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Heterogeneity, Ribonuclease Activity of PR-10 Gene Products and its Induction by Abscisic Acid and Methyl Jasmonate in the Tapetum of Lily Anthers
|關鍵字:||Tapetum;花藥絨氈層;RNase activity;PR-10 proteins;RNase活性;PR-10蛋白||出版社:||農業生物科技學研究所||摘要:||
百合花藥絨氈層的發育和小孢子及花粉的成熟有密切的關係。百合PR-10蛋白為花藥絨氈層專一性蛋白，本實驗從2個子群的PR-10中各取一個cDNA clone，分別為PR-10a及PR-10c，轉殖到大腸桿菌中表現其蛋白。經由雙向電泳及免疫轉漬分析，發現兩個子群表現不同型的蛋白，PR-10a蛋白的等電點為5.9，而PR-10c為5.7。利用formaldehyde洋菜瓊膠電泳及膠體活性分析証實百合PR-10蛋白會分解yeast RNA。在溶液中，百合PR-10蛋白會降解百合花藥RNA。百合PR-10蛋白的活性也會受到RNasin抑制。證明百合PR-10蛋白具有 RNase的活性。但是百合PR-10蛋白不會分解雙股或單股DNA。在百合花藥發育過程中，當花苞的長度到1.4公分可以偵測到PR-10蛋白，PR-10蛋白的量會隨著花苞長度（到2公分）而增加。取1.4公分以前的花苞，經過100 μM ABA和10 μM MeJA的處理，發現百合PR-10 mRNA有表現，但是沒有偵測到PR-10蛋白。顯示在花藥內百合PR-10基因的表現是受後轉錄機制的調控。雖然百合PR- 10蛋白在早期的花藥中沒有累積，但是同時期的花苞，經ABA或MeJA處理後，仍可看到其他的花藥蛋白被誘導表現出來。在1公分的花苞，經過100 μM ABA的處理後，有18個百合花藥蛋白被誘發出來，其中包括7個經過誘導處理才出現的蛋白及11個累積量顯著增加的蛋白，分子量為15-44 kDa。經10 μM MeJA處理後，有11個百合花藥蛋白被誘發出來，其中包括6個處理後才出現的蛋白，5個累積量有顯著增加的蛋白，分子量為17-55 kDa。不過也有一些百合花藥蛋白會受到ABA及MeJA的抑制，蛋白的量明顯減少或消失。
There is a close relationship between the development of lily tapetum and maturation of microspore and pollen. The lily PR-10 protein is a tapetum-specific protein. We chose one clone from each of the two subclasses of lily PR-10 cDNAs and overexpressed its protein in E. coli. The expressed proteins from each subclass are different when analyzed by 2D-PAGE and immunoblotting. The pI value of PR-10a protein is 5.9 whereas PR-10c is 5.7. Analysis of formaldehyde agarose gel and gel activity assay revealed that the lily PR-10 proteins can degrade yeast RNA and lily anther RNA. The activity of lily PR-10 proteins can also be inactivated with the addition of RNasin. The lily PR-10 proteins showed no nuclease activity on double or single-stranded DNA. The lily PR-10 proteins were initially detected in 14-mm lily buds during anther development. The protein increase with increasing growth of lily buds. The lily PR-10 genes can be induced by either the addition of 100 μM abscisic acid (ABA) or 10 μM methyl jasmonate (MeJA). While the lily PR-10 mRNA is induced in the anthers, the lily PR-10 protein remain undetectable in the anthers of early stage of lily buds. Thus, post-transcriptional regulation exists in the anthers. Except for the absent accumulation of lily PR-10 proteins, other proteins accumulated upon ABA or MeJA treatment. Eighteen proteins were induced by ABA, seven of them only appear with the treatment of ABA, and eleven proteins obviously increased in quanity. We also obtained eleven MeJA-induced proteins, six of which only appear with the treatment of MeJA, and five obviously increased in quanity. On the contrary, some proteins in the anther were inhibited by treatment of ABA and MeJA.
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