STRUCTURE OF CUTINASE GENE, CDNA, AND THE DERIVED AMINO-ACID-SEQUENCE FROM PHYTOPATHOGENIC FUNGI
STRUCTURE OF CUTINASE GENE, CDNA, AND THE DERIVED AMINO-ACID-SEQUENCE FROM PHYTOPATHOGENIC FUNGI
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DOI：
10.1021/bi00398a052
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发表时间：
1987-12-01
期刊：
影响因子：
2.9
通讯作者：
KOLATTUKUDY, PE
中科院分区：
文献类型：
--
作者：
ETTINGER, WF;THUKRAL, SK;KOLATTUKUDY, PE
Cutinase是一种胞外真菌酶，在真菌感染的最初阶段，它允许病原真菌穿过角质层屏障进入寄主植物。利用从葡萄糖培养的辣椒炭疽菌中提取的经角质水解物诱导产生角质酶的mRNA，制备cDNA，并克隆到表达载体lambda中。Gt11.根据克隆的角质酶基因的核苷酸序列，推测了辣椒角质酶的一级结构。从辣椒菌产生的角质酶中分离到的两个胰蛋白酶多肽的氨基酸序列与由核苷酸序列推导出的两个氨基酸序列完全匹配，这有力地表明所克隆的cDNA是真正的角质酶基因。以该克隆为探针，分别筛选构建于Charon35和EMBL3中的辣椒炭疽菌和辣椒炭疽菌基因组文库。测定了辣椒炭疽菌和胶孢霉菌的角质酶结构基因的核苷酸序列。通过S1定位揭示了辣椒链霉菌初级转录本的转录起始点和多聚腺苷化位点。比较了炭疽菌的酶的一级结构和基因结构与茄病镰刀菌角质酶的一级结构和基因结构。皮西。对推导出的酶一级结构的比较表明，角质酶催化三联体和二硫键交联的残基是非常保守的。然而，只有43%的残基在所有三种酶之间都是保守的。对这三个基因的结构进行比较，发现单个内含子的位置是保守的。该基因的转录起始点位于序列TCCAGACCA上，其核心(CAGAC)在21个核苷酸之后重复。在球孢霉和立枯丝核菌基因的5‘端非翻译区也发现了相同的核心序列，在11个核苷酸之后重复。
Cutinase is an extracellular fungal enzyme that allows pathogenic fungi to penetrate through the cuticular barrier into the host plant during the initial stages of the fungal infection. mRNA isolated from glucose-grown Colletotrichum capsici, induced to produce cutinase by the addition of cutin hydrolysate, was used to prepare cDNA which was cloned in the expression vector .lambda. gt11. The primary structure of the cutinase from C. capsici was deduced from the nucleotide sequence of the cloned cutinase cDNA. Amino acid sequences of two tryptic peptides isolated from cutinase produced by C. capsici completely matched with two segments of the amino acid sequence deduced from the nucleotide sequence, strongly suggesting that the cloned cDNA was authentic cutinase cDNA. The cDNA clone was used as probe to screen C. capsici and Colletotrichum gloeosporioides genomic libraries constructed in Charon 35 and EMBL 3, respectively. The nucleotide sequences of the cutinase structural genes from C. capsici and C. gloeosporioides were also determined. S1 mapping was used to reveal the transcriptional start sites and polyadenylation site of the primary transcript from C. capsici. The primary sequences and gene structure of the enzymes from the Colletotrichum species were compared with the primary structure and gene structure of a cutinase from Fusarium solani f. sp. pisi. A comparison of the deduced primary structures of the enzymes showed that residues involved in the catalytic triad and disulfide cross-linking of cutinase are strongly conserved. Yet, only 43% of the residues are conserved between all three enzymes. A comparison of the structure of the three genes revealed the location of the single intron has been conserved. The transcriptional start site of the C. capsici gene was centered on the sequence TCCAGACCA, the core of which (CAGAC) is found repeated after 21 nucleotides. The same core sequence, repeated after 11 nucleotides, was also identified in the 5'' nontranslated regions of the C. gloeosporioides and F. solani genes.