龙眼焦腐病菌（Lasiodiplodia theobromae）转化体系的建立
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摘要
龙眼(Phyllanthus reticulatus Pior)采后贮藏期真菌性病害主要是由于几种病原真菌的潜伏侵染造成的,其中龙眼焦腐病菌[Lasiodiplodia theobromae (Pat.) Griff. &Maubl.]分离频率最高(46%)。为进一步研究龙眼焦腐病菌致病的分子机制,本文致力于该菌遗传转化系统的建立。
首先,为获得大量分生孢子以培养细嫩菌丝用于原生质体制备,比较了龙眼焦腐病菌在不同培养条件下产生子座的能力,结果表明燕麦和米糠培养基最适于该菌产生子座;培养基pH6.0,连续光照15d,子座产生量最大。
其次,对龙眼焦腐病菌原生质体的制备和再生条件进行了的研究,结果是新鲜的分生孢子在CM液体培养基中培养30h左右的菌丝,采用Sorbital(1M,pH 5.5～6)作为酶液稳渗剂,用组合酶:1%纤维素酶+1%溶壁酶+1%蜗牛酶+1%崩溃酶作为裂解酶,30℃裂解3～3.5h,原生质体生成量最大,其再生率也最高,达到40%。
最后,采用PEG介导转化法将pEGFP-H3质粒转化龙眼焦腐病菌原生质体,得到35个转化子,随机挑取6个转化子经PCR验证和测序结果确认均为阳性克隆,说明了我们已经初步建立了龙眼焦腐病菌的遗传转化体系。
Several patogenic fungi cause post-harvest diseases of longan (Phyllanthus reticu latus Pior), in which the latent infection by Lasiodiplodia theobromae (Pat.) Griff. & Maubl is major one. It has the highest frequency (up to 46%) in fungal isolation from the post-harvest logan fruits. To further study the molecular mechanisms of disease, we tried to establish the transformation system of Lasiodiplodia theobromae in this study.
First, we studied the capacity of conidiation of Lasiodiplodia theobromae (Pat.) Griff & Maubl under different culture conditions in order to obtain a large number of conidia for cultivating delicate mycelia to prepare for the protoplast. The results showed that oat and rice polish media are suitable for the fungus to produce stromata, and the quantity of stromata can reach the maximum when cultured at the pH 6.0 in media and under successive light for 15 days.
Second, we optimized the conditions of the protoplast preparation and its regeneration. The fresh conidia were incubated in CM liquid medium for 30h and then the hyphae were collected, and digested by combination of enzyme (1%Nailase+1%Lyticase+1%Cellulase+1%Driselase) at 30℃by using sorbital (1M, pH 5.5) as buffer. The quantity and quality of the protoplast reached the maximum under this optimized condition.
Last, we transformed plasmid pEGFP-H3 into the fungal protoplast with PEG-mediated transformation method. In total, 35 transformants were obtained, and 6 transformants were randomly selected to conduct further study. These clones were confirmed to be positive transformants by PCR verification and sequencing.
In a summary, our results showed that we had primarily established a genetic transformation system of the fungus Lasiodiplodia theobromae.
首先,为获得大量分生孢子以培养细嫩菌丝用于原生质体制备,比较了龙眼焦腐病菌在不同培养条件下产生子座的能力,结果表明燕麦和米糠培养基最适于该菌产生子座;培养基pH6.0,连续光照15d,子座产生量最大。
其次,对龙眼焦腐病菌原生质体的制备和再生条件进行了的研究,结果是新鲜的分生孢子在CM液体培养基中培养30h左右的菌丝,采用Sorbital(1M,pH 5.5～6)作为酶液稳渗剂,用组合酶:1%纤维素酶+1%溶壁酶+1%蜗牛酶+1%崩溃酶作为裂解酶,30℃裂解3～3.5h,原生质体生成量最大,其再生率也最高,达到40%。
最后,采用PEG介导转化法将pEGFP-H3质粒转化龙眼焦腐病菌原生质体,得到35个转化子,随机挑取6个转化子经PCR验证和测序结果确认均为阳性克隆,说明了我们已经初步建立了龙眼焦腐病菌的遗传转化体系。
Several patogenic fungi cause post-harvest diseases of longan (Phyllanthus reticu latus Pior), in which the latent infection by Lasiodiplodia theobromae (Pat.) Griff. & Maubl is major one. It has the highest frequency (up to 46%) in fungal isolation from the post-harvest logan fruits. To further study the molecular mechanisms of disease, we tried to establish the transformation system of Lasiodiplodia theobromae in this study.
First, we studied the capacity of conidiation of Lasiodiplodia theobromae (Pat.) Griff & Maubl under different culture conditions in order to obtain a large number of conidia for cultivating delicate mycelia to prepare for the protoplast. The results showed that oat and rice polish media are suitable for the fungus to produce stromata, and the quantity of stromata can reach the maximum when cultured at the pH 6.0 in media and under successive light for 15 days.
Second, we optimized the conditions of the protoplast preparation and its regeneration. The fresh conidia were incubated in CM liquid medium for 30h and then the hyphae were collected, and digested by combination of enzyme (1%Nailase+1%Lyticase+1%Cellulase+1%Driselase) at 30℃by using sorbital (1M, pH 5.5) as buffer. The quantity and quality of the protoplast reached the maximum under this optimized condition.
Last, we transformed plasmid pEGFP-H3 into the fungal protoplast with PEG-mediated transformation method. In total, 35 transformants were obtained, and 6 transformants were randomly selected to conduct further study. These clones were confirmed to be positive transformants by PCR verification and sequencing.
In a summary, our results showed that we had primarily established a genetic transformation system of the fungus Lasiodiplodia theobromae.
引文
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