家蚕蛋白酶抑制剂抵御昆虫致病性真菌入侵的分子机理研究
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摘要
家蚕是一种具有很高经济价值的绢丝昆虫,有大量的基础研究积累,已经逐渐发展成鳞翅目的模式生物。然而,家蚕易受到环境中各种病原微生物和寄生虫等威胁,发生疾病。昆虫致病性真菌,如绿僵菌、球孢白僵菌、黄曲霉菌和米曲霉菌,都可能引发高致病性蚕病,严重影响茧丝产量和质量,给整个桑蚕行业造成重大经济损失。昆虫致病性真菌,作为一种新型生物农药,已被广泛应用于农林害虫防治和蚊虫控制。真菌生物农药的使用,不可避免的会与家蚕发生交叉感染,可能导致蚕病爆发。因此,阐明昆虫致病性真菌与家蚕相互作用的分子机制,发现新的抗真菌分子,不仅有利于提高家蚕抗真菌能力,而且对农林害虫防治及人类真菌疾病治疗也具有重要意义。
昆虫致病性真菌,通过利用丰富的水解酶来穿透昆虫体壁。这些水解酶包括体壁降解蛋白酶和几丁质酶,它们都是重要的毒力因子。过表达这些毒性蛋白酶,可以显著增强致病性真菌的毒力。虽然真菌穿透昆虫体壁的基本模式已经提出,但昆虫是怎样抵御真菌入侵以及与真菌之间具体的相互作用如何都不甚明了,有待于进一步研究。
昆虫体壁和血液中含有丰富的蛋白酶抑制剂,这些抑制剂与抵御病原微生物入侵密切相关。为了阐明昆虫抵御真菌入侵的分子机制以及为提高家蚕抗真菌能力寻找新的分子靶标,我们重点对丝氨酸蛋白酶抑制剂进行深入研究。本研究室利用基因芯片技术对家蚕在微生物诱导下的基因表达变化进行调查,发现了19个家蚕丝氨酸蛋白酶抑制剂在微生物经口感染家蚕后,上调或者下调表达,初步表明这些丝氨酸蛋白酶抑制剂可能参与防御病原微生物入侵的过程。特别是TIL(trypsin inhibitor-like cysteine rich domain)家族的蛋白酶抑制剂在球孢白僵菌感染后明显上调表达。因此,可以从家蚕蛋白酶抑制剂中寻找能够有效对抗致病性真菌入侵的蛋白分子,以提高家蚕抗真菌能力。基于此,本论文对TIL家族的BmSPI38和BmSPI39进行了全长克隆、原核表达和活性测定,并对其抵御真菌入侵的分子机理进行了深入的研究；利用3D结构预测和定点突变技术,对BmSPI38和BmSPI39的抑制活性的关键位点进行了分析；并对BmSPI38和BmSPI39在家蚕中的生理存在形式及BmSPI39在茧丝保护中的功能进行了系统的研究。本论文获得的主要研究结果如下：
1.丝氨酸蛋白酶抑制剂的全长克隆、原核表达及活性测定
基于BmSPI38和BmSPI39(?)勺EST序列,我们对这两个基因分别设计了4个基因特异引物,用于该基因的5'RACE扩增和3'RACE扩增,获得了这两个基因的全长cDNA序列。BmSPI38和BmSPI39都具有一个含有八个半胱氨酸残基的TIL结构域。多序列比对结果表明,与其它物种的丝氨酸蛋白酶抑制剂相比,BmSPI38和BmSPI39的TIL结构域中有2个Cys发生了替换’(Cys2th和Cys6th),且活性位点也发生了明显变化。基于独特的活性位点和保守的Cys数目,我们认为BmSPI38和BmSPI39是TIL家族丝氨酸蛋白酶抑制剂的新成员。
系统发生树分析显示,所有TIL类蛋白酶抑制剂聚为3支,家蚕BmSPI35、 BmSPI36、BmSPI37、BmSPI38、BmSPI39、BmSPI40和BmFPI-F(家蚕真菌蛋白酶抑制剂-F)聚为一支(Group Ⅲ),铃蟾BbBSTI独自聚为一支(Group Ⅱ),其余丝氨酸蛋白酶抑制剂聚为一支(Group Ⅰ)。家蚕BmSPI38、BmSPI39与其它物种的TIL家族的蛋白酶抑制剂进化关系较远,而与BmFPI-F的序列相似性较高,推测它们与BmFPI-F具有相似的功能,参与家蚕的免疫过程。
利用原核表达技术,我们获得了具有生物活性的BmSPI38和BmSPI39重组蛋白。通过对不同温度或pH条件下的抑制剂活性进行测定,我们发现BmSPI38和BmSPI39具有很高的热和酸碱稳定性。为了确定BmSPI38和BmSPI39所抑制的蛋白酶的类型,我们选择了几种蛋白酶来进行酶活测定实验,结果表明BmSPI38和BmSPI39能够强烈地抑制微生物蛋白酶,如枯草杆菌蛋白酶A和蛋白酶K,但对胰蛋白酶、胰凝乳蛋白酶和弹性蛋白酶都没有抑制活性。
2.BmSPI38和BmSPI393(?)寸真菌体壁降解蛋白酶的抑制及其动力学特征分析
为了探讨BmSPI38和BmSPI39是否参与抵御昆虫致病性真菌,我们选取了球孢白僵菌Prl蛋白酶(CDEP-1)来进行酶活抑制和体外结合实验。结果表明,BmSPI38和BmSPI39能够强烈地抑制球孢白僵菌的毒力蛋白酶CDEP-1。RT-PCR分析表明,BmSPI38在头、体壁和脂肪体等免疫相关组织中高量表达,暗示其可能通过抑制真菌分泌的体壁降解蛋白酶,进而阻止真菌穿透表皮侵染家蚕；BmSPI39在家蚕前部和中部丝腺中特异表达,可能参与丝腺相关的某种过程。与绿僵菌的Prl蛋白酶相似,CDEP-1能够诱导家蚕的黑化反应。而BmSPI38和BmSPI39能阻断CDEP-1诱导的黑化反应。这表明,家蚕蛋白酶抑制剂BmSPI38和BmSPI39(?)能够抑制CDEP-1蛋白酶诱导的杀虫性黑化,进而保护家蚕。酚氧化酶前体体外激活实验表明,CDEP-1不能直接激活PPO,可能是通过激活PPO上游的某种蛋白因子,间接激活PPO。本研究阐明了家蚕应对真菌伤害的另一种途径,即阻断昆虫致病性真菌分泌的蛋白酶引发的有害黑化。球孢白僵菌感染后108h,家蚕血液发生了一定程度的黑化,且黑化程度与分生孢子浓度在一定程度上具有正相关性,表明酚氧化酶级联的黑化反应,参与对真菌入侵的免疫应答。
本研究中使用的枯草杆菌蛋白酶A、蛋白酶K和体壁降解蛋白酶CDEP-1都属于Peptidases_S8家族,具有一个Peptidases_S8结构域。序列分析表明,Peptidases_S8家族相似性很高,已报道的体壁降解蛋白酶主要是PCSK9_ProteinaseK_like亚家族与Subtilisin_subset亚家族中成员,是真菌重要的毒力因子。鉴于枯草杆菌蛋白酶类的相似性很高,且BmSPI38和BmSPI39能够强烈地抑制这些微生物蛋白酶活性,我们认为蛋白酶抑制剂BmSPI38和BmSPI39有助于提高家蚕的抗真菌能力。
3.蛋白酶抑制剂抑制球孢白僵菌的入侵
为了研究BmSPI39等蛋白酶抑制剂是否参与家蚕对真菌的免疫应答,我们利用不同浓度的球孢白僵菌感染家蚕,并对攻毒后不同时间点的体壁中的BmSPI39进行Western blot分析。结果表明,家蚕蛋白酶抑制剂BmSPI39以四聚体形式在体壁中表达,在球孢白僵菌感染后,先上调表达,后下调表达,参与家蚕对真菌的免疫应答。免疫荧光实验表明,BmSPI39主要定位于内表皮和中表皮中。鉴于该抑制剂能够抑制真菌的体壁降解蛋白酶活性,我们认为体壁中的蛋白酶抑制剂可能是通过抑制真菌的体壁降解蛋白酶活性,来减缓真菌对昆虫体壁的穿透过程。
为了进一步研究蛋白酶抑制剂能否增强家蚕的抗真菌能力,我们将蛋白酶抑制剂BmSPI38和BmSPI39重组蛋白作为球孢白僵菌孢子悬液的添加剂,通过体表浸泡法处理家蚕。单独应用球孢白僵菌或与BSA、BmSPI38或BmSPI39等一起处理家蚕。实验结果表明,随着攻毒后时间的增加,家蚕的累积发病率逐渐增加。截止到第5天,单独使用球孢白僵菌攻毒的家蚕累积发病率为64.67%,而使用BSA、BmSPI38或BmSPI39重组蛋白添加剂的的家蚕累积发病率分别为44.44%、18%和8%。我们对攻毒处理第5天的家蚕进行观察,发现单独使用球孢白僵菌和以BSA作为添加剂的处理组,多数家蚕表现为：体壁出现黑色破损病斑,体色加深无光泽,体表脱水下陷,活动性差,触摸感觉迟钝等现象。而蛋白酶抑制剂BmSPI38或BmSPI39的重组蛋白的处理组中,多数家蚕没有明显的发病征兆。家蚕体壁出现黑色斑点,说明家蚕体壁中存在酚氧化酶级联系统,当球孢白僵菌感染家蚕时,会激活家蚕的黑化反应,从而参与抵御真菌的免疫应答。意外的是,蚕蛹节间膜没有发现明显的黑色斑点,这可能是由于节间膜处缺乏酚氧化酶级联系统,不能有效激活黑化反应。通过对五个不同处理组的生存率进行统计分析,发现BmSPI38和BmSPI39都能够显著提高家蚕的生存率,表明蛋白酶抑制剂能够增强家蚕的抗真菌能力,可以作为提高家蚕抗性的靶标基因。
4.蛋白酶抑制剂BmSPI38和BmSPI39的抑制特异性关键位点分析
家蚕蛋白酶抑制剂与其它物种的TIL类蛋白酶抑制剂的多序列比对结果表明,BmSPI38和BmSPI39的氨基酸序列中有两个半胱氨酸发生了替换,因此,我们推测这两个位置的氨基酸残基是决定其抑制活性的关键位点。利用定点突变技术将这两个位置的Asp和Leu分别突变为Cys。我们对BmSPI39突变前后的三维结构进行了建模预测。结果表明,BmSPI39中只含有2个反平行的β折叠片和一个小α螺旋,其余区域都是柔性的loop区域。整个结构中含有四对二硫键(Cys29-Cys62、 Cys40-Cys5、Cys44-Cys92和Cys64-Cys76),它们维持了蛋白刚性结构的稳定,使大片的柔性loop区域形成一个相对紧凑而又具有一定柔性的稳定结构。将BmSPI39与突变后的BmSPI39Mu的三维结构进行叠合比较发现,新突变的两个Cys分别位于相邻的两个loop区域,它们会形成一个分子内二硫键,并将相邻的两个loop区域拉近,同时,Thr36m-O和Thr61m-N形成一对氢键以稳定该结构的变化。新生成的二硫键(Cys38m-Cys58m)与相邻的二硫键(Cys40m-Cys54m)一起形成一个稳定的刚性区域,P1残基Ala56m正好处于这个刚性区域中间。
将BmSPI38和BmSPI39中的两个氨基酸定点突变为半胱氨酸,突变后的BmSPI38Mu和BmSPI39Mu对枯草杆菌蛋白酶类的抑制活性大大降低,表明这两个位置的半胱氨酸替换是BmSPI38和BmSPI39获得抑制微生物蛋白酶能力的一个重要原因。然而,BmSPI38Mu和BmSPI39Mu没有获得对胰蛋白酶、胰凝乳蛋白酶或弹性蛋白酶等的抑制能力,推测P1残基在决定其抑制特异性中起重要作用。通过对TIL家族蛋白酶抑制剂活性位点及半胱氨酸数目进行分析,蛋白酶抑制剂的抑制特异性与TIL结构域中的半胱氨酸数目和P1残基性质及大小存在很大的关联。本研究表明,在进化过程中,家蚕TIL家族蛋白酶抑制剂中P1残基替换为小分子的中性氨基酸,使其获得了一定程度的抑制微生物蛋白酶活性的能力,半胱氨酸的替换增强了其抑制的活性和特异性。
5.BmSPI38和BmSPI39体内存在形式的分析及在茧壳保护中的功能研究
利用还原与非还原SDS-PAGE电泳、MALDI-TOF MS和胶内活性染色技术,我们对BmSPI38和BmSPI39重组蛋白的存在形式进行了分析。结果发现,BmSPI38和BmSPI39重组蛋白主要以多聚体形式存在。Western blot结果表明,BmSPI38和BmSPI39主要以四聚体形式分布于各组织器官中,可能以多聚体形式在家蚕中执行功能。
RT-PCR及Western blot结果表明,BmSPI39在5龄第5天家蚕的前部和中部丝腺特异表达,推测BmSPI39可能参与丝腺相关的某种过程。BmSPI39在上蔟前的丝腺中高量表达,并随着泌丝过程进入茧壳中。我们的研究表明,蛋白酶抑制剂BmSPI39能够通过抑制微生物蛋白酶对茧壳蛋白的水解过程,从而为预蛹和蛹提供长效有力的保护。
Bombyx mori is a silk-producing insect with tremendous economic value. Based on the immense accumulation of fundamental research, B. mori has gradually developed into a model organism of Lepidoptera. However, silkworm is subject to environmental threats of various pathogenic microorganisms and parasites, which lead to serious diseases. Entomopathogenic fungi such as Metarhizium anisopliae, Beauveria bassiana, Aspergillus flavus, and Aspergillus oryzae can cause various communicable diseases, which seriously affect the production and quality of cocoon, and cause enormous economic losses in the silkworm industry. Entomopathogenic fungi, as a new biopesticide, has been widely used in agriculture and forestry pest control and mosquito control. The use of fungal biological pesticide, would trigger inevitable cross infection in silkworm, and lead to silkworm disease outbreak. Therefore, elucidation of the molecular mechanism of the interaction between entomopathogenic fungi and B. mori, and discovering novel antifungal molecules, would be beneficial to raising the antifungal ability of B. mori and has vast importance to agricultural pest control and cures for human fungal diseases.
Entomopathogenic fungi penetrate the insect cuticle using its abundant hydrolases. These hydrolases, including cuticle-degrading protease and chitinase, both of them are important virulence factors. Overexpression of toxic protease can significantly enhance the virulence of pathogenic fungi. Although the basic model of fungal penetration of insect integument has been proposed, how do insects protect against fungal invasion and specific interactions between insects and fungi has yet to be further studied.
The insect integument and hemolymph contain a large number of protease inhibitors, which are closely related to resistance against pathogenic microorganisms. To elucidate the molecular mechanism of insect resistance to fungal invasion and seek novel targets to improve silkworm antifungal ability, we focus on in-depth study of the serine protease inhibitors. In our previous study, microarray was used to screen expression-changed genes after microbial challenge. At least19serine protease inhibitors were up-regulated or down-regulated after microbial induced via oral. The preliminal results indicated that these serine protease inhibitors may be involved in resistance against pathogenic microorganisms. In particular, the expression level of various TIL-type protease inhibitors were significantly up-regulated after B. bassiana infection. Hence we can look for effective protein molecules against pathogenic fungus in order to enhance the antifungal ability of B. mori. In view of this, we cloned the full length cNDA of BmSPI38and BmSPI39from TIL family, expressed them in the prokaryotic cells, measured their activities and further studied the molecular mechanism of silkworm protease inhibitors against fungal invasion.3D structure prediction and site-directed mutagenesis were used to analyse the key sites for the inhibitory activity of BmSPI38and BmSPI39. Their physiological existence in the silkworm and the protection function of BmSPI39in the cocoon was researched systematically. The major results in this study are as follows:
1. Full length cNDA clone, prokaryotic expression and activity assay of serine protease inhibitors
Based on the known EST sequences of BmSPI38and BmSPI39, we designed respectively four gene-specific primers for3'-and5'-RACE PCR. Finally, we obtained their fall length cDNA sequences. Both of BmSPI38and BmSPI39contain a single TIL domain with eight cysteines. Multiple sequence alignment showed that two cysteine(Cys2th and Cys6th) substitutions occurred in TIL domain, and the active sites are significantly different from protease inhibitors of other species. BmSPI38and BmSPI39were considered to be new members of the TIL family of SPIs due to its unique reactive sites and the numbers of conserved cysteines.
Phylogenetic tree showed that all the TIL-type protease inhibitors were clustered into3branches, B. mori BmSPI35, BmSPI36, BmSPI37, BmSPI38, BmSPI39, BmSPI40and BmFPI-F (Fungal protease inhibitor-F) clustered into one clade (Group Ⅲ), Bombina bombina BbBSTI clustered into one clade (Group Ⅱ), other protease inhibitors clustered into one clade (Group Ⅰ). BmSPI38and BmSPI39have a farther evolutionary relationship to TIL-type protease inhibitors of other species, and shared high sequence identity with the BmFPI-F, suggesting that they might have similar functions to BmFPI-F in silkworm immunity.
We obtained recombinant BmSPI38and BmSPI39with biological activity using prokaryotic expression technology. The inhibitory activity assay under different conditions of temperature or pH indicated that BmSPI38and BmSPI39have very high thermal and pH stability. To investigate the type of proteases that could be suppressed by recombinant BmSPI38and BmSPI39, various proteases were used in in vitro tests. The results showed that BmSPI38and BmSPI39can significantly suppress microbial proteases, such as subtilisin A and proteinase K, whereas it could not inhibit bovine pancreas trypsin, bovine pancreas chymotrypsin and elastase.
2. Inhibitory activity of BmSPI38and BmSPI39against cuticle-degrading protease from fungi and dynamic characteristic analysis
In order to explore whether BmSPI38and BmSPI39are involved in protection from Entomopathogenic fungi, we choose the cuticle-degrading protease (CDEP-1) from B. bassiana for activity assays and in vitro binding experiments. The results showed that BmSPI38and BmSPI39can potently inhibit virulence protease CDEP-1of B. bassiana. RT-PCR analysis showed that BmSPI38was expressed highly in immune related tissues, such as head, integument and fatbody, suggesting that might suppress the integument penetration and invasion of silkworm by inhibiting cuticle-degrading proteases secreted by fungi. BmSPI39expressed specifically in the anterior and middle silk gland, may participate in some silk gland-related processes.
Similar to Prl from M. anisopliae, CDEP-1induced the melanization of silkworm. Thus, CDEP-1-induced melanization can be blocked using BmSPI38or BmSPI39. This indicates that inhibitors could inhibit Prl-induced insecticidal melanization of silkworm and protect it. Precursor activation of phenoloxidase in vitro showed that CDEP-1cannot directly activate PPO, it may indirectly activate PPO by activating an upstream protein factor. Our results clarified another way to cope with fungal damage to silkworms, by blocking the harmful melanization caused by the protease produced by entomopathogenic fungi. A certain degree of melanization occurred in the hemolymph of silkworm108h after B. bassiana infection, and there was a postive correlation between blackening degree and concentration of conidia to some extent. Melanization associated with the phenoloxidase cascade, was involved in the immune response to fungal invasion.
Subtilisin, protease K and cuticle-degrading protease CDEP-1used in this study belong to the Peptidases_S8family which contain a Peptidases_S8domain. Sequence analysis showed that members of Peptidases_S8family were highly similarity. The reported cuticle-degrading proteases are mainly belong to the PCSK9_ProteinaseK_like subfamily and Subtilisin_subset subfamily, are important fungal virulence factors. Based on the high similarity of subtilisin-like proteases and the intensive inhibitory activity of BmSPI38and BmSPI39against microbial proteases, these inhibitors might be useful for enhancing the anti-fungi ability of the silkworm.
3. Protease inhibitors inhibit the invasion ofB. bassiana
To investigate whether BmSPI39is involved in the immune response to fungi, we use different concentrations of conidia suspension to infect silkworms, and performed a Western blot analysis of BmSPI39in the integument at different points after B. bassiana infection. The results showed that the protease inhibitor BmSPI39of silkworm was expressed in integument as tetramer. The expression of BmSPI39was up-regulated first, and then down-regulated, indicating that BmSPI39was involved in the immune response to fungi. Immunofluorescence experiments indicated that BmSPI39is mainly located in endocuticle and mesocuticle. In the light of the activity of fungal cuticle-degrading protease can be inhibited by BmSPI39, suggesting that protease inhibitors in integument can depress the penetration of fungi through inhibiting its cuticle-degrading proteases.
In order to investigate whether protease inhibitors can enhance the antifungal ability of B. mori, recombinant BmSPI38and BmSPI39was used as an additive to spore suspension of B. bassiana, and treat silkworm by surface immersion. Each assay was done by application of B. bassiana alone or in conjunction with BSA, BmSPI38and BmSPI39. The experimental result indicated that the cumulative incidence of silkworm increased gradually with time after inoculation. By the fifth day, cumulative incidence rate of silkworm using B. bassiana alone was64.67%, and that using BSA, BmSPI38, BmSPI39as an additive was44.44%,18%and8%, respectively. We make observations on the silkworm at the5th day after infection. Most silkworm treated with B. bassiana alone or BSA as the additive displayed black damaged spots on the integument, darkened and matt, surface dehydration subsidence, inactivity and insensitivity to touch. The majority of silkworm treated with recombinant BmSPI38or BmSPI39as the additive have no obvious disease symptoms. Black spots emerged in the integument indicating that there is a cascade of phenoloxidase in silkworm, and the melanization reaction will be activated to participate in immune response against fungi, when infected by B. bassiana. Surprisingly, there were no obvious black spots on the intersegmental membrane of silkworm. This may be due to lack of the phenoloxidase system in intersegmental membrane, in which can not effeciently activate melanization. Through statistical analysis of survival rates of the five different treatment groups, BmSPI38and BmSPI39are able to significantly improve the survival rate of silkworm, suggesting that protease inhibitors could enhance the antifungal ability of silkworm, and can be used as a target resistant gene of silkworm.
4. Analysis of key sites for inhibitory activity of BmSPI38and BmSPI39
Multiple sequence alignment of TIL-type protease inhibitors from B. mori and other species showed that there are two cysteine substitutions in amino acid sequences of BmSPI38and BmSPI39. Therefore, we speculate that the amino acid residues in the two positions are the key sites to determine its inhibitory activity. Asp and Leu in the two positions were mutated to cysteines using site-directed mutagenesis technology. Structural models were constructed for three-dimensional structure of BmSPI39before and after mutation. The result shown that BmSPI39contains only2antiparallel (3sheets, as well as a small a helix, the rest are flexible loop regions. The whole structure contains four disulfide bonds (Cys29-Cys62, Cys40-Cys54, Cys44-Cys92, Cys64-Cys76), which maintain a rigid structure of protein stability and make the large flexible loop regions form a relatively compact and stable structure with certain flexibility. The superimposed structural models of BmSPI39and mutated BmSPI39Mu revealed that the two different mutagenic Cys are located in two adjacent loop regions, and they form an intramolecular disulfide bonds and make the two loop regions closer together. At the same time, Thr36m-O and Thr61m-N form a pair of hydrogen bonds to stabilize the structure changes. The disulfide bonds (Cys38m-Cys58m) and the adjacent new generating disulfide bonds bond (Cys40m-Cys54m) together to form a stable rigid region, and PI residue (Ala56m) is in the middle of the rigid region.
The two amino acids in the BmSPI38and BmSPI39were mutated to cysteines using site-directed mutagenesis technology. The inhibitory activity of mutant BmSPI38Mu and BmSP139Mu against subtilisins was greatly reduced, indicating that cysteine substitutions in the two positions are an important cause of access to inhibitory ability against microbial protease. However. BmSPI38Mu and BmSPI39Mu didn't obtain the ability to inhibit trypsin, chymotrypsin and elastase, suggesting that P1residues play an important role in determining its specificity. Base on the analysis of active sites and the number of cysteine in TIL-type protease inhibitors, we found that there is a great correlation of protease inhibitors specificity with the numbers of cysteine in TIL domain, properties and size of P1amino-acid residue. This study shows that replacement of P1residues by small molecules of neutral amino acids in silkworm TIL-type protease inhibitor, during the course of evolution, enable the inhibitors to gain a certain degree of inhibition against microbial protease, and cysteine substitutions enhance their inhibitory activity and specificity.
5. Analysis of existing forms of the BmSPI38and BmSPI39in vivo and functions in cocoon shell protection
We conducted an analysis of the recombinant BmSPI38and BmSPI39, using reducing and non-reducing SDS-PAGE electrophoresis, MALDI-TOF MS and activity staining. The results showed recombinant BmSPI38and BmSPI39were existing predominantly as polymers. Western blot analysis indicated that BmSPI38and BmSPI39expressed in various tissues and organs mainly in the form of tetramer, and they may execute function as polymers in silkworm.
The RT-PCR and Western blot analysis revealed that BmSPI39is expressed specifically in the anterior and middle silk gland on the5th day of the fifth instar. It is supposed that BmSPI39was involved in some silk gland-related processes. BmSPI39was highly expressed in the silk gland prior to mounting, and secreted into the cocoon shell in the process of spinning silk. Our study showed that, protease inhibitor BmSPI39can inhibit microbial protease hydrolysis process of cocoon shell protein, hence provide a long-term effective protection for the prepupa and pupa.
昆虫致病性真菌,通过利用丰富的水解酶来穿透昆虫体壁。这些水解酶包括体壁降解蛋白酶和几丁质酶,它们都是重要的毒力因子。过表达这些毒性蛋白酶,可以显著增强致病性真菌的毒力。虽然真菌穿透昆虫体壁的基本模式已经提出,但昆虫是怎样抵御真菌入侵以及与真菌之间具体的相互作用如何都不甚明了,有待于进一步研究。
昆虫体壁和血液中含有丰富的蛋白酶抑制剂,这些抑制剂与抵御病原微生物入侵密切相关。为了阐明昆虫抵御真菌入侵的分子机制以及为提高家蚕抗真菌能力寻找新的分子靶标,我们重点对丝氨酸蛋白酶抑制剂进行深入研究。本研究室利用基因芯片技术对家蚕在微生物诱导下的基因表达变化进行调查,发现了19个家蚕丝氨酸蛋白酶抑制剂在微生物经口感染家蚕后,上调或者下调表达,初步表明这些丝氨酸蛋白酶抑制剂可能参与防御病原微生物入侵的过程。特别是TIL(trypsin inhibitor-like cysteine rich domain)家族的蛋白酶抑制剂在球孢白僵菌感染后明显上调表达。因此,可以从家蚕蛋白酶抑制剂中寻找能够有效对抗致病性真菌入侵的蛋白分子,以提高家蚕抗真菌能力。基于此,本论文对TIL家族的BmSPI38和BmSPI39进行了全长克隆、原核表达和活性测定,并对其抵御真菌入侵的分子机理进行了深入的研究；利用3D结构预测和定点突变技术,对BmSPI38和BmSPI39的抑制活性的关键位点进行了分析；并对BmSPI38和BmSPI39在家蚕中的生理存在形式及BmSPI39在茧丝保护中的功能进行了系统的研究。本论文获得的主要研究结果如下：
1.丝氨酸蛋白酶抑制剂的全长克隆、原核表达及活性测定
基于BmSPI38和BmSPI39(?)勺EST序列,我们对这两个基因分别设计了4个基因特异引物,用于该基因的5'RACE扩增和3'RACE扩增,获得了这两个基因的全长cDNA序列。BmSPI38和BmSPI39都具有一个含有八个半胱氨酸残基的TIL结构域。多序列比对结果表明,与其它物种的丝氨酸蛋白酶抑制剂相比,BmSPI38和BmSPI39的TIL结构域中有2个Cys发生了替换’(Cys2th和Cys6th),且活性位点也发生了明显变化。基于独特的活性位点和保守的Cys数目,我们认为BmSPI38和BmSPI39是TIL家族丝氨酸蛋白酶抑制剂的新成员。
系统发生树分析显示,所有TIL类蛋白酶抑制剂聚为3支,家蚕BmSPI35、 BmSPI36、BmSPI37、BmSPI38、BmSPI39、BmSPI40和BmFPI-F(家蚕真菌蛋白酶抑制剂-F)聚为一支(Group Ⅲ),铃蟾BbBSTI独自聚为一支(Group Ⅱ),其余丝氨酸蛋白酶抑制剂聚为一支(Group Ⅰ)。家蚕BmSPI38、BmSPI39与其它物种的TIL家族的蛋白酶抑制剂进化关系较远,而与BmFPI-F的序列相似性较高,推测它们与BmFPI-F具有相似的功能,参与家蚕的免疫过程。
利用原核表达技术,我们获得了具有生物活性的BmSPI38和BmSPI39重组蛋白。通过对不同温度或pH条件下的抑制剂活性进行测定,我们发现BmSPI38和BmSPI39具有很高的热和酸碱稳定性。为了确定BmSPI38和BmSPI39所抑制的蛋白酶的类型,我们选择了几种蛋白酶来进行酶活测定实验,结果表明BmSPI38和BmSPI39能够强烈地抑制微生物蛋白酶,如枯草杆菌蛋白酶A和蛋白酶K,但对胰蛋白酶、胰凝乳蛋白酶和弹性蛋白酶都没有抑制活性。
2.BmSPI38和BmSPI393(?)寸真菌体壁降解蛋白酶的抑制及其动力学特征分析
为了探讨BmSPI38和BmSPI39是否参与抵御昆虫致病性真菌,我们选取了球孢白僵菌Prl蛋白酶(CDEP-1)来进行酶活抑制和体外结合实验。结果表明,BmSPI38和BmSPI39能够强烈地抑制球孢白僵菌的毒力蛋白酶CDEP-1。RT-PCR分析表明,BmSPI38在头、体壁和脂肪体等免疫相关组织中高量表达,暗示其可能通过抑制真菌分泌的体壁降解蛋白酶,进而阻止真菌穿透表皮侵染家蚕；BmSPI39在家蚕前部和中部丝腺中特异表达,可能参与丝腺相关的某种过程。与绿僵菌的Prl蛋白酶相似,CDEP-1能够诱导家蚕的黑化反应。而BmSPI38和BmSPI39能阻断CDEP-1诱导的黑化反应。这表明,家蚕蛋白酶抑制剂BmSPI38和BmSPI39(?)能够抑制CDEP-1蛋白酶诱导的杀虫性黑化,进而保护家蚕。酚氧化酶前体体外激活实验表明,CDEP-1不能直接激活PPO,可能是通过激活PPO上游的某种蛋白因子,间接激活PPO。本研究阐明了家蚕应对真菌伤害的另一种途径,即阻断昆虫致病性真菌分泌的蛋白酶引发的有害黑化。球孢白僵菌感染后108h,家蚕血液发生了一定程度的黑化,且黑化程度与分生孢子浓度在一定程度上具有正相关性,表明酚氧化酶级联的黑化反应,参与对真菌入侵的免疫应答。
本研究中使用的枯草杆菌蛋白酶A、蛋白酶K和体壁降解蛋白酶CDEP-1都属于Peptidases_S8家族,具有一个Peptidases_S8结构域。序列分析表明,Peptidases_S8家族相似性很高,已报道的体壁降解蛋白酶主要是PCSK9_ProteinaseK_like亚家族与Subtilisin_subset亚家族中成员,是真菌重要的毒力因子。鉴于枯草杆菌蛋白酶类的相似性很高,且BmSPI38和BmSPI39能够强烈地抑制这些微生物蛋白酶活性,我们认为蛋白酶抑制剂BmSPI38和BmSPI39有助于提高家蚕的抗真菌能力。
3.蛋白酶抑制剂抑制球孢白僵菌的入侵
为了研究BmSPI39等蛋白酶抑制剂是否参与家蚕对真菌的免疫应答,我们利用不同浓度的球孢白僵菌感染家蚕,并对攻毒后不同时间点的体壁中的BmSPI39进行Western blot分析。结果表明,家蚕蛋白酶抑制剂BmSPI39以四聚体形式在体壁中表达,在球孢白僵菌感染后,先上调表达,后下调表达,参与家蚕对真菌的免疫应答。免疫荧光实验表明,BmSPI39主要定位于内表皮和中表皮中。鉴于该抑制剂能够抑制真菌的体壁降解蛋白酶活性,我们认为体壁中的蛋白酶抑制剂可能是通过抑制真菌的体壁降解蛋白酶活性,来减缓真菌对昆虫体壁的穿透过程。
为了进一步研究蛋白酶抑制剂能否增强家蚕的抗真菌能力,我们将蛋白酶抑制剂BmSPI38和BmSPI39重组蛋白作为球孢白僵菌孢子悬液的添加剂,通过体表浸泡法处理家蚕。单独应用球孢白僵菌或与BSA、BmSPI38或BmSPI39等一起处理家蚕。实验结果表明,随着攻毒后时间的增加,家蚕的累积发病率逐渐增加。截止到第5天,单独使用球孢白僵菌攻毒的家蚕累积发病率为64.67%,而使用BSA、BmSPI38或BmSPI39重组蛋白添加剂的的家蚕累积发病率分别为44.44%、18%和8%。我们对攻毒处理第5天的家蚕进行观察,发现单独使用球孢白僵菌和以BSA作为添加剂的处理组,多数家蚕表现为：体壁出现黑色破损病斑,体色加深无光泽,体表脱水下陷,活动性差,触摸感觉迟钝等现象。而蛋白酶抑制剂BmSPI38或BmSPI39的重组蛋白的处理组中,多数家蚕没有明显的发病征兆。家蚕体壁出现黑色斑点,说明家蚕体壁中存在酚氧化酶级联系统,当球孢白僵菌感染家蚕时,会激活家蚕的黑化反应,从而参与抵御真菌的免疫应答。意外的是,蚕蛹节间膜没有发现明显的黑色斑点,这可能是由于节间膜处缺乏酚氧化酶级联系统,不能有效激活黑化反应。通过对五个不同处理组的生存率进行统计分析,发现BmSPI38和BmSPI39都能够显著提高家蚕的生存率,表明蛋白酶抑制剂能够增强家蚕的抗真菌能力,可以作为提高家蚕抗性的靶标基因。
4.蛋白酶抑制剂BmSPI38和BmSPI39的抑制特异性关键位点分析
家蚕蛋白酶抑制剂与其它物种的TIL类蛋白酶抑制剂的多序列比对结果表明,BmSPI38和BmSPI39的氨基酸序列中有两个半胱氨酸发生了替换,因此,我们推测这两个位置的氨基酸残基是决定其抑制活性的关键位点。利用定点突变技术将这两个位置的Asp和Leu分别突变为Cys。我们对BmSPI39突变前后的三维结构进行了建模预测。结果表明,BmSPI39中只含有2个反平行的β折叠片和一个小α螺旋,其余区域都是柔性的loop区域。整个结构中含有四对二硫键(Cys29-Cys62、 Cys40-Cys5、Cys44-Cys92和Cys64-Cys76),它们维持了蛋白刚性结构的稳定,使大片的柔性loop区域形成一个相对紧凑而又具有一定柔性的稳定结构。将BmSPI39与突变后的BmSPI39Mu的三维结构进行叠合比较发现,新突变的两个Cys分别位于相邻的两个loop区域,它们会形成一个分子内二硫键,并将相邻的两个loop区域拉近,同时,Thr36m-O和Thr61m-N形成一对氢键以稳定该结构的变化。新生成的二硫键(Cys38m-Cys58m)与相邻的二硫键(Cys40m-Cys54m)一起形成一个稳定的刚性区域,P1残基Ala56m正好处于这个刚性区域中间。
将BmSPI38和BmSPI39中的两个氨基酸定点突变为半胱氨酸,突变后的BmSPI38Mu和BmSPI39Mu对枯草杆菌蛋白酶类的抑制活性大大降低,表明这两个位置的半胱氨酸替换是BmSPI38和BmSPI39获得抑制微生物蛋白酶能力的一个重要原因。然而,BmSPI38Mu和BmSPI39Mu没有获得对胰蛋白酶、胰凝乳蛋白酶或弹性蛋白酶等的抑制能力,推测P1残基在决定其抑制特异性中起重要作用。通过对TIL家族蛋白酶抑制剂活性位点及半胱氨酸数目进行分析,蛋白酶抑制剂的抑制特异性与TIL结构域中的半胱氨酸数目和P1残基性质及大小存在很大的关联。本研究表明,在进化过程中,家蚕TIL家族蛋白酶抑制剂中P1残基替换为小分子的中性氨基酸,使其获得了一定程度的抑制微生物蛋白酶活性的能力,半胱氨酸的替换增强了其抑制的活性和特异性。
5.BmSPI38和BmSPI39体内存在形式的分析及在茧壳保护中的功能研究
利用还原与非还原SDS-PAGE电泳、MALDI-TOF MS和胶内活性染色技术,我们对BmSPI38和BmSPI39重组蛋白的存在形式进行了分析。结果发现,BmSPI38和BmSPI39重组蛋白主要以多聚体形式存在。Western blot结果表明,BmSPI38和BmSPI39主要以四聚体形式分布于各组织器官中,可能以多聚体形式在家蚕中执行功能。
RT-PCR及Western blot结果表明,BmSPI39在5龄第5天家蚕的前部和中部丝腺特异表达,推测BmSPI39可能参与丝腺相关的某种过程。BmSPI39在上蔟前的丝腺中高量表达,并随着泌丝过程进入茧壳中。我们的研究表明,蛋白酶抑制剂BmSPI39能够通过抑制微生物蛋白酶对茧壳蛋白的水解过程,从而为预蛹和蛹提供长效有力的保护。
Bombyx mori is a silk-producing insect with tremendous economic value. Based on the immense accumulation of fundamental research, B. mori has gradually developed into a model organism of Lepidoptera. However, silkworm is subject to environmental threats of various pathogenic microorganisms and parasites, which lead to serious diseases. Entomopathogenic fungi such as Metarhizium anisopliae, Beauveria bassiana, Aspergillus flavus, and Aspergillus oryzae can cause various communicable diseases, which seriously affect the production and quality of cocoon, and cause enormous economic losses in the silkworm industry. Entomopathogenic fungi, as a new biopesticide, has been widely used in agriculture and forestry pest control and mosquito control. The use of fungal biological pesticide, would trigger inevitable cross infection in silkworm, and lead to silkworm disease outbreak. Therefore, elucidation of the molecular mechanism of the interaction between entomopathogenic fungi and B. mori, and discovering novel antifungal molecules, would be beneficial to raising the antifungal ability of B. mori and has vast importance to agricultural pest control and cures for human fungal diseases.
Entomopathogenic fungi penetrate the insect cuticle using its abundant hydrolases. These hydrolases, including cuticle-degrading protease and chitinase, both of them are important virulence factors. Overexpression of toxic protease can significantly enhance the virulence of pathogenic fungi. Although the basic model of fungal penetration of insect integument has been proposed, how do insects protect against fungal invasion and specific interactions between insects and fungi has yet to be further studied.
The insect integument and hemolymph contain a large number of protease inhibitors, which are closely related to resistance against pathogenic microorganisms. To elucidate the molecular mechanism of insect resistance to fungal invasion and seek novel targets to improve silkworm antifungal ability, we focus on in-depth study of the serine protease inhibitors. In our previous study, microarray was used to screen expression-changed genes after microbial challenge. At least19serine protease inhibitors were up-regulated or down-regulated after microbial induced via oral. The preliminal results indicated that these serine protease inhibitors may be involved in resistance against pathogenic microorganisms. In particular, the expression level of various TIL-type protease inhibitors were significantly up-regulated after B. bassiana infection. Hence we can look for effective protein molecules against pathogenic fungus in order to enhance the antifungal ability of B. mori. In view of this, we cloned the full length cNDA of BmSPI38and BmSPI39from TIL family, expressed them in the prokaryotic cells, measured their activities and further studied the molecular mechanism of silkworm protease inhibitors against fungal invasion.3D structure prediction and site-directed mutagenesis were used to analyse the key sites for the inhibitory activity of BmSPI38and BmSPI39. Their physiological existence in the silkworm and the protection function of BmSPI39in the cocoon was researched systematically. The major results in this study are as follows:
1. Full length cNDA clone, prokaryotic expression and activity assay of serine protease inhibitors
Based on the known EST sequences of BmSPI38and BmSPI39, we designed respectively four gene-specific primers for3'-and5'-RACE PCR. Finally, we obtained their fall length cDNA sequences. Both of BmSPI38and BmSPI39contain a single TIL domain with eight cysteines. Multiple sequence alignment showed that two cysteine(Cys2th and Cys6th) substitutions occurred in TIL domain, and the active sites are significantly different from protease inhibitors of other species. BmSPI38and BmSPI39were considered to be new members of the TIL family of SPIs due to its unique reactive sites and the numbers of conserved cysteines.
Phylogenetic tree showed that all the TIL-type protease inhibitors were clustered into3branches, B. mori BmSPI35, BmSPI36, BmSPI37, BmSPI38, BmSPI39, BmSPI40and BmFPI-F (Fungal protease inhibitor-F) clustered into one clade (Group Ⅲ), Bombina bombina BbBSTI clustered into one clade (Group Ⅱ), other protease inhibitors clustered into one clade (Group Ⅰ). BmSPI38and BmSPI39have a farther evolutionary relationship to TIL-type protease inhibitors of other species, and shared high sequence identity with the BmFPI-F, suggesting that they might have similar functions to BmFPI-F in silkworm immunity.
We obtained recombinant BmSPI38and BmSPI39with biological activity using prokaryotic expression technology. The inhibitory activity assay under different conditions of temperature or pH indicated that BmSPI38and BmSPI39have very high thermal and pH stability. To investigate the type of proteases that could be suppressed by recombinant BmSPI38and BmSPI39, various proteases were used in in vitro tests. The results showed that BmSPI38and BmSPI39can significantly suppress microbial proteases, such as subtilisin A and proteinase K, whereas it could not inhibit bovine pancreas trypsin, bovine pancreas chymotrypsin and elastase.
2. Inhibitory activity of BmSPI38and BmSPI39against cuticle-degrading protease from fungi and dynamic characteristic analysis
In order to explore whether BmSPI38and BmSPI39are involved in protection from Entomopathogenic fungi, we choose the cuticle-degrading protease (CDEP-1) from B. bassiana for activity assays and in vitro binding experiments. The results showed that BmSPI38and BmSPI39can potently inhibit virulence protease CDEP-1of B. bassiana. RT-PCR analysis showed that BmSPI38was expressed highly in immune related tissues, such as head, integument and fatbody, suggesting that might suppress the integument penetration and invasion of silkworm by inhibiting cuticle-degrading proteases secreted by fungi. BmSPI39expressed specifically in the anterior and middle silk gland, may participate in some silk gland-related processes.
Similar to Prl from M. anisopliae, CDEP-1induced the melanization of silkworm. Thus, CDEP-1-induced melanization can be blocked using BmSPI38or BmSPI39. This indicates that inhibitors could inhibit Prl-induced insecticidal melanization of silkworm and protect it. Precursor activation of phenoloxidase in vitro showed that CDEP-1cannot directly activate PPO, it may indirectly activate PPO by activating an upstream protein factor. Our results clarified another way to cope with fungal damage to silkworms, by blocking the harmful melanization caused by the protease produced by entomopathogenic fungi. A certain degree of melanization occurred in the hemolymph of silkworm108h after B. bassiana infection, and there was a postive correlation between blackening degree and concentration of conidia to some extent. Melanization associated with the phenoloxidase cascade, was involved in the immune response to fungal invasion.
Subtilisin, protease K and cuticle-degrading protease CDEP-1used in this study belong to the Peptidases_S8family which contain a Peptidases_S8domain. Sequence analysis showed that members of Peptidases_S8family were highly similarity. The reported cuticle-degrading proteases are mainly belong to the PCSK9_ProteinaseK_like subfamily and Subtilisin_subset subfamily, are important fungal virulence factors. Based on the high similarity of subtilisin-like proteases and the intensive inhibitory activity of BmSPI38and BmSPI39against microbial proteases, these inhibitors might be useful for enhancing the anti-fungi ability of the silkworm.
3. Protease inhibitors inhibit the invasion ofB. bassiana
To investigate whether BmSPI39is involved in the immune response to fungi, we use different concentrations of conidia suspension to infect silkworms, and performed a Western blot analysis of BmSPI39in the integument at different points after B. bassiana infection. The results showed that the protease inhibitor BmSPI39of silkworm was expressed in integument as tetramer. The expression of BmSPI39was up-regulated first, and then down-regulated, indicating that BmSPI39was involved in the immune response to fungi. Immunofluorescence experiments indicated that BmSPI39is mainly located in endocuticle and mesocuticle. In the light of the activity of fungal cuticle-degrading protease can be inhibited by BmSPI39, suggesting that protease inhibitors in integument can depress the penetration of fungi through inhibiting its cuticle-degrading proteases.
In order to investigate whether protease inhibitors can enhance the antifungal ability of B. mori, recombinant BmSPI38and BmSPI39was used as an additive to spore suspension of B. bassiana, and treat silkworm by surface immersion. Each assay was done by application of B. bassiana alone or in conjunction with BSA, BmSPI38and BmSPI39. The experimental result indicated that the cumulative incidence of silkworm increased gradually with time after inoculation. By the fifth day, cumulative incidence rate of silkworm using B. bassiana alone was64.67%, and that using BSA, BmSPI38, BmSPI39as an additive was44.44%,18%and8%, respectively. We make observations on the silkworm at the5th day after infection. Most silkworm treated with B. bassiana alone or BSA as the additive displayed black damaged spots on the integument, darkened and matt, surface dehydration subsidence, inactivity and insensitivity to touch. The majority of silkworm treated with recombinant BmSPI38or BmSPI39as the additive have no obvious disease symptoms. Black spots emerged in the integument indicating that there is a cascade of phenoloxidase in silkworm, and the melanization reaction will be activated to participate in immune response against fungi, when infected by B. bassiana. Surprisingly, there were no obvious black spots on the intersegmental membrane of silkworm. This may be due to lack of the phenoloxidase system in intersegmental membrane, in which can not effeciently activate melanization. Through statistical analysis of survival rates of the five different treatment groups, BmSPI38and BmSPI39are able to significantly improve the survival rate of silkworm, suggesting that protease inhibitors could enhance the antifungal ability of silkworm, and can be used as a target resistant gene of silkworm.
4. Analysis of key sites for inhibitory activity of BmSPI38and BmSPI39
Multiple sequence alignment of TIL-type protease inhibitors from B. mori and other species showed that there are two cysteine substitutions in amino acid sequences of BmSPI38and BmSPI39. Therefore, we speculate that the amino acid residues in the two positions are the key sites to determine its inhibitory activity. Asp and Leu in the two positions were mutated to cysteines using site-directed mutagenesis technology. Structural models were constructed for three-dimensional structure of BmSPI39before and after mutation. The result shown that BmSPI39contains only2antiparallel (3sheets, as well as a small a helix, the rest are flexible loop regions. The whole structure contains four disulfide bonds (Cys29-Cys62, Cys40-Cys54, Cys44-Cys92, Cys64-Cys76), which maintain a rigid structure of protein stability and make the large flexible loop regions form a relatively compact and stable structure with certain flexibility. The superimposed structural models of BmSPI39and mutated BmSPI39Mu revealed that the two different mutagenic Cys are located in two adjacent loop regions, and they form an intramolecular disulfide bonds and make the two loop regions closer together. At the same time, Thr36m-O and Thr61m-N form a pair of hydrogen bonds to stabilize the structure changes. The disulfide bonds (Cys38m-Cys58m) and the adjacent new generating disulfide bonds bond (Cys40m-Cys54m) together to form a stable rigid region, and PI residue (Ala56m) is in the middle of the rigid region.
The two amino acids in the BmSPI38and BmSPI39were mutated to cysteines using site-directed mutagenesis technology. The inhibitory activity of mutant BmSPI38Mu and BmSP139Mu against subtilisins was greatly reduced, indicating that cysteine substitutions in the two positions are an important cause of access to inhibitory ability against microbial protease. However. BmSPI38Mu and BmSPI39Mu didn't obtain the ability to inhibit trypsin, chymotrypsin and elastase, suggesting that P1residues play an important role in determining its specificity. Base on the analysis of active sites and the number of cysteine in TIL-type protease inhibitors, we found that there is a great correlation of protease inhibitors specificity with the numbers of cysteine in TIL domain, properties and size of P1amino-acid residue. This study shows that replacement of P1residues by small molecules of neutral amino acids in silkworm TIL-type protease inhibitor, during the course of evolution, enable the inhibitors to gain a certain degree of inhibition against microbial protease, and cysteine substitutions enhance their inhibitory activity and specificity.
5. Analysis of existing forms of the BmSPI38and BmSPI39in vivo and functions in cocoon shell protection
We conducted an analysis of the recombinant BmSPI38and BmSPI39, using reducing and non-reducing SDS-PAGE electrophoresis, MALDI-TOF MS and activity staining. The results showed recombinant BmSPI38and BmSPI39were existing predominantly as polymers. Western blot analysis indicated that BmSPI38and BmSPI39expressed in various tissues and organs mainly in the form of tetramer, and they may execute function as polymers in silkworm.
The RT-PCR and Western blot analysis revealed that BmSPI39is expressed specifically in the anterior and middle silk gland on the5th day of the fifth instar. It is supposed that BmSPI39was involved in some silk gland-related processes. BmSPI39was highly expressed in the silk gland prior to mounting, and secreted into the cocoon shell in the process of spinning silk. Our study showed that, protease inhibitor BmSPI39can inhibit microbial protease hydrolysis process of cocoon shell protein, hence provide a long-term effective protection for the prepupa and pupa.
引文
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