建立T-DNA插入點分析與親和性抗體純化技術探討HC-Pro誘導細胞自噬途徑降解AGO1之機制

Abstract

多基因轉殖技術常用於研究植物基因調控與交互作用之關係，須克服轉殖植物基因表現不穩定之障礙。多基因轉植株可經多次轉殖或雜交獲得，但轉殖基因共同分離 (cosegregation)、多拷貝數 (copy number)、或是轉殖基因結構複雜皆可能造成後生遺傳調控現象而干擾轉殖基因表現。因此，我們建立再定序(resequencing) 的分析技術以確認農桿菌轉殖殖株之T-DNA插入點。欲驗證輔助性蛋白酶 (helper component-proteinase, HC-Pro) 利用細胞自噬途徑 (autophagy) 降解 AGRONAUTE1 (AGO1)之假說，以雜交方式將HC-Pro基因導入不同autophagy-related (atgs) 突變株。利用熱不對稱性交錯PCR (Thermal asymmetric interlaced-polymerase chain reaction; TAIL-PCR) 與再定序技術確認P1/HC21-21轉植株的T-DNA插入點，結果顯示P1/HC21-21轉植株基因中含有多個插入片段，且HC-Pro基因與GUS基因鏈鎖。假設中預期atg/P1/HC21-21植物中因細胞自噬缺陷，HC-Pro無法促進AGO1降解，導致AGO1累積且表現較微弱的鋸齒葉葉形。然而，雜交atg突變株與P1/HC21-21轉植株結果顯示，atgs × P1/HC21-21 F2子代可能發生轉錄時基因默化現象 (transcriptional gene silencing)。此外，AGO1與其他相關蛋白之抗體經由親和性層析純化出IgG後，可增加靈敏性與專一性並用於蛋白質分析實驗。了解P1/HC與atg/ P1/HC植物之AGO1表現量可驗證HC-Pro誘導細胞自噬降解AGO1之假說。

In plant science, the major obstacle of the exploration of gene relationship in regulation and interaction is the difficulty to manipulate multiple transgenes. Multiple transgenes can be introduced either by retransformation or by sexual crossing, but transgenic loci cosegregation, copy number, the structure of transgene may cause epigenetic regulation and sometimes interrupt the expression level of transgenes. Here, we established a procedure to identify the T-DNA insertion site in Agrobacterium- mediated transgenic plants by resequencing. The study of helper component-proteinase (HC-Pro), a viral gene silencing suppressor, triggering autophagic AGRONAUTE1 (AGO1) degradation is evaluated through expressing HC-Pro gene in different genetic backgrounds of autophagy-related gene T-DNA insertion mutants (atgs) by crossing. Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) and resequencing approaches were used for identification of the T-DNA insertion sites in various transgenic plants. The results indicated that multiple insertions and the linkage between HC-Pro and GUS gene showed in P1/HC21-21 plants. We expected that autophagy-deficiency in atg/P1/HC21-21 plants enhanced AGO1 accumulation and showed milder serrated phenotype. However, normal leaf phenotype in all of the atg8a × P1/HC21-21, atg5 × P1/HC21-21 F2 progenies suggested that transcriptional gene silencing might occur. Besides, antibodies against AGO1, HEN1 and another silencing suppressor p19 were produced with affinity purification for protein assay. Verifying AGO1 expression in P1/HC and atg/ P1/HC plants will help to prove our hypothesis that HC-Pro triggers autophagic AGO1 degradation.