以轉基因植物分析水稻花粉結鈣激活酵素之功能

Abstract

鈣離子於植物的各種生長、發育過程乃至於應對逆境具有調控的功能，其訊息傳遞經常藉由活化植物所特有的多種結鈣激活酵素(calcium-dependent calmodulin-independent protein kinases，簡稱CDPKs)而執行。CDPKs屬於絲氨酸(serine)/蘇氨酸(threonine)蛋白激酶的基因家族，阿拉伯芥中具有34個成員蛋白，而水稻中則估計至少有29個成員蛋白。這些蛋白的結構相似，但卻常具有高度專一的基因表現模式，或是特定的蛋白分布位置，藉此專一性之機制參與調控各種生理活動，雖然關於CDPKs的分生或生化研究報告頗多，然而除了逆境反應外，少見對其生理角色的探討，絕大多數CDPK蛋白的功能與其下游受質均未明朗。本實驗室曾自水稻中篩選出於花粉成熟期大量表現的兩個CDPK基因- OSCK1與OSCK2，分別構築可大量表現OSCK1(pOSCK1-31)或可同時干擾兩個基因表現(pOSCK1-37)的質體，轉殖水稻並已獲得轉殖株。本實驗乃針對上述兩種構築之T2代與T3代轉殖株，分析其生殖發育期的種種性狀，以探討OSCK1與OSCK2基因的生理功能。首先進行Genomic Southern雜合實驗，以確認轉殖株中含有插入片段，另進行RT-PCR實驗，發現pOSCK1-37轉殖植物中OSCK1與OSCK2的轉錄量多為同步下降，惟OSCK2轉錄量下降的程度不一。詳細檢查多種性狀後發現，除了穗數、單穗平均穎花數並無顯著差異外，OSCK1基因表現被降低的轉殖株具有株高下降、抽穗提早以及稔實率降低等現象；相反的，大量表現OSCK1的轉殖株則有株高增加及延遲抽穗的現象。進一步利用掃描式電子顯微鏡檢查花粉的形態，發現OSCK1表現量降低的植株其花粉表面呈現嚴重皺縮之性狀，雖然存活率並未改變，但花粉萌發率降為未轉殖株的65~80%，應是導致稔實率降低的主因，此證據支持OSCK1基因表現與調控花粉發育以及花粉萌發過程相關。相反的，由於已知OSCK1基因主要於成熟花粉中大量表現，故轉殖株中株高及抽穗日性狀的改變是否與OSCK1基因功能相關仍有疑問。

Ca2+ signals play important roles in many aspects of plant growth and development, including the responses of plants to biotic and abiotic stresses. The CDPKs (calcium-dependent calmodulin-independent protein kinases) which comprise a large family of serine/threonine kinases are often responsible for transducing the calcium signals. Thirty-four and at least twenty-nine members of CDPKs were found in Arabidopsis and rice, respectively. Structures of CDPK proteins are conserved except for their N-terminal domains. CDPKs protein had been found in specific subcellular locations or expressed with distinct profiles, which may consequently conduct their specific roles in response to various stimuli. Although reports concerning the biochemical or molecular characterization of CDPK are plenty, the biological functions of most of the CDPKs in plants and their target proteins are still unclear. We have isolated two CDPK genes, OSCK1 and OSCK2, sharing high sequence similarity and the same pollen-predominant expression patterns. Overexpression and gene-silencing plasmids were constructed and independent lines of transgenic rice were obtained. Aims in this study are to investigate the biological role of OSCK1 by analyzing transgenic rice, with emphasis on characterization of the reproductive phenotypes. Genomic Southern hybridization was used to confirm the transgenic status. RT-PCR experiments revealed that both OSCK1 and OSCK2 were simultaneously down-regulated in most lines analyzed. Except for numbers of panicle or spikelet that remain unchanged, OSCK1-silenced plants exhibited reduced plant height, early flowering, and reduced fertility rate. Oppositely, OSCK1-overexpressed rice was taller and delayed in flowering. Further examination by scanning electron microscopy (SEM) revealed that the surface of mature pollen was severely creased and wrinkled. Although the viability seem to be normal, pollen germination ratio was reduced to 65~80% of the un-transformed TNG67 and may result in the low fertility phenotype. This result suggests that OSCK1 plays critical roles in pollen development and germination process. On the other hand, as OSCK1 is known to express predominantly in mature pollen, it is not convincing that change of OSCK1 gene expression have effect on plant height and heading date. More experiments need to be carried out to address this phenomenon.