本期重点推介

昆虫抗菌肽因其重要的可开发利用价值愈来愈受到人们的重视。家蚕Bombyx mori基因组全测序完成后,经注释发现了35条拟抗微生物肽基因序列,但是这些拟抗微生物肽基因是否具有抗菌活性,是否与其他昆虫中鉴定报道的同种抗菌肽具有相似的抗菌谱和抗菌活性,在同一基因组中的同种抗菌     

中国野蚕一种强抗病毒蛋白的基因分析和活性鉴定

以最近报道的家蚕抗病毒蛋白基因为线索,从中国野蚕（Bombyx mandarina Moore）中肠内克隆了抗家蚕BmNPV病毒的SP-2 cDNA（GenBank登录号：AY945210）,基因大小855bp,编码284个氨基酸的蛋白质,分子量29．6kD,基因组全长1376bp,包含5个外显子和4个内含子．该基因的表达仅限于中肠,具有组织特异性,在幼虫龄中表达水平较高,而在眠期和熟蚕没有表达．推导其氨基酸序列,发现其C端氨基酸序列与已报道的家蚕相应序列差别较大,有8个氨基酸完全不同．通过体外重组技术,由高效基因表达系统获得大量重组蛋白,发现该蛋白具有很强的抗家蚕BmNPV活性,与家蚕对应的抗病毒蛋白BmSP-2相比,其抗BmNPV活性高1．6倍．初步认为,该蛋白质C端序列差异可能是造成家蚕与野蚕抗病毒活性差别的主要原因．     

家蚕瘫痪肽结合蛋白基因克隆与分析

ENF肽家族具有保守的N末端结构（Glu—Asn—Phe-）。该家族成员肽大多具有重叠功能活性,在鳞翅目昆虫的免疫反应,生长调控和自体调节等方面都发挥着重要的作用。在昆虫的免疫反应中,血细胞尤其是淋巴液的黏附性是针对外来侵入物的免疫应答过程中的重要因素。家蚕瘫痪肽（paralytic peptide）是ENF肽家族的一种,其具有多种的生物学活性,包括致瘫痪性及在家蚕血细胞免疫反应中的促吞噬细胞扩散活性。ENF肽家族的另一成员,粘虫（Pseudaletia separata）的生长阻抑肽（Growth-blocking peptide）,同家蚕瘫痪肽一样能够在粘虫的血细胞免疫反应中起到调节吞噬细胞的功能。目前,关于昆虫细胞免疫应答的终端调控分子机制的研究还比较少,有文献报道粘虫的生长阻抑肽结合蛋白（GBP—BP）能够起到沉默生长阻抑肽活性的功能,从而可能参与调节细胞免疫应答的终端调控。在本研究中,利用荧光差异显示技术（FDD）分析了家蚕感染BmNPV病毒后基因表达差异情况,在血淋巴中获得了一条差异条带G12782＊通过5＇-RACE技术,首次在家蚕中克隆得到了该基因的全长cDNA序列。通过同源性分析得知,该基因所编码的蛋白质与粘虫的生长阻抑肽结合蛋白具有很大的同源性,并被命名为家蚕瘫痪肽结合蛋白（Bmori paralytic peptide binding protein,PP-BP）。通过RT-PCR研究发现,该蛋白基因在血淋巴中大量表达。同时,利用实时荧光定量PCR（Real-time quantitative PCR）技术分析了该基因在正常饲养家蚕与添食BmNPV病毒的家蚕中的表达差异,结果显示该基因在家蚕添食BmNPV病毒后的表达量大大增强,这就暗示该基因可能与BmNPV病毒刺激后所引起的家蚕血液细胞免疫反应相关。利用生物信息学方法对该基因的结构进行了分析,发现该基因具有两个外显子和一个内含子。这个基因已经登入GenBank数据库,收入号为DQ306881。     

《生命科学》：研究发现细胞周期必需因子

来自西南大学家蚕基因组生物学国家重点实验室的研究人员利用家蚕EST数据,成功克隆了家蚕CyclinB3基因（ElJ074796）,并通过家蚕卵巢细胞系BmN—SWUl的BmCyclinB和BmCyclinB3基因RNA干涉研究,     

黑腹果蝇抗真菌肽基因Drs和Drs-lC 的原核可溶性表达及抗真菌活性测定

Drosomycin （Drs）是第1个从黑腹果蝇Drosophila melanogaster体内鉴定发现的昆 虫抗真菌肽因子。它对细菌无明显的抗性,但对丝状真菌具有高效广谱的抑杀作用。此外, 在黑腹果蝇基因组还存在着Drs的另外6个同系物的基因序列,其中同系物Drosomycin-lC（Drs-lC）的抗真菌谱仅次于Drs。将Drs抗真菌肽基因(Drs)和同系物Drs-lC基因（Drs-lC）进行可溶性表达,对果蔬等农产品防腐保鲜的研究有应用前景。本实验将Drs和Drs-lC分别克隆到硫氧还蛋白（Trx）融合表达载体pThiohis A中,转化宿主菌TOP10,进行可溶性表达,并从诱导表达的菌液起始浓度、IPTG的诱导浓度及诱导时间等方面进行了表达条件的优化。结果表明2种融合蛋白Trx-Drs和Trx-Drs-lC大部分以可溶形式表达,可溶性表达的Trx-Drs在上清液中约占菌体总蛋白的22％。2种融合蛋白的表达产物经 Ni-NTA亲和层析得到纯化。生测结果表明, 2种融合蛋白分别对8种供试真菌中的5种真菌显示明显的抗性。     

家蚕Hox基因研究进展

Hox基因（homeobox genes）在昆虫躯体模式（body plan）的发育调控机制中扮演着重要角色,其表达具有严格的组织特异性和胚胎发育的程序性。家蚕Bombyx mori作为鳞翅目昆虫的代表,其Hox基因也陆续得到鉴定。在家蚕中存在一个拟复等位基因群--E群基因,其突变表型均与过剩斑纹和过剩附肢有关,这可能与Hox基因有着密切联系。家蚕全基因组测序完成后,发现其Hox基因簇中存在12个特有的homeobox基因（Bmshx1~Bmshx12）, 说明家蚕Hox基因可能具有独特的生物学意义。我们还利用家蚕基因芯片数据分析了Bmlab与Bmpb基因的组织表达特征。通过对家蚕Hox基因的研究,探索家蚕躯体模式建立机制,可望为解析其他鳞翅目昆虫的躯体模式的建立机制提供理论依据。本文就家蚕Hox基因的表达、功能及其与E群突变的关系等方面进行了综述。     

家蚕CRISPR/Cpf1基因编辑系统建立

自CRISPR/Cas9基因编辑系统成功应用于模式生物以来,因其快速、高效、便捷等特点,广泛应用于基因功能研究、基因治疗和基因工程等研究领域。与此同时,CRISPR/Cas系统不断在微生物界的发现也加速了新的基因编辑工具的不断涌现。CRISPR/Cpf1是第二类 (Ⅴ型) 能够编辑哺乳动物基因组的CRISPR系统,相比于CRISPR/Cas9基因编辑系统,能够利用5′T-PAM富集区增加基因组覆盖率,具有其切割位点为粘性末端和更不易同源重组修复等诸多优势。基于此,本研究构建了能够在家蚕细胞表达的3个不同来源的CRISPR/Cpf1 (AsCpf1、FnCpf1和LbCpf1) 表达载体,选择高度保守的家蚕热休克蛋白基因BmHSP60和家蚕ATP酶家族BmATAD3A基因分别设计靶标gRNA,构建gHSP60-266R和gATAD3A-346R基因编辑载体。通过T7E1酶切分析和T克隆测序,鉴定3个Cpf1基因编辑系统AsCpf1、FnCpf1和LbCpf1对靶标基因BmHSP60和BmATAD3A的编辑效率。同时,利用Western blotting分析不同基因编辑系统敲除靶基因后对其BmATAD3A和BmHSP60蛋白翻译的影响。本研究成功构建了家蚕CRISPR/Cpf1基因编辑系统,能够在家蚕细胞中有效编辑家蚕基因组,为家蚕基因功能研究、基因工程和遗传育种开发了新技术与新方法。     

家蚕胚胎期重力相关基因的抑制消减杂交分析

目的：筛选家蚕胚胎期重力相关基因。方法：对模拟失重与正常重力条件下的家蚕胚胎cDNA进行抑制消减杂交（Suppression subtractive hybridization,SSH）,并对模拟失重过程中家蚕胚胎期表达发生变化的基因进行克隆、测序及同源性分析。结果：获得了34个与重力有关的序列标签。在模拟失重条件下有16个基因表达上调,其中15个为未知基因,1个为已知基因,其作用是维持mRNA的稳定性。在模拟失重条件下有18个基因表达下调,其中4个为未知基因,6个为蛋白合成相关基因,3个为基因组contig基因,5个为家蚕est库中功能未知基因。结论：模拟失重环境影响了家蚕胚胎发育期与mRNA稳定性和蛋白质合成相关基因的表达。     

家蚕微孢子虫丝氨酸蛋白酶抑制剂蛋白serpin基因NbSPN106的鉴定

摘要：【目的】Serpin在病原与宿主互作中起着重要的作用。本研究旨在分析在家蚕微孢子虫中的丝氨酸蛋白酶抑制剂蛋白（Serpin）的结构特征,原核克隆表达以及Western blotting检测。【方法】 基于家蚕微孢子虫全基因组序列,同源序列比对搜索获得serpin基因序列。利用在线软件分析基因的序列特征,ClustalX对氨基酸序列进行多重序列比对。构建含有GST标签的pGEX4T1-NbSPN106原核重组表达载体,在大肠杆菌BL21（DE3）诱导表达并进行纯化。纯化的重组蛋白免疫小鼠,制备抗体,并与家蚕微孢子虫总蛋白进行Western blotting免疫杂交。【结果】比对搜索在家蚕微孢子虫基因组中发现一个新的serpin基因NbSPN106。NbSPN106蛋白序列长度为384 aa,N端具有信号肽,编码一个42 kDa左右的成熟蛋白。多重序列比对说明NbSPN106具有保守的serpin位点,可能具有抑制功能。免疫杂交在家蚕微孢子虫总蛋白检测到一条45 kDa左右的特异条带。【讨论】生物信息学分析以及免疫杂交结果说明在家蚕微孢子虫中存在NbSPN106。这是在微孢子虫中首次报道发现serpin基因。对研究家蚕微孢子虫与宿主家蚕的互作具有一定的参考价值。     

Genome duplications within the Xenopodinae do not increase the multiplicity of antimicrobial peptides in Silurana paratropicalis and Xenopus andrei skin secretions

A putative genome duplication event within the Silurana lineage has given rise to the tetraploid frog S. paratropicalis and a second polyploidization within the Xenopus lineage has produced the octoploid frog X. andrei. Peptidomic analysis of norepinephrine-stimulated skin secretions of S. paratropicalis and X. andrei led to identification of multiple peptides with growth-inhibitory activity against Escherichia coli and Staphylococcus aureus. Structural characterization demonstrated that the S. paratropicalis components comprised three peptides belonging to the caerulein-precursor fragment family (CPF-SP1, -SP2 and -SP3), two peptides from the xenopsin-precursor fragment family (XPF-SP1 and -SP2), and one peptide orthologous to peptide glycine-leucine-amide (PGLa-SP1). The CPF peptides showed potent, broad-spectrum antimicrobial activity. The X. andrei components comprised two peptides from the magainin family, (magainin-AN1 and -AN2), two from the XPF family (XPF-AN1 and -AN2), two from the PGLa family(PGLa-AN1 and -AN2), and one caerulein-precursor fragment (CPF-AN1).The primary structures of these peptides indicate a close phylogenetic relationship between X. andrei and the octoploid frog X. amieti. Under the same experimental conditions, seven orthologous antimicrobial peptides were previously isolated from the diploid frog S. tropicalis, nine from the tetraploid frog X. borealis, and five from the tetraploid frog X. clivii. The data indicate, therefore, that nonfunctionalization (gene deletion) has been the most common fate of duplicated antimicrobial peptide genes following polyploidization events in the Silurana and Xenopus lineages.     

Identification and expression pattern of the chemosensory protein gene family in the silkworm, Bombyx mori

Insect chemosensory proteins (CSPs) as well as odorant-binding proteins (OBPs) have been supposed to transport hydrophobic chemicals to receptors on sensory neurons. Compared with OBPs, CSPs are expressed more broadly in various insect tissues. We performed a genome-wide analysis of the candidate CSP gene family in the silkworm. A total of 20 candidate CSPs, including 3 gene fragments and 2 pseudogenes, were characterized based on their conserved cysteine residues and their similarity to CSPs in other insects. Some of these genes were clustered in the silkworm genome. The gene expression pattern of these candidates was investigated using RT-PCR and microarray, and the results showed that these genes were expressed primarily in mature larvae and the adult moth, suggesting silkworm CSPs may be involved in development. The majority of silkworm CSP genes are expressed broadly in tissues including the antennae, head, thorax, legs, wings, epithelium, testes, ovaries, pheromone glands, wing disks, and compound eyes.     

Molecular evolution of the cecropin multigene family in silkworm Bombyx mori

Cecropins constitute one of the largest and most potent immune protein families found in insect species with diversified numbers and features. In view of the large number of cecropin proteins existing with much sequence variations among them, an overview of the multigene cecropin family in silkworm Bombyx mori was attempted in this study. Cecropin encodes an inducible 64 residue anti-bacterial peptide and was clustered into two groups; first group viz. A and second group including B, D, E and Enbocin. Cecropin A consisted of two sub-groups located on chromosome number 6 of B.mori genome. Cecropin B consisted of six sub-groups, cecropin D and E of one each and Enbocin of two. The second sub-group formed in tandem array of multigene family locus over a length of 78.62 kb on chromosome number 26 in B.mori genome and was organized in positive as well as opposite orientation. The results indicated that cecropin B genes were organized in a close cluster with the intergenic sequence ranging from 1366 bp to 23526 bp. Interestingly a distantly related cecropin E was also located within the cecropin B multigene locus. Similarly distant members like cecropin D and Enbocin were also located in the 3' region of cecropin B locus. The maximum intergenic region of 23526 bp observed between Cecropin D and Enbocin indicates that the two genes were distantly evolved. The phylogenetic analysis clearly indicates a positive correlation between the clusters and physical location on the chromosome, as the length of the intergenic region plays a major role to create newer cecropin families. EST database analysis suggests that most of the cecropin A members were expressed in the microbial fat body while, the cecropin B was equally expressed in fat body and other target tissues. The signal peptides were conserved in all the twelve paralogous gene sequences.