长柄链格孢（Alternaria longipes）蛋白激酶基因克隆及功能研究
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摘要
信号转导是外界同生物体或生物体细胞间的信息传递的重要手段。大量研究表明蛋白的可逆磷酸化是细胞信号传递系统的重要组成部分,也是生物体内存在的一种最为普遍的调节方式,在许多生命活动中扮演重要角色,几乎涉及所有的生理及病理过程。蛋白质可逆磷酸化是由蛋白激酶(protein kinase,PK)和蛋白磷酸酶(protein phosphates,PP)催化完成。蛋白激酶作为重要的信号分子逐渐被广大学者所认识和接受。
本研究以长柄链格孢(Alternaria longipes)为研究对象,研究了A. longipes原生质体制备和再生的最佳方法和条件,采用10mg/mL的溶壁酶,选择菌龄为24h的菌体,酶解温度为30℃,酶解时间为3h,制备选择0.7M的NaCl,再生选择0.7M的蔗糖为稳渗剂,可以得到高浓度且再生能力强的原生质体。
利用限制性内切酶介导的整合技术成功的转化了A. longipes原生质体,并对转化体系进行了优化,包括限制性内切酶的种类和使用量、原生质体的浓度、质粒的形态和用量、PEG的浓度。本实验中用2μg HindⅢ线性化的质粒pUCATPH,转化过程中加入20U限制性内切酶HindⅢ,原生质体浓度为107个/mL,60%的PEG3350,共获得600多个转化子,转接5代能稳定遗传。随机挑选转化子进行PCR验证,结果证明潮霉素基因已经整合到受体菌的基因组中。
研究并优化了农杆菌介导的A. longipes分生孢子转化,包括AS的使用状态和使用浓度、共培养时间、受体菌孢子浓度、农杆菌浓度。在诱导过程和共培养过程中加200μmol/L AS处理,共培养48h,农杆菌OD600为0.25,受体孢子浓度为106个/mL时,共得到了85个转化子,转化效率明显低于REMI。
利用蛋白激酶同源保守序列设计兼并寡核苷酸引物,结合RT-PCR和RACE技术从A. longipes中克隆得到了一种蛋白激酶,全长cDNA1905bp,包含有一个1476bp的开放阅读框,编码491个氨基酸残基。该基因的cDNA和DNA序列已经在GenBank中注册,登录号分别为EU381116和EU3811167。运用生物信息学软件对A. longipes蛋白激酶编码基因进行碱基组成和氨基酸组成特征、功能位点、跨膜结构、保守区结构以及一级,二级结构等方面进行了分析,并与其他来源的蛋白激酶编码基因进行了同源比较。aapk1基因的催化域具有蛋白激酶催化域一般结构的特征。亚区Ⅰ的富含甘氨酸的区域(GTGSFGRV),亚区Ⅱ的K72和亚区Ⅲ的E91的保守碱基,亚区VIB的YRDLKPEN,亚区Ⅶ的DFG,亚区Ⅷ的GTPDYLAPE,亚区Ⅸ的D220,亚区XI的R280都是蛋白激酶中高度保守的碱基,并且在催化蛋白质的磷酸化过程中扮演着重要的角色。
采用同源重组利用双连接PCR(double-joint PCR)构建了aapk1基因的打靶载体,分别取基因上游652bp和下游620bp片段作为同源重组的两臂。对A. longipes原生质体进行了转化,采用PCR方法筛选到aapk1基因缺失突变体,并利用southern杂交和RT-PCR的方法对筛选的转化子△aapk1进行了验证,结果表明我们筛选的转化子就是aapk1基因缺失突变体。对△aapk1转化子和野生菌进行了比较,结果显示△aapk1转化子的产孢量和孢子的形状、大小、颜色都没有明显的差异,但△aapk1转化子在PDA上生长速度明显慢,PDB培养后测干重△aapk1转化子也低于野生菌,我们认为aapk1基因对于A. longipes的菌丝生长起着调控作用。对致病性的研究结果显示,△aapk1转化子孢子悬浮液接种NC89烟草的离体叶片,病斑出现晚,但病斑大小没有明显的差异。
Signal transduction plays an important role in the processes of environment-cell or cell-cell interaction. A lot of studies have resulted that kinases and phosphatases are likely to be important mediators of signal transduction.Through phosphrylation and dephosphorylation, the activity and structure of protein changed. External signal are transducted into the cell nucleolus by the signal cascade.
The best methods and conditions were studied for protoplast preparation and regeneration of Alternaria longipes. Protoplast with high concentration and strong ability can be obtained when the hypha were digested with 10 mg / mL Lywallzyme at 30℃for 3 h.The hypha used in this process were cultivate for 24h in PDB,and the osmotic stabilizers used in digestion and regeneration were NaCl and sucrose.
The protoplast of Alternaria longipes were successfully transformed with restriction enzyme-mediated integration and the transformation system has been examined, including the enzymes and their concentration, Alternaria longipes of protoplast, concentration of plasmid, concentration of PEG. Over 600 transformants were obtained when protoplasts were transformed with 2μg linear pUCATPH (digested by HindⅢ),20 U HindⅢ, 107 / mL protoplast and 60% of PEG3350。It indicated that the hygromycin B-resistant gene have been integrated into their genome by PCR amplification.Most of the transformants were fully stable after five rounds successive culture.
By using the system of Agrobacterium tumefacines-mediated transformation,we successfully transformed Alternaria longipes and obtained T-DNA insertion mutants.Under an optimum condition(Alternaria longipes concentration:106/mL;A.tumefacinesOD600=0.25; Acetosyringone concentration: 300μg/mL, co-cultivationtime: 48h), 85 transformants have been obtained and most of them were quite stable after five rounds successive culture.PCR amplification showed that the T-DNA was integrated into the genome, and was stable through mitotic cell division.
Degenerate primers designed on the conserved domain of other reported serine proteases, and a cDNA fragment encoding the protease gene was obtained through RT-PCR. The RACE was used to generate full-length cDNA clones. The full length of aapk1 cDNA gene is 1905bp, which contained an ORF of 1476bp encoding 491amino acids. The cDNA and DNA sequence of gene aapk1 has been registered in Genbank with accession number EU381116 and EU381117 respectively. The catalytic domain of aapk1 was compared with the common protein kinase,and the result showed that aapk1 has the common features,Such as GTGSFGRV in subdomainⅠ,K72 in subdomainⅡ,E91 in subdomainⅢ,YRDLKPEN in subdomainVIB,DFG in subdomainⅦ,GTPDYLAPE in subdomainⅧ,D220 in subdomainⅨ,R280 in subdomain XI。
The aapk1 gene targeting vector was constructed by double-joint PCR (DJ-PCR), and the homologous fragment was 652 bp and 620 bp, respectively. Protoplasts of A. longipes were transformed, and PCR was used to screen aapk1 gene deletion mutants(△aapk1). Southern blot and RT-PCR method were used to verify△aapk1 mutant. The wild type of A. longipes and△aapk1 mutants were compared and contrasted. the resulted showed that the differences between the conidia sporulation and the shape, size, color of conidia were not obvious, but△aapk1 transformat showed slow growth rate on PDA .Also the dry weight of△aapk1 mutants was lower than that of wild type of A. longipes.From these result we concluded that the aapk1 gene plays an important role in the mycelia growth of A. longipes.The pathogenicity test showed that the lesion which caused by inoculation with spores suspension of△aapk1 mutant occurred later than that of wild type of A. longipes,but the lesion size showed no significant difference.
本研究以长柄链格孢(Alternaria longipes)为研究对象,研究了A. longipes原生质体制备和再生的最佳方法和条件,采用10mg/mL的溶壁酶,选择菌龄为24h的菌体,酶解温度为30℃,酶解时间为3h,制备选择0.7M的NaCl,再生选择0.7M的蔗糖为稳渗剂,可以得到高浓度且再生能力强的原生质体。
利用限制性内切酶介导的整合技术成功的转化了A. longipes原生质体,并对转化体系进行了优化,包括限制性内切酶的种类和使用量、原生质体的浓度、质粒的形态和用量、PEG的浓度。本实验中用2μg HindⅢ线性化的质粒pUCATPH,转化过程中加入20U限制性内切酶HindⅢ,原生质体浓度为107个/mL,60%的PEG3350,共获得600多个转化子,转接5代能稳定遗传。随机挑选转化子进行PCR验证,结果证明潮霉素基因已经整合到受体菌的基因组中。
研究并优化了农杆菌介导的A. longipes分生孢子转化,包括AS的使用状态和使用浓度、共培养时间、受体菌孢子浓度、农杆菌浓度。在诱导过程和共培养过程中加200μmol/L AS处理,共培养48h,农杆菌OD600为0.25,受体孢子浓度为106个/mL时,共得到了85个转化子,转化效率明显低于REMI。
利用蛋白激酶同源保守序列设计兼并寡核苷酸引物,结合RT-PCR和RACE技术从A. longipes中克隆得到了一种蛋白激酶,全长cDNA1905bp,包含有一个1476bp的开放阅读框,编码491个氨基酸残基。该基因的cDNA和DNA序列已经在GenBank中注册,登录号分别为EU381116和EU3811167。运用生物信息学软件对A. longipes蛋白激酶编码基因进行碱基组成和氨基酸组成特征、功能位点、跨膜结构、保守区结构以及一级,二级结构等方面进行了分析,并与其他来源的蛋白激酶编码基因进行了同源比较。aapk1基因的催化域具有蛋白激酶催化域一般结构的特征。亚区Ⅰ的富含甘氨酸的区域(GTGSFGRV),亚区Ⅱ的K72和亚区Ⅲ的E91的保守碱基,亚区VIB的YRDLKPEN,亚区Ⅶ的DFG,亚区Ⅷ的GTPDYLAPE,亚区Ⅸ的D220,亚区XI的R280都是蛋白激酶中高度保守的碱基,并且在催化蛋白质的磷酸化过程中扮演着重要的角色。
采用同源重组利用双连接PCR(double-joint PCR)构建了aapk1基因的打靶载体,分别取基因上游652bp和下游620bp片段作为同源重组的两臂。对A. longipes原生质体进行了转化,采用PCR方法筛选到aapk1基因缺失突变体,并利用southern杂交和RT-PCR的方法对筛选的转化子△aapk1进行了验证,结果表明我们筛选的转化子就是aapk1基因缺失突变体。对△aapk1转化子和野生菌进行了比较,结果显示△aapk1转化子的产孢量和孢子的形状、大小、颜色都没有明显的差异,但△aapk1转化子在PDA上生长速度明显慢,PDB培养后测干重△aapk1转化子也低于野生菌,我们认为aapk1基因对于A. longipes的菌丝生长起着调控作用。对致病性的研究结果显示,△aapk1转化子孢子悬浮液接种NC89烟草的离体叶片,病斑出现晚,但病斑大小没有明显的差异。
Signal transduction plays an important role in the processes of environment-cell or cell-cell interaction. A lot of studies have resulted that kinases and phosphatases are likely to be important mediators of signal transduction.Through phosphrylation and dephosphorylation, the activity and structure of protein changed. External signal are transducted into the cell nucleolus by the signal cascade.
The best methods and conditions were studied for protoplast preparation and regeneration of Alternaria longipes. Protoplast with high concentration and strong ability can be obtained when the hypha were digested with 10 mg / mL Lywallzyme at 30℃for 3 h.The hypha used in this process were cultivate for 24h in PDB,and the osmotic stabilizers used in digestion and regeneration were NaCl and sucrose.
The protoplast of Alternaria longipes were successfully transformed with restriction enzyme-mediated integration and the transformation system has been examined, including the enzymes and their concentration, Alternaria longipes of protoplast, concentration of plasmid, concentration of PEG. Over 600 transformants were obtained when protoplasts were transformed with 2μg linear pUCATPH (digested by HindⅢ),20 U HindⅢ, 107 / mL protoplast and 60% of PEG3350。It indicated that the hygromycin B-resistant gene have been integrated into their genome by PCR amplification.Most of the transformants were fully stable after five rounds successive culture.
By using the system of Agrobacterium tumefacines-mediated transformation,we successfully transformed Alternaria longipes and obtained T-DNA insertion mutants.Under an optimum condition(Alternaria longipes concentration:106/mL;A.tumefacinesOD600=0.25; Acetosyringone concentration: 300μg/mL, co-cultivationtime: 48h), 85 transformants have been obtained and most of them were quite stable after five rounds successive culture.PCR amplification showed that the T-DNA was integrated into the genome, and was stable through mitotic cell division.
Degenerate primers designed on the conserved domain of other reported serine proteases, and a cDNA fragment encoding the protease gene was obtained through RT-PCR. The RACE was used to generate full-length cDNA clones. The full length of aapk1 cDNA gene is 1905bp, which contained an ORF of 1476bp encoding 491amino acids. The cDNA and DNA sequence of gene aapk1 has been registered in Genbank with accession number EU381116 and EU381117 respectively. The catalytic domain of aapk1 was compared with the common protein kinase,and the result showed that aapk1 has the common features,Such as GTGSFGRV in subdomainⅠ,K72 in subdomainⅡ,E91 in subdomainⅢ,YRDLKPEN in subdomainVIB,DFG in subdomainⅦ,GTPDYLAPE in subdomainⅧ,D220 in subdomainⅨ,R280 in subdomain XI。
The aapk1 gene targeting vector was constructed by double-joint PCR (DJ-PCR), and the homologous fragment was 652 bp and 620 bp, respectively. Protoplasts of A. longipes were transformed, and PCR was used to screen aapk1 gene deletion mutants(△aapk1). Southern blot and RT-PCR method were used to verify△aapk1 mutant. The wild type of A. longipes and△aapk1 mutants were compared and contrasted. the resulted showed that the differences between the conidia sporulation and the shape, size, color of conidia were not obvious, but△aapk1 transformat showed slow growth rate on PDA .Also the dry weight of△aapk1 mutants was lower than that of wild type of A. longipes.From these result we concluded that the aapk1 gene plays an important role in the mycelia growth of A. longipes.The pathogenicity test showed that the lesion which caused by inoculation with spores suspension of△aapk1 mutant occurred later than that of wild type of A. longipes,but the lesion size showed no significant difference.
引文
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