生物防治菌木黴菌屬(Trichoderma spp.)之原生質體融合

Abstract

本論文主要是以木黴菌屬(Trichoderma spp.)為材料，希望能建立異種間 的融合系統，並得到較高的融合頻率，以便提高生物防治的效果。首先吾 人利用 PEG 和 CaCl2 兩種藥劑的轉形系統，促使原生質體吸收外來的 DNA 。將帶有抗 benomyl 基因的質體，轉形到康寧木黴菌 (Trichoderma koningii) 中 ，以 benomyl 來篩選而得到的轉形株，配 合本實驗室已經轉形成功的轉形株〔 帶有抗 hygromycin B 基因之質體 pUCH1 被轉形到哈氏木黴菌 (Trichoderma harzianum ) 中，經篩選後所 得之轉形株〕，將這兩種轉形株，利用 Novozym 234來分解其菌絲之細胞 壁，可獲得大約每200μl 穩定劑中5× 106-108個原生質體，且加前處理 則濃度更高。將這兩不同種原生質體融合在終濃度為33% 的 PEG 溶液中( 含有 10mM Tris -HCl 及 10mM CaCl2，pH 7.5)，然後培養於再生培養基 ，加入benomyl與 hygromycin B 來篩選，經此種方式進行原生質體融合 ，其融合頻率約為2 -5% 。在轉形株與融合株之進一步研究中，在拮抗作 用表現方面，乃利用土媒病原菌─立枯絲核菌之對峙培養，來評估其拮抗 作用能力，發現融合株之差異頗大，後經多次的次培養，拮抗能力已有明 顯提升；而經統計分析得知融合株以及轉形株與親本之拮抗能力無異。在 抗生素敏感試驗方面，可以得知融合株間表現差異性頗大，但與親本大致 相似，故知無突變株產生。在遺傳分離分析中，顯示融合株沒有分離現象 且應為同質核。再根據細胞DNA含量測定結果，融合株應為單倍體，而非 雙倍體。最後經過DNA南方氏雜交實驗，顯示篩選所得菌株，確實含有二 種外來質體 DNA，顯示融合成功而非突變株。

The objective of this paper was to set up the protoplast fusion technique for Trichoderma spp.,to obtain the stable and high frequency fusants,and to improve biocontrol effect.At first, pSV50 with anti-bonomyl gene was transfered to T.koningii and stable transformants were selected. Two kinds of protoplasts were fused by combining the selected anti-hygromycin B transformant from T. harzianum and the anti-benomyl transformant. Protoplasts from two transformants of T. koningii and T. harzianum, obtained from young thalli following cell wall digestion by Novozym 234, were fused in final 33% PEG suspended in 10mM Tris-HCl and 10mM CaCl2, pH 7.5. The frequency of fusion between anti-hygromycin B and anti-benomyl transformants was about 2-5% .The ability of all fusants, transformants and wild type to overgrow the soil-borne pathogenic fungi Rhizoctonia solani in dual culture was used to evaluate their antagonistic capability. The antagonistic ability of the fusant strains were found to vary with pathogenic fungi. The fusants were improved for their antagonistic ability by genetic purification (subculture).By statistical analysis,we could find that fuants and transformants had no distinct difference from parental strains. As to the effect of antibiotics on growth ,it exhibited some varities in fusants and fusants were similiar with parental strains. The results of DNA content and segregation analysis indicted that the interspecific funants had no segreation ,homologous, and haploid. DNA-DNA Southern hybridization revealed the integration of the pUCH1 and pSV50 plasmids into the fusant chromosome DNA .It indicated that a sucessful fusion was no longer a mutation.