菸草毛狀根形成機制之探討

The Study on Formation Mechanism of Tobacco Hairy Root

郭翰蓉(Han-Jung Kuo)

指導教授 ： 李昆達

國立臺灣大學/生命科學院/生化科技學系/博士(2021年)

https://doi.org/10.6342/NTU202104293

摘要

根毛農桿菌會將T-DNA插入植物染色體並誘導植物生成毛狀根，T-DNA上的rolB與rolC基因在毛狀根誘導與側根生長中扮演重要角色。RolC之交互作用蛋白篩選無法在現成之酵母菌雙雜合系統中進行，因為RolC蛋白本身會自活化影響篩選，因而建立一系列的RolC突變，篩選具有較低自活化能力之突變。A119K+P120D突變有最低的自活化能力，N91K與G76K亦具有不錯之效果，將A119K+P120D突變RolC置換回根毛農桿菌中，其生長與野生型農桿菌相同，另外也以突變RolC農桿菌誘導菸草毛狀根，其根生長形態與野生型相近。接著以A119K+P120D突變RolC進行酵母菌雙雜合篩選，發現兩個可能與RolC有交互作有的蛋白質，分別為quinolinate合成酶與延伸因子1a (elongation factor 1-a)，在酵母菌單雜交實驗中發現延伸因子1可能與RolC有交互作用，後續將進一步驗證此交互作用之真偽。
在先前研究中本實驗室發現一群可能受rolB或rolC調控之脂質轉移蛋白(LTP)，找出這些LTP全長cDNA並檢驗這些LTP、rolB與rolC在不同荷爾蒙處理之毛狀根中的表現，我們發現LTP之表現與rolB或rolC之表現關聯性並不大，顯示LTP可能並不直接受到rolB或rolC之調控。而LTP6與LTP7表現量調降之毛狀根，與rolB或rolC缺失之毛狀根有類似的側根生長抑制之現象。

關鍵字

根毛農桿菌 ； 毛狀根 ； rolC ； 酵母菌雙雜合系統 ； 脂質轉移蛋白

並列摘要

Agrobacterium rhizogenes is a plant pathogen inducing hairy root disease with the insertion of T-DNA. rolB and rolC, the genes located on A. rhizogenes T-DNA, play important roles in hairy root induction and lateral root growth. Screening of RolC interacting proteins using the well-established yeast two-hybrid system is not workable due to autoactivation of RolC. A set of RolC random mutants were screened and the mutants with lower autoactivation activity were selected. The mutant A119K+P120D were identified with the lowest autoactivation and N91K and G76K mutants showed relatively lower autoactivation. A. rhizogenes with RolC mutant A119K+P120D was established, and the growth of this mutant strain was equal to wild-type. The architecture of hairy roots induced by this mutant A. rhizogenes was similar to wild-type. Yeast two-hybrid screen was performed by using RolC A119K+P120D mutant with a tobacco hairy root cDNA library. Two potential interacting proteins of RolC were identified: quinolinate synthase and elongation factor 1-a. Elongation factor 1-a was found having weak interaction with RolC. Further experiment would be performed to confirm the interaction.
A group of tobacco lipid transfer proteins (LTPs) which could be regulated by the expression of rolB or rolC was identified according to our previous study. The full-length cDNA of the LTPs was identified. The expression levels of LTPs, rolB and rolC under a variety of phytohormone treatment in hairy root were analyzed. The correlation between the expression of rolB/rolC and LTPs under different conditions was not high. The results suggested LTPs may not be regulated by rolB or rolC directly. The hairy roots of the tobacco plants with LTP6 or LTP7 knock-down showed lateral root growth inhibition which was similar to those of rolB or rolC deficient mutant.

並列關鍵字

Agrobacterium rhizogenes ； hairy root ； rolC ； yeast two-hybrid ； lipid transfer proteins

參考文獻

Cardarelli M, Spano L, De Paolis A, Mauro ML, Vitali G, Costantino P: Identification of the genetic locus responsible for non-polar root induction by Agrobacterium rhizogenes 1855. Plant Mol Biol 1985, 5(6):385-391.

Wang JH, Lin HH, Liu CT, Lin TC, Liu LY, Lee KT: Transcriptomic analysis reveals that reactive oxygen species and genes encoding lipid transfer protein are associated with tobacco hairy root growth and branch development. Mol Plant Microbe Interact 2014, 27(7):678-687.

Palazon J, Mallol A, Eibl R, Lettenbauer C, Cusido RM, Pinol MT: Growth and ginsenoside production in hairy root cultures of Panax ginseng using a novel bioreactor. Planta Med 2003, 69(4):344-349.

Cai G, Li G, Ye H, Li G: Hairy root culture of Artemisia annua L. by Ri plasmid transformation and biosynthesis of artemisinin. Chin J Biotechnol 1995, 11(4):227-235.

Patra N, Srivastava AK: Artemisinin production by plant hairy root cultures in gas- and liquid-phase bioreactors. Plant Cell Rep 2016, 35(1):143-153.