以分裂泛素酵母菌雙雜交系統篩選與RolB具交互作用之蛋白質

Abstract

毛狀根為植物受根毛農桿菌感染所生成的特化組織，被視為植物次級代謝物生產的潛力平台。農桿菌TL-DNA上之rolB 基因對毛狀根的生長與代謝物之累積均有重大影響，然而rolB 基因在毛狀根生理中之角色仍然未知。為了解RolB是否與宿主之膜蛋白交互作用，本研究以RolB 蛋白質作為分裂泛素酵母菌雙雜交系統的餌蛋白來篩選阿拉伯芥中可能與RolB具交互作用之膜蛋白質。系統建構過程中，控制組之酵母轉形株均建構與測試完成，且已知的RolB與Nt14-3-3 ωII蛋白質之交互作用可被重現並作為蛋白質交互作用之正控制組。此外，RolB 蛋白質可在篩選系統中大量表現，且無強烈的自活化HIS3報導基因現象，篩選的嚴苛度也以HIS3報導基因之抑制劑濃度調整完畢。轉形效率為1.46 x 105 / μg 之質體。總計130 酵母菌轉形株在二次104篩選池大小之篩選後，β-半乳糖苷酶測試呈現陽性反應，且篩選結果顯示二個位於質粒的蛋白質LIL3:1及AT1G44920可能與RolB有交互作用，且此二基因之酵母菌轉形株之β-半乳糖苷酶活性與陽性控制組相當。LIL3:1被報導可促進葉綠素、α-生育酚及植醇之生合成，AT1G44920為一功能未知之穿膜蛋白質。然而RolB於酵母菌中之表現位置需進一步確認，且篩選反應之轉形效率須優化至106/ μg 之質體。為進一步確定此二個位於質粒的蛋白質與RolB蛋白質是否具有交互作用及其作用位置，本研究建構紅花菸草N. tabacum毛狀根之原生質體分離方法，以及LIL3:1及AT1G44920之青色螢光融合蛋白表現載體。多個分離參數包括組織處理、酵素裂解前處理、纖維素酶及離析酶之比例與濃度，以及酵素液培養時間皆進行測試。利用新調整之組織去皮法及2:1之酵素比，原生質體之產率達2.02 * 105 / 每克之毛狀根根尖。

Hairy root, a syndrome induced by Agrobacterium rhizogenes, can be a potential platform for phytochemical production. rolB on Agrobacterium rhizogenes TL-DNA is crucial for hairy root growth and phytochemical accumulation. However, how rolB affect hairy root physiology remains elusive. In this study, a split-ubiquitin based membrane yeast two-hybrid system (MYTH) was constructed to screen the possible interacting proteins of RolB in Arabidopsis thaliana. Yeast co-transformants were constructed, and the known interaction between Nt14-3-3 ωII and RolB is reproducible as a positive control for protein-protein interaction in control test. RolB can express in MYTH abundantly without strong self-activation of HIS3 reporter gene, and the screening stringency was optimized by adjusting the 3-AT concentration. The transformation efficiency reached 1.46 x 105 /μg of plasmid. Total 130 yeast transformants were found by β-galactosidase assay after two 104-scale screening reactions. Two plastid-localized protein, LIL3:1 and AT1G44920 were identified, and the β-galactosidase activity of LIL3:1 and AT1G44920 yeast candidates were as higher as positive controls. LIL3:1 has been shown to promote the biosynthesis of chlorophyll, α-tocopherol and phytol, and AT1G44920 is a transmembrane protein with unknown function. However, the RolB expression site in yeast needs verification, and the transformation efficiency should reached 106/μg of plasmid. To examine if RolB can interact with LIL3:1 and AT1G44920 in plant cell, the isolation procedure for Nicotiana tabacum hairy root protoplast were established, and the cyan fluorescent protein (CFP) expression vector for LIL3:1 and AT1G44920 were constructed. Several crucial parameters for isolation including the tissue processing, pre-treatments, enzyme ratio and incubation time have been optimized. The modified peeled method has been developed, and the yield of hairy root protoplast reached 2.02 * 105 / g of hairy root tip.