本文選題：飛蝗 + 幾丁質(zhì)脫乙�；�　； 參考：《中國農(nóng)業(yè)科學(xué)》2017年06期

【摘要】：【目的】體外真核表達(dá)飛蝗(Locusta migratoria)幾丁質(zhì)脫乙�；�1和2(chitin deacetylase 1and 2,LmCDA1和LmCDA2)并測定其酶活性,為進(jìn)一步明確飛蝗LmCDA1和LmCDA2在幾丁質(zhì)降解途徑中的生理功能及研發(fā)新型綠色環(huán)保殺蟲劑提供依據(jù)。【方法】使用BLASTP和SMART軟件在線預(yù)測LmCDA1、LmCDA2a和LmCDA2b的結(jié)構(gòu)域;PCR克隆獲得目的基因LmCDA1、LmCDA2a和LmCDA2b的全長序列,并分別構(gòu)建p Fast Bac-LmCDAs重組質(zhì)粒,轉(zhuǎn)化獲得Bacmid重組質(zhì)粒后,轉(zhuǎn)染至昆蟲Sf9細(xì)胞進(jìn)行目的蛋白的體外表達(dá)。采用Western blot技術(shù)對目的蛋白表達(dá)情況進(jìn)行檢測,并通過Ni-NTA親和層析柱和陰離子(Q-Sepharose)交換層析柱對蛋白產(chǎn)物進(jìn)行純化。12%SDS-PAGE檢測蛋白純度后,采用分光光度法以對硝基乙酰苯胺為底物檢測目的蛋白的酶活性,T檢驗(yàn)法對LmCDA2a和LmCDA2b酶活力進(jìn)行差異顯著性分析。【結(jié)果】BLASTP和SMART軟件預(yù)測結(jié)果顯示LmCDA1、LmCDA2a和LmCDA2b均含有4個(gè)結(jié)構(gòu)域:N-端信號肽(signal peptide)、幾丁質(zhì)結(jié)合域(chitin binding peritrophin-A,Ch BD)、A型低密度脂蛋白受體結(jié)構(gòu)域(low-density lipoprotein receptor class A,LDLa)和脫乙�；复呋Y(jié)構(gòu)域(catalytic domain,CDA)。3個(gè)基因的幾丁質(zhì)結(jié)合域中均包含6個(gè)保守的半胱氨酸。LmCDA2a和LmCDA2b兩個(gè)剪切子除在其第3個(gè)半胱氨酸和第4個(gè)半胱氨酸之間(67—84 aa)的氨基酸數(shù)目和組成及在第4和第6個(gè)半胱氨酸(84—106 aa)之間的序列存在差異外,其余部分完全一致。Western blot結(jié)果顯示LmCDA1、LmCDA2a和LmCDA2b的蛋白分子量約為61 k D左右,與預(yù)測的蛋白分子量大小一致,表明Bacmid重組質(zhì)粒在昆蟲Sf9細(xì)胞中成功表達(dá)。采用12%SDS-PAGE膠電泳對各蛋白純化組分進(jìn)行檢測,結(jié)果顯示Ni-NTA親和層析柱可將大部分雜蛋白洗脫,而Q-Sepharose交換層析柱可對蛋白進(jìn)行更徹底地純化。酶活檢測結(jié)果顯示LmCDA1、LmCDA2a和LmCDA2b的酶活力分別為0.268、0.354、0.228 U·μL~(-1),并且LmCDA2a和LmCDA2b的酶活力存在顯著差異。【結(jié)論】體外真核表達(dá)LmCDA1、LmCDA2a和LmCDA2b蛋白并進(jìn)行酶活測定后發(fā)現(xiàn)三者均具有幾丁質(zhì)脫乙�；富盍�,且LmCDA2a和LmCDA2b的酶活力具有顯著性差異,推測前期研究中沉默LmCDA2a和LmCDA2b后分別出現(xiàn)不同飛蝗表型的原因可能是由于它們酶活力存在顯著性差異。
[Abstract]:[objective] to express chitin deacetylase 1 and 2(chitin deacetylase 1and 2 (LmCDA1 and LmCDA2) in eukaryotes of Locusta migratoriain vitro, and to determine the enzyme activity of LmCDA1 and LmCDA2. In order to further clarify the physiological function of Locust Locust LmCDA1 and LmCDA2 in chitin degradation pathway and to develop new green insecticides. [methods] using BLASTP and smart software on-line to predict LmCDA1LmCDA2a and LmCDA2b domain PCR cloning to obtain the order of LmCDA1LmCDA2a and LmCDA2b. [methods] using BLASTP and smart software on-line to predict LmCDA1LmCDA2a and LmCDA2b. The full-length sequence of LmCDA2a and LmCDA2b, The recombinant plasmids of p Fast Bac-LmCDAs were constructed and transformed into Bacmid recombinant plasmids. The recombinant plasmids were transfected into insect Sf9 cells for expression of the target protein in vitro. Western blot technique was used to detect the expression of the target protein, and the protein product was purified by Ni-NTA affinity chromatography column and anionic Q-Sepharose exchange chromatography. SDS-PAGE was used to detect the protein purity. The difference of enzyme activity between LmCDA2a and LmCDA2b was analyzed by spectrophotometric method using p-nitroacetanilide as substrate to detect the target protein. [results] BLASTP and smart software predicted that LmCDA1LmCDA2a and LmCDA2b both contained LmCDA2a and LmCDA2b, and the results of BLASTP and smart software showed that LmCDA1LmCDA2a and LmCDA2b both contained LmCDA2a and LmCDA2b. Four structural domains: N-terminal signal peptide, chitin binding rophin-Ach receptor domain low-density lipoprotein receptor class ALDLaand deacetylase catalytic domain, catalytic domain of deacetylase, catalytic domain, CDA. The chitin binding domains of the three genes all contain six conserved domains. The number and composition of amino acids of cysteine. LmCDA2a and LmCDA2b between the third cysteine and the fourth cysteine, and the sequence between the 4th and 6th cysteine (84-106aaa) were different. The results of Western blot showed that the molecular weight of LmCDA1a and LmCDA2b was about 61 KD, which was consistent with the predicted molecular weight of protein, indicating that Bacmid recombinant plasmid was successfully expressed in insect Sf9 cells. The purified proteins were detected by SDS-PAGE gel electrophoresis. The results showed that most of the proteins could be eluted by Ni-NTA affinity chromatography column, while the protein could be purified more thoroughly by Q-Sepharose exchange chromatography column. The enzyme activity of LmCDA1a and LmCDA2b were 0.268U 0.354U 渭 L ~ (-1) and 0.228U / L ~ (-1), respectively, and there were significant differences between LmCDA _ 2a and LmCDA _ 2b. [conclusion] the eukaryotic expression of LmCDA1-LmCDA2a and LmCDA2b protein in vitro were found to have the activity of chitinyldeacetylase, and the results showed that the enzyme activity of LmCDA2a and LmCDA2b were higher than that of LmCDA2b. [conclusion] the enzyme activity of LmCDA1a and LmCDA2b were determined in vitro. The enzyme activities of LmCDA2a and LmCDA2b were significantly different. It is speculated that the reasons of different phenotypes of LmCDA2a and LmCDA2b after silencing LmCDA2a and LmCDA2b in previous studies may be due to the significant differences in their enzyme activities.
【作者單位】： 山西大學(xué)應(yīng)用生物學(xué)研究所;山西大學(xué)生命科學(xué)學(xué)院;
【基金】：國家自然科學(xué)青年基金(31402020) 山西省基礎(chǔ)研究計(jì)劃(2015011070) 山西省回國留學(xué)人員科研資助項(xiàng)目(2015-007)
【分類號】：S433.2

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