，其萌發和生長速率與新鮮收集的花粉則無差別。許多文獻指出在花粉成熟期間許多蛋白質和mRNAs可預先合成並儲存於花粉粒中以支援其後花粉管的快速成長。因此一些對於正常花粉管萌發和生長所必需的蛋白質和mRNAs，可能在低溫度儲存過程中逐漸降解，然而這些降解的mRNAs和蛋白質在花粉粒遇到適當環境時會重新轉錄和轉譯以供正常生長之用。為了測試這個假設，首先利用新鮮收集花粉及低溫儲存二個月花粉之mRNA，經由suppression subtractive hybridization方式，將這些可能參與花粉管正常萌發及mRNA片段進行選殖及定序以確定該基因為何。結果共得63個基因片斷，並對其30個基因片斷進行北方墨點檢測，了解其mRNA於低溫儲存時之變化。結果顯示大部份mRNA確實會於低溫儲存期間逐漸分解，但將低溫儲存二個月花粉於體外培養液中浸泡二小時，其mRNA組成則恢復與新鮮花粉無顯著不同。在此次實驗發現一與COP8相似基因，並試圖以北方墨點法及5’與3’-RACE找尋其在花粉中的表現及cDNA全長，但皆無任何結果。之後，以阿拉伯芥COP8抗體卻能找尋到百合花粉COP8相似蛋白，並於免疫組織化學定位分析中發現其在細胞核有大量的累積，再經萌發反應後會在細胞質中的特殊胞器有大量堆積現象。除了COP8蛋白外，本實驗中所得到的許多基因亦第一次在花粉中發現，值得進一步的研究。

The normal germination of pollen tube is essential for successful double fertilization during plant sexual reproduction. As worked on the lily (Lilium lotigiflorhim Thunb. cv Avita) pollen tube germination, one interesting phenomenon was observed. Pollen grains stored at -20˚C for certain periods would delay their germination and growth rates in the first two hours during in vitro culture. Once they germinated, there was no difference in germination and growth rates as compared to the fresh-collected pollen grains. It has been documented that a lot of proteins and mRNAs were pre-synthesized during pollen maturation to support rapid growth of pollen tube during fertilization. We proposed that some proteins and mRNAs, which are essential for normal pollen tube germination and growth, were gradually degraded during the low temperature storage. These degraded mRNAs and proteins are expected to be de novo transcribed and translated for normal pollen germination and growth. To test this hypothesis, mRNAs of -20˚C-stored and fresh pollen grains were subtracted by suppression subtractive hybridization, and then cloning and sequencing these clones which maybe involve in pollen germination. Sixty-three clones were obtained, thirty of them were analyzed by Northern blotting. For the expression pattern, most of them were significantly reduced during low temperature storage, but recovered by incubating the stored pollen grain in germination medium for 2 hours. In this experiment, we found a COP8-like gene and tried to clone its full-length by 5'/3' RACE, but did not have any result. By western blotting, we can found lily COP8-like protein by Arabidopsis COP8 antibody. By immunolocalization, we found COP8 protein mainly located in pollen nucleus. After pollen germination, we also found COP8 protein in special organelle. In this experiment, a lot of genes were first time identified in pollen grain and worth further charactering.