华北农学报
華北農學報
화북농학보
ACTA AGRICULTURAE BOREALI-SINICA
2015年
2期
78-86
,共9页
王凯%赵洋%朱先婷%才秀华%武侠
王凱%趙洋%硃先婷%纔秀華%武俠
왕개%조양%주선정%재수화%무협
虫草棒束孢%南方根结线虫卵%克隆%几丁质酶基因%序列分析
蟲草棒束孢%南方根結線蟲卵%剋隆%幾丁質酶基因%序列分析
충초봉속포%남방근결선충란%극륭%궤정질매기인%서렬분석
Isaria farinosa%Meloidogyne incognita%Cloning%Chitinase gene%Sequence analysis
为研究卵寄生真菌在寄生并破坏卵的过程中几丁质酶的作用，本研究从烟草南方根结线虫卵上分离得到虫草棒束孢 HNE3，采用 RT-PCR 及 RACE 技术，首次克隆得到一个几丁质酶基因 IFCHI1。该基因 DNA 序列全长1417 bp，具有1个内含子，长度为61 bp，包含1个1185 bp的开放阅读框（ ORF），编码1个由394个氨基酸组成的蛋白。通过在线软件分析，该蛋白理论分子量为44.1 kDa，等电点为4.88。通过 GenBank 比对，几丁质酶 IFCHI1属于第18家族糖基水解酶，比对分析不同来源真菌几丁质酶，发现该几丁质酶具有典型的催化区保守序列 SIGGW 和FDGIDIDWE及结合区保守序列 GTWEQGVYDY和 GLGGAMWWESS。同源性比对发现，该几丁质酶同昆虫寄生真菌几丁质酶同源关系近。结果表明，从虫草棒束孢 HNE3克隆得到几丁质酶基因 IFCHI1，为进一步明确该菌产生的几丁质酶对南方根结线虫卵壳降解及降低孵化率的作用机理提供理论依据。
為研究卵寄生真菌在寄生併破壞卵的過程中幾丁質酶的作用，本研究從煙草南方根結線蟲卵上分離得到蟲草棒束孢 HNE3，採用 RT-PCR 及 RACE 技術，首次剋隆得到一箇幾丁質酶基因 IFCHI1。該基因 DNA 序列全長1417 bp，具有1箇內含子，長度為61 bp，包含1箇1185 bp的開放閱讀框（ ORF），編碼1箇由394箇氨基痠組成的蛋白。通過在線軟件分析，該蛋白理論分子量為44.1 kDa，等電點為4.88。通過 GenBank 比對，幾丁質酶 IFCHI1屬于第18傢族糖基水解酶，比對分析不同來源真菌幾丁質酶，髮現該幾丁質酶具有典型的催化區保守序列 SIGGW 和FDGIDIDWE及結閤區保守序列 GTWEQGVYDY和 GLGGAMWWESS。同源性比對髮現，該幾丁質酶同昆蟲寄生真菌幾丁質酶同源關繫近。結果錶明，從蟲草棒束孢 HNE3剋隆得到幾丁質酶基因 IFCHI1，為進一步明確該菌產生的幾丁質酶對南方根結線蟲卵殼降解及降低孵化率的作用機理提供理論依據。
위연구란기생진균재기생병파배란적과정중궤정질매적작용，본연구종연초남방근결선충란상분리득도충초봉속포 HNE3，채용 RT-PCR 급 RACE 기술，수차극륭득도일개궤정질매기인 IFCHI1。해기인 DNA 서렬전장1417 bp，구유1개내함자，장도위61 bp，포함1개1185 bp적개방열독광（ ORF），편마1개유394개안기산조성적단백。통과재선연건분석，해단백이론분자량위44.1 kDa，등전점위4.88。통과 GenBank 비대，궤정질매 IFCHI1속우제18가족당기수해매，비대분석불동래원진균궤정질매，발현해궤정질매구유전형적최화구보수서렬 SIGGW 화FDGIDIDWE급결합구보수서렬 GTWEQGVYDY화 GLGGAMWWESS。동원성비대발현，해궤정질매동곤충기생진균궤정질매동원관계근。결과표명，종충초봉속포 HNE3극륭득도궤정질매기인 IFCHI1，위진일보명학해균산생적궤정질매대남방근결선충란각강해급강저부화솔적작용궤리제공이론의거。
In order to explore the function of chitinase in parasitizing and destroying egg,a nematode egg-paraastic fungus Isaria farinosa HNE3 was isolated from Meloidogyne incognita infecting tobacco in Henan Prov-ince. By using RT-PCR and RACE,we had cloned a chitinase gene IFCHI1 for the first time. The full-length DNA of IFCHI1 was 1 417 bp,containing a intron with the length of 61 bp,ORF was 1 185 bp and it encoded 394 amino acids. Analyzing chitinase IFCHI1 by online biological software deduced that its molecular mass and isoelecftric point were 44. 1 kDa and 4. 88,especially. By compared with other chitinase from different fungus in GenBank,IF-CHI1 belongs to glycosyl hydrolase family 18 chitinase. We found two prevalent conserved catalytic domains with the sequence of SIGGW and FDGIDIDWE and two binding domains with the sequence of GTWEQGVYDY and GLG-GAMWWESS. Comparison of chitinase IFCHI1 with other chitinases from entomopathogenic fungi revealed that en-zymes were highly similar. It was concluded that the chitinase coded by gene IFCHI1 provided theoretical basis for damaging on eggshell structure and inhibiting on egg-hatching of M. incognita.
