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Molecular Biology of Tapetal Proteins and Pollen-Specific Proteins Associated with Desiccation in Lily Anther
花藥的發育可分成三個連續的時期：第一個時期包含早期的細胞增殖和藥囊腔的分化；第二時期是小孢子的形成，從減數分裂開始一直到小孢子成熟，此時期花藥的絨氈層細胞呈極化高度分泌；最後是花粉成熟時期，小孢子行有絲分裂到花粉形成。本研究建立了百合花藥小孢子時期的cDNA集合庫，利用免疫篩選法得到7個選殖體（PR-10）。依照序列比對以及南方墨跡法的分析，可大致將此7個PR-10選殖體分為兩個族群。兩族群之間的序列相似性高達82%（以PR-10c以及PR-10d為代表）。兩個cDNA中都有一段長474 bp可轉譯157個氨基酸的open reading frame。在5′端以及3′端的非轉譯區相似性不高，但在轉譯區相似性非常高。由北方墨跡法的結果，得知百合PR-10基因具專一性，只會在花藥表現，而不會在其他的器官中表現，而且在花粉發育過程中，只在小孢子時期的花藥中表現。百合PR-10 cDNA所推測而得的蛋白質序列經比對後，發現與其他PR-10蛋白質，如蘆筍AoPR1，馬鈴薯STH2，樺樹花粉Betv1，香菜PcPR1，豆類PvPR2，羽扁豆LIR18B 以及豌豆I49等均具有顯著的相似性。百合基因組DNA的南方墨跡法分析得知百合PR-10為一多基因族。利用PCR找到一族群PR-10的部分基因。此PR-10基因包括270 bp的轉譯區和991 bp的5′端啟動子區。已知的轉譯區內有一67 bp的插入子。5′端啟動子區含有CACATG序列，可能為ABA調控的區域。另外，含有兩個E-box motif，可能與轉錄因子結合，藉以調控基因的表現。
百合PR-10基因會受到abscisic acid (ABA) 以及methyl jasmonate（MeJA）的誘發而在花藥和其他器官中表現。在百合花藥內ABA和MeJA所誘發PR-10基因的表現是經由兩條不同的訊息傳遞路徑。蛋白質磷酸酶抑制劑okadaic acid會在MeJA的下游抑制MeJA誘發PR-10基因表現。而且，蛋白質激酶抑制劑staurosporine也會抑制MeJA誘發PR-10基因表現，顯示花藥中對staurosporine敏感的蛋白質激酶也會在MeJA的下游參與MeJA訊息傳遞路徑。Okadaic acid不會抑制ABA所誘發的PR-10基因表現，但是staurosporine會，顯示除了已知ABA會經由刺激JA或MeJA來誘發基因的表現外，在花藥中ABA可以不經由MeJA來誘發PR-10基因表現。而這一條不同的ABA訊息傳遞路徑，至少有一個對staurosporine敏感的蛋白質激酶參與訊息傳遞。
此外，本研究亦從百合成熟花粉的cDNA 集合庫中篩選出一個與乾燥有關蛋白質（LLA23）的cDNA選殖體。北方墨跡法的分析顯示，LLA23基因具花粉專一性，並且只在開花前的花粉成熟期才開始出現。從序列分析中知道LLA23氨基酸序列與一群water-deficit / ripening—induced蛋白質，尤其是序列後半端的部分，有很高的相似性，包括了松樹 Lp3-1蛋白質、番茄的Asr1/2蛋白質、pummelo Asr1同源蛋白質以及馬鈴薯的DS2蛋白質。百合基因組DNA的南方墨跡法分析得知LLA23為一多基因族。未成熟的花粉先行乾燥（premature drying）實驗證實LLA23基因的表現與乾燥有關。當花粉於萌發溶液萌發時，LLA23蛋白質的量會逐漸減少，若萌發溶液中加入10 μM ABA或20% PEG-8000時，LLA23蛋白質及mRNA減少的速度會減緩。在in vitro或in vivo的萌發環境下，花粉LLA23 mRNA的量都會減少，但在in vivo萌發環境下LLA23 mRNA消失的速度比in vitro的萌發環境緩慢。利用免疫金粒定位法所完成的電顯切片觀察中，證實LLA23蛋白質存在於花粉粒的細胞質中。依本研究結果推測與乾燥有關的LLA23蛋白質可能在花粉的細胞質中扮演著保護的角色。
Anther ontogeny is histologically divided into three consecutive phases. The first phase encompasses early proliferative stages and differentiation of the locules. The second phase concerns microspore development from the onset of meiosis through microspore maturation. The final phase involves pollen maturation, originating with microspore mitosis through pollen formation. We have constructed the lily(Lilium longiflorum)anther cDNA library at the stage of microspore development during which tapetum is highly secretory. Seven cDNA clones (PR-10) have been identified by immunoscreening. These PR-10 clones can be divided into two subclasses based on sequence comparison and Southern hybridization. An 82% overall sequence similarity was found between the two subclasses (represented by PR-10c and d). The two cDNAs include an open reading frame of 474 bp encoding 157 amino acids. 5'- and 3'-untranslated regions exhibit low similarity, but similarity is high in the coding region. The lily PR-10 transcripts are anther-specific and temporally expressed only at the phase of microspore development. The predicted amino acid sequence of PR-10 reveals significant resemblance to a group of IPR(intracellular pathogenesis-related) proteins, including asparagus AoPR1, potato STH2, birch Betv1, parsley PcPR1, bean PvPR2, lupin LIR18B and pea I49. Genomic Southern analysis indicates that the lily PR-10 proteins are encoded by a family of multiple genes. A PR-10 gene identified by PCR includes a coding region of 270 bp and a 5'- region of 991 bp. The known coding region is interrupted by an intron of 67 bp. The 5'-region contains the segment of CACATG, a core segment of ABA-responsive element. In addition, the 5'-region contains two E-box motifs that may interact with transcription factors for gene regulation.
The lily PR-10 genes are induced by abscisic acid (ABA) and methyl jasmonate (MeJA) in the anther and various other organs of lily plants. The induction of PR-10 genes by ABA and MeJA in lily anthers occurs by two separate signal transduction pathways. The protein phosphotase inhibitor okadaic acid inhibits the MeJA-induced expression of PR-10 genes downstream of MeJA. In addition, the protein kinase inhibitor staurosporine inhibits the MeJA-induced expression of PR-10 genes, implying that an activity of staurosporine-sensitive protein kinase exists downstream of MeJA in the anther. However, okadaic acid does not inhibit the ABA-induced expression of PR-10 genes whereas staurosporine does. These observations suggest that, in addition to the known pathway that ABA induces gene expression by activating JA or MeJA, a MeJA-independent pathway of ABA induction exists in the anther. The alternative pathway of ABA induction involves a staurosporine-sensitive protein kinase activity downstream of ABA.
Besides, a cDNA clone encoding a desiccation-induced protein(LLA23)has been isolated from the mature pollen of a lily cDNA library. Sequence analysis revealed significant similarity between the predicted LLA23 polypeptide, particularly at the C-terminal half of the sequence and a group of water-deficit/ripening-induced proteins. The expression of LLA23 gene is pollen-specific and the transcript accumulates only at the later stage of pollen maturation prior to anthesis. Genomic Southern analysis indicates that LLA23 proteins are encoded by a family of multiple genes. Premature drying of developing pollen confirmed that accumulation of LLA23 transcripts was associated with desiccation. The LLA23 proteins decreased their levels when pollen/pollen tubes grew in the germination buffer. Treatments of pollen with abscisic acid(ABA)and polyethylene glycol(PEG)-8000 during germination greatly retarded the disappearance of LLA23 proteins and mRNAs. The LLA23 transcripts decreased their levels in pollen tubes grown both in vitro and in vivo, but the disappearance of LLA23 transcripts in tubes cells grown in vivo was slower than those grown in vitro. Immuno-localization using anti-chicken immunoglobulin G conjugated with gold particles confirmed that LLA23 was located in the cytoplasm of pollen grains. The protective function of the desiccation-related proteins in the cytoplasm of pollen grains is discussed.
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