Biodegradation of Paclobutrazol, a Plant Growth Regulator, by Serratia Marcescens

Abstract

Paclobutrazol is a recalcitrant plant growth retardant that is used worldwide, for such purposesas increasing the yield of cereal crops and enhancing seed production in eucalypt orchards.Paclobutrazol has been widely used in wax-apple cultivation in Taiwan to depress new shootgrowing and promote early flowering. However, paclobutrazol is xenobiotic compound thatremains active in soil for several years and can severely affect the growth and development ofsubsequent crops, mainly by reducing vegetative vigor. This compound is quite resistant tobiodegradation by soil microbes. The use of recombinant microorganisms is expected to be aneffective tool for remediation of polluted environments. In previous work, we have established aquick method for the isolation of bacterium with capability of degrading the paclobutrazol. Serratiamarcescens strains NCHU 4-3 and NCHU 5-3 were isolated, to which about 9% of paclobutrazolsupplemented in the culture medium was degraded in 30 days. In this study, we are going to find outthe pathway for the catabolism of paclobutrazol by S. marcescens, clone the genes related to thedegradation of paclobutrazol, construct a soil bacterium capable of degrading the paclobutrazol, andevaluate the feasibility of using the recombinant soil bacteria in the remediation of the soils pollutedwith paclobutrazol.First grant year1. Determine the effect of cultural conditions on the biodegradation of paclobutrazol by S.marcescens NCHU 4-3 and S. marcescens NCHU 5-3.2. To predict the putative degradation pathway of paclobutrazol and the enzymes involvedin the pathway.3. Establishment of the methods for the extraction and chemical analysis of metabolitesfrom paclobutrazol.4. Cloning of the gene(s) involved in the first step of paclobutrazol biodegradation pathwayfrom S. marcescens and expression of cloned genes in E. coli.5. Create paclobutrazol-negative mutants (PBZ- strain) of S. marcescens using EZ-Tn5TM Tnp TransposomeTM Kit.Second grant year1. Isolation and identification of metabolites from paclobutrazol biodegradation by S.marcescens.2. Cloning and sequencing of the transposon-disrupted genes to elucidate the possible rolesof the genes in the paclobutrazol biodegradation.3. Cloning and expression of transposon-disrupted genes in E. coli and S. marcescens todetermine the possible roles of the genes in the paclobutrazol biodegradation.4. To find out the major microbial flora in the rhizosphere of wax-apple garden.Third grand year1. To develop a gene expression vector for the transformation of the major microbes in therhizosphere of wax-apple garden without the use of drug-resistant selective marker.2. To construct a soil bacterium capable of degrading the paclobutrazol by DNArecombination using 16S rDNA as homologous gene.3. To evaluate the feasibility of using recombinant paclobutrazol-degradative bacteria inthe bioremediation of paclobutrazol-polluted soils.4. Patent application.

Paclobutrazol 為一種人工合成的植物生長調節劑，其主要作用方式為抑制植物的吉貝素(gibberellin)生合成，使得植株節間縮短達到矮化的效果；對於果樹也具有抑制新梢形成的能力，因此可以調整開花結果的時期。由於paclobutrazol 的使用劑量低且效果顯著，因此廣泛被農民使用於園藝作物之生產。然而paclobutrazol 在自然環境中非常穩定，半衰期超過兩年，水溶性非常差，因此施用後會長期累積於土壤中。如果農民不當超量使用，短期內雖然可以明顯的改進果樹生產，但是數年後常因為土壤中累積過多的paclobutrazol，使得植株根部生長被抑制而枯萎，改種其它果樹亦無法生長，造成農地廢耕。而這樣的問題確實已經出現在台灣南部地區的部分蓮霧果園中。因此，如何有效的處理土壤中殘留的paclobutrazol，恢復果園土地的生產能力，已成為一個相當重要的課題。Paclobutrazol 的穩定性極佳，一般微生物難以分解，截至目前為止，其代謝途徑也不清楚。四年前曾獲得農業國家型計畫的補助，當年曾經建立paclobutrazol 的分析方法及paclobutrazol 分解菌的大量篩選方法。由於研發主題不合農業國家型計畫之產業導向，隨後自行投入研究工作，很幸運的，由被paclobutrazol 汙染的土壤中分離到兩株Serratia marcescens菌株(NCHU 4-3 及NCHU 5-3)，對paclobutrazol 具有較強的分解能力，這是S. marcescens 首次被發現能分解paclobutrazol。因此，本研究的目的在於釐清S. marcescens 分解paclobutrazol的代謝途徑，選殖代謝paclobutrazol 之相關酵素基因，並將這些基因利用非抗藥性基因篩選標誌，選殖入非病源性的根圈細菌，利用具有新的代謝途徑的重組菌體，測試復育被paclobutrazol 汙染之土壤的可行性。本研究擬在三年內完成，研究工作如下：第一年一、分析S. marcescens NCHU 4-3 及S. marcescens NCHU 5-3 在不同培養條件下對paclobutrazol 之分解能力。二、預測可能參與paclobutrazol 生物分解之酵素及途徑。三、建立paclobutrazol 經菌株分解或修飾後之代謝產物之萃取(液相-液相萃取及固相萃取)與分析方法(包括氣相層析質譜、液相層析質譜及核磁共振分析)。四、從S. marcescens NCHU 4-3 及S. marcescens NCHU 5-3 選殖可能參與paclobutrazol 生物分解之第一個步驟的基因，並轉形至E. coli 中進行表現及分析。五、使用汎用型跳躍子(transposon)系統(EZ-Tn5TM Tnp TransposomeTM Kit)任意插入S. marcescens 菌株NCHU 4-3 或NCHU 5-3 的染色體中，篩選無paclobutrazol 分解能力的菌株(PBZ- 菌株)。第二年一、分離及純化paclobutrazol 之分解或修飾產物，並進行鑑定。二、針對被跳躍子破壞所產生的S. marcescens PBZ-菌株，利用跳躍子上之篩選標誌，分析插入位置的序列，並推斷其可能參與paclobutrazol 分解的角色。三、Paclobutrazol 分解或修飾途徑中，重要步驟的基因之選殖與表現，並分析其可能之角色。四、分析蓮霧根圈之主要微生物族群，俾建立環保微生物非抗藥性基因轉型系統。第三年一、使用非抗藥性基因篩選系統- lysine racemase，建立土壤微生物基因表現平台。二、以土壤細菌利用16S rDNA 之homologous recombintion 方式構築paclobutrazol 分解菌。三、構築完成之轉型菌株在實驗室內分別測試對培養基中之paclobutrazol 及土壤內外加paclobutrazol 之分解能力。四、申請專利。