家蚕羧酸酯酶基因克隆及差异表达

家蚕浓核病毒 (Bombyx mori densonucleosis virus,BmDNV)是蚕业生产上危害比较严重的一类病毒。用完全抗浓核病中国镇江株(BmDNV-Z)的家蚕品系秋丰、感性品系华八及以华八为轮回亲本回交8代和自交8代构建的近等基因系BC₈为材料,采用mRNA荧光差显技术首次分离克隆了家蚕羧酸酯酶（B. mori carboxylesterase,BmCarE）基因全长cDNA,并用实时荧光定量PCR检测了添毒后12 h、36 h、72 h BmCarE在感、抗BmDNV-Z家蚕品系中肠内的表达差异。结果表明： （1）添毒后12 h不同品系家蚕中肠BmCarE表达差异最大,抗性品系BC₈和秋丰分别是感性品系华八的17.714倍和3.602倍,三者彼此间的差异达到极显著水平;（2）同一品系添毒后12 h与添清水后12 h BmCarE表达也有较大差异, BC₈添毒是BC₈添清水的15.08倍, 秋丰添毒是秋丰添清水的3.39倍, 差异达到极显著水平,而华八添毒和添清水的BmCarE表达量均低,二者差异不显著;（3）同一品系添毒后不同时间BmCarE表达也有较大差异, BC₈和秋丰添毒后12 h BmCarE表达量最高,显著高于各自添毒后36 h和72 h表达水平,而添毒后36 h与72 h表达无显著差异;华八添毒后12 h、36 h和72 h,BmCarE表达无显著差异。上述结果提示羧酸酯酶基因可能与家蚕抗浓核病毒有一定关系。     

Cloning and characterization of nanos gene in silkworm Bombyx mori

Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Drosophila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5＇-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mor/and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5.38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee （Apis mellifera）. This gene has been registered in the GenBank under the accession number EF647589. One encoding sequence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells.     

家蚕细胞周期蛋白基因BmCcnl1的克隆及表达分析

家蚕Bombyx mori由受精卵到完成胚胎发育孵化的过程中, 细胞进行大量的分裂和分化, 然而滞育性卵的胚胎细胞分化至G2期便停滞在此阶段。为了探索这一发育阶段细胞内的分子调控, 本研究以人Homo sapiens的细胞周期蛋白基因cyclin L1为模板, 成功克隆了家蚕同源基因BmCcnl1（GenBank登录号: FJ889988）。BmCcnl1基因开放阅读框（open reading frame, ORF）全长1 254 bp, 编码417个氨基酸。利用Protean软件分析得出BmCcnl1蛋白预测分子量为49 kDa, 等电点为9.84。利用DNA重组技术构建了BmCcnl1基因的重组表达载体pET-21d-BmCcnl1, 对其进行原核表达, 其表达的蛋白以包涵体形式存在。利用RT-PCR技术分析了BmCcnl1基因在胚胎发育过程中的转录水平, BmCcnl1基因在非滞育性卵的胚胎发育阶段基本保持相对稳定的转录表达, 而滞育性卵从蛾体产下经过72 h后已经检测不到BmCcnl1基因的转录。结果提示, BmCcnl1基因与胚胎期滞育及非滞育性卵的发育调控相关。对该基因的克隆和表达分析为今后研究家蚕胚胎发育及细胞周期调控奠定了基础。     

家蚕Caspase家族基因 BmCaspase-X 的克隆与功能分析

【目的】本研究旨在克隆获得家蚕 Bombyx mori Caspase家族基因,并通过RNAi技术初步分析其在家蚕细胞水平细胞凋亡中的功能。【方法】 用cDNA末端快速扩增方法（rapid amplification of cDNA ends, RACE）克隆家蚕Caspase家族一个新基因,用RNAi和流式细胞术初步分析该基因在家蚕细胞凋亡中的功能。【结果】将克隆获得的家蚕Caspase家族基因命名为 BmCaspase-X,其全长为2 105 bp,开放阅读框长为1 494 bp,编码497 aa,分子量为57.8 kDa,等电点为5.29。BmCaspase-X含有Caspase特有的结构位点。系统进化分析表明,BmCaspase-X与家蚕ICE同其他昆虫Caspase-4同源物先聚在一起,再与具有长前体结构域的昆虫其他因子聚为一类。RNAi初步结果显示该基因干扰后没有出现明显的细胞凋亡。【结论】BmCaspase-X具有典型Caspase特征,属于Caspase家族成员。目前RNAi实验结果表明 BmCaspase-X 未能引起家蚕细胞凋亡的改变。本文为进一步研究该基因的功能奠定基础。     

Cloning and characterization of the highly polymorphic Ser2 gene of Bombyx mori

Three alleles of the sericin (Ser) 2-encoding gene (Ser2), called L, C and mC, were isolated from a Bombyx mori genomic library, and two related ones, called mCL and Cv, were also characterized in B. mori European strains. The Ser2 gene gives rise to two middle silk gland mRNAs by differential splicing. The size of a short mRNA (3.1 kb) is constant, but the length of a longer one ranges from 5 to 6.4 kb depending on the Ser2 allele. These length variations probably result from unequal recombinations in a region which contains about 30 well conserved 45-bp repeats coding for a Ser-like peptide. Furthermore, the L allele (and probably the mCL one) contains a 4.4-kb retrotransposon, resembling the copia-like ones of Drosophila.     

家蚕小热激蛋白家族成员Hsp23.7基因的克隆与分析

Hsp23.7基因是小热激蛋白家族的成员,本文研究了家蚕BombyxmoriL.的Hsp23.7基因,并对其进行了原核表达,获得了相应分子量的表达产物。推导的开放阅读框编码210个氨基酸,分子量为23.7ku,等电点为5.17。同时,利用实时定量PCR技术对Hsp23.7基因在家蚕不同组织的表达谱进行了鉴定。结果显示Hsp23.7基因在5龄幼虫时期的各组织中都有表达,在卵巢中表达量最高,达到3.64×107拷贝数/μg,其次在脂肪体,翅原基,马氏管中表达量也较高,在血淋巴中表达量最低,仅为7.11×103拷贝数/μg。     

Degenerate PCR method for identification of an antiapoptotic gene in BHV-1

To investigate on the hypothetical presence of an antiapoptotic gene, we utilized the CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primers) strategy amplifying unknown sequences from a background of genomic (bovine herpesvirus type-1) BHV-1 DNA. An alignment of carboxyl-terminal domains belonging to three proteins encoded by gamma34.5, MyD116 and GADD34 genes, was carried out to design degenerate PCR primers in highly conserved regions. This allowed the amplification of a 110 bp fragment. This fragment was subjected to automatic sequencing and DNA sequence analysis revealed that its position resided between the nt 14363 and the nt 14438 in bovine herpesvirus type-1 (BHV-1) Cooper strain sharing an identity of 86% (UL14). Transient transfections showed that UL14 protein is efficient in protecting MDBK and K562 cells from sorbitol induced apoptosis. The protein's anti-apoptotic function may derive from its heat shock protein-like properties.     

家蚕先天免疫基因 Bmimd 的克隆、表达及序列分析

昆虫的先天免疫应答由一组基因通过级联网络调控实现。果蝇 Drosophila 免疫缺陷(immune deficiency, imd)基因在体液免疫信号传递途径中起着重要的作用。我们利用生物信息学方法进行电子克隆,成功地找到了 imd 基因在家蚕 Bombyx mori 中的同源体,命名为 Bmimd。该基因全长1 092 bp,由 4 个外显子和 3 个内含子组成,开放阅读框(open reading frame, ORF)长 750 bp,编码 250 个氨基酸,预测蛋白质分子量为 28.6 kD。Bmimd 序列中含有一个致死结构域,经聚类分析表明该结构域与哺乳动物的受体相互作用蛋白(receptor interacting protein, RIP)相似。将该基因亚克隆到 PET-50b 载体进行原核表达,表达出了带有 2 个 6×His tag 和 1 个 Nus·Tag 标签的重组蛋白。Western blotting 结果表明 Bmimd 蛋白在 5 龄 4 天家蚕的头、脂肪体 、生殖腺、表皮和中肠中都有表达,但丝腺中没有检测到表达。     

家蚕丙氨酸转氨酶的纯化与鉴定

应用细胞匀浆,硫酸铵分段盐析,DEAE-纤维素柱层析和羟基磷灰石柱层析等方法,从家蚕后部丝腺中成功地分离制备了高纯度的丙氨酸转氨酶,经聚丙烯酰胺凝胶电泳分析鉴定,本法制备的丙氨酸转氨酶已达到均一的纯度。     

家蚕丝素蛋白轻链基因（fib-L）启动子序列的克隆及其活性分析

家蚕Bombyx mori丝素蛋白轻链（fibroin light-chain, fib-L）基因fib-L具有在后部丝腺组织专一性、高效性表达的特点。为了利用其启动子构建能够表达外源基因的丝腺生物工厂,本实验对fib-L启动子活性进行了研究。通过PCR法克隆了fib-L启动子元件,序列分析显示fib-L启动子由位于-33 ～ -25处的TATA盒元件和位于－128～－121处的特征性序列GTCAATTT共同组成。用fib-L启动子控制报告基因DsRed进行家蚕BmN细胞和蚕体内的瞬时表达研究,结果表明fib-L启动子可以驱动DsRed报告基因在BmN细胞和家蚕后部丝腺组织中瞬时表达。     

家蚕BmBrat基因的克隆与鉴定

NHL家族蛋白具有调控细胞增殖与分化的功能,在哺育动物中被广泛研究。本文克隆得到家蚕NHL蛋白家族成员BmBrat基因,通过RACE技术获得该基因cDNA全长序列为3 614 bp,其ORF为2 580 bp,编码859个氨基酸,预测其蛋白分子量为94.3 kDa,等电点为6.65。利用RT-PCR技术检测其在五龄3 d家蚕各组织表达情况,结果表明其在幼虫各组织均有表达,包括丝腺、中肠、脂肪体、马氏管等,且卵巢和头部表达量最高;胚胎时期表达谱分析显示其在胚胎发育第4天和第5天有高量表达。经原核表达、蛋白纯化及免疫小鼠后获得家蚕BmBrat多克隆抗体,且Western blotting及免疫荧光检测显示该抗体可以特异检测家蚕BmBrat蛋白;免疫荧光结果表明BmBrat蛋白定位于家蚕血细胞胞质中,为进一步研究BmBrat基因的生物学功能奠定了基础。     

Sperm-mediated gene transfer in the silkworm Bombyx mori

Plasmid DNA containing the CAT reporter gene was injected into the testis of V instar silkworm larvae. The persistence, expression, and transmission of the injected DNA were monitored in the injected individuals till eclosion as well as in the progeny. The DNA injected into the testis persisted extrachromosomally during the entire period of metamorphosis and was also transferred into the egg via sperm during fertilization. Injected plasmids were rescued from the moths that emerged from the injected larvae and also from the eggs laid by the moths that copulated with injected males. Positive signals for CAT assay in the experimental samples suggested that the injected DNA was internalized in the testicular cells and sperm. The persistence, expression, and transmission of the DNA injected into the testes indicate that sperm-mediated gene transfer is possible in the silkworm, Bombyx mori. Arch. Insect Biochem. Physiol. 37:168–177, 1998. © 1998 Wiley-Liss, Inc.     

家蚕CyPA基因的克隆、表达谱及进化分析

CyP蛋白家族在蛋白质折叠过程中起着重要作用。本研究克隆了家蚕Bombyx mori CyPA基因(BmCyPA),该基因由2个外显子和1个内含子组成,推导开放阅读框编码165个氨基酸,分子量为19.4 kD,等电点为8.79。序列分析表明BmCyPA在不同物种间具有高度的保守性,含有肽酰脯氨酸顺反异构酶活性位点及与CsA侧链结合的氨基酸,提示BmCyPA可能具有肽酰脯氨酸顺反异构酶活性和与CsA结合的特性。组织表达谱及EST数据分析显示,BmCyPA在丝腺中高丰度表达。通过对不同物种来源的CyP基因的进化分析,进一步预测了BmCyP基因的功能,BmCyP可能与丝蛋白的正确折叠相关。     

家蚕酪氨酸羟化酶基因BmTh的表达及功能

酪氨酸羟化酶作为儿茶酚胺合成的限速酶, 广泛存在于昆虫、哺乳动物和人类中, 是其新陈代谢不可缺少的酶类。在其他昆虫中, 酪氨酸羟化酶参与了黑色素的合成, 并在昆虫外骨骼的硬化过程中发挥关键作用。为了研究家蚕Bombyx mori酪氨酸羟化酶基因的生理生化功能, 本文对其基因结构、表达特征及功能进行了研究。基于家蚕基因组和基因芯片数据的生物信息学分析表明, BmTh位于家蚕1号染色体上, 含有8个外显子, 编码561个氨基酸。基因芯片数据显示在家蚕5龄第3天的头部和体壁组织中的表达量较高, RT-PCR验证结果与此一致。利用石蜡组织切片材料和RNA探针对BmTh进行表达定位, 原位杂交结果显示在家蚕头部边缘和体壁上有明显的杂交信号。在幼虫发育至熟蚕时注射酪氨酸羟化酶抑制剂3-indole-L-tyrosine （3-IT）, 20 mmol/L的浓度对幼虫几乎没有影响, 50 mmol/L的浓度导致幼虫变态不完全和化蛹困难, 100 mmol/L的浓度使幼虫致死且体色变黑。结果提示, BmTh对家蚕变态发育起重要作用, 是家蚕正常发育不可缺少的关键基因。     

家蚕MLP基因的克隆及其结构分析

利用生物信息学的方法快速获得家蚕MLP (Muscle LIM protein, MLP)基因cDNA电子序列, 经RT-PCR生物验证正确, 登录GenBank (No. DQ311195)。MLP基因cDNA长2 327 bp, ORF全长1 485 bp, 编码产生494个氨基酸。该MLP基因组DNA含有11个外显子, 10个内含子, 所有内含子/外显子边界都符合典型的GT/AG剪切模式。MLP基因编码的蛋白富含Gly (14.4%), 分子量约为53.03 kDa, 等电点(PI)为8.29。通过BLAST分析发现该基因编码的家蚕肌肉LIM蛋白, 含有5个保守的LIM结构域, 家蚕的另一种LIM蛋白(AAR23823)含一个LIM结构域, 两者可能是通过可变剪切产生; 后者可能通过竞争作用调节前者在肌细胞中的功能。MLP的克隆为进一步研究其体内功能奠定了基础。     

Stimulation of flap endonuclease-1 by the Bloom's syndrome protein

Bloom's syndrome (BS) is a rare autosomal recessive genetic disorder associated with genomic instability and an elevated risk of cancer. Cellular features of BS include an accumulation of abnormal replication intermediates and increased sister chromatid exchange. Although it has been suggested that the underlying defect responsible for hyper-recombination in BS cells is a temporal delay in the maturation of DNA replication intermediates, the precise role of the BS gene product, BLM, in DNA metabolism remains elusive. We report here a novel interaction of the BLM protein with the human 5'-flap endonuclease/5'-3' exonuclease (FEN-1), a genome stability factor involved in Okazaki fragment processing and DNA repair. BLM protein stimulates both the endonucleolytic and exonucleolytic cleavage activity of FEN-1 and this functional interaction is independent of BLM catalytic activity. BLM and FEN-1 are associated with each other in human nuclei as shown by their reciprocal co-immunoprecipitation from HeLa nuclear extracts. The BLM-FEN-1 physical interaction is mediated through a region of the BLM C-terminal domain that shares homology with the FEN-1 interaction domain of the Werner syndrome protein, a RecQ helicase family member homologous to BLM. This study provides the first evidence for a direct interaction of BLM with a human nucleolytic enzyme. We suggest that functional interactions between RecQ helicases and Rad2 family nucleases serve to process DNA substrates that are intermediates in DNA replication and repair.     

家蚕磷酸吡哆醛激酶cDNA的克隆和酶活表征

吡哆醛激酶 (EC 2.7.1.35) 在 ATP 和 Zn2 的存在下,催化吡哆醛的磷酸化反应生成磷酸吡哆醛 (PLP)。在生物体内许多酶促反应中,PLP 是一种重要的辅酶因子。家蚕和哺乳动物一样,需依赖食物中的维生素 B6前体来合成 PLP。文章描述了利用家蚕基因组数据库序列信息及使用 PCR 方法,克隆出编码家蚕吡哆醛激酶的 cDNA (GenBank 登录号:DQ452397)。克隆到的 cDNA 含有一个 894 bp 的完整可读框,编码一条分子量为 33.1 kDa,含 298 个氨基酸残基的蛋白质。序列比对显示此蛋白质序列与人类吡哆醛激酶蛋白序列具有 48.6%的同一性,包含吡哆醛激酶家族共有的特征保守序列,但其氨基酸残基数比哺乳动物和植物克隆到的吡哆醛激酶残基数均少 10 多个残基。多序列比对结果显示,吡哆醛激酶中几个有关键功能且在哺乳动物和植物中均保守的氨基酸残基在此蛋白中被替换为其他种类氨基酸残基。采用 T7 启动子和 T7 聚合酶表达系统对克隆到的 cDNA 进行了原核表达并对表达粗提产物进行了酶活检测。实验结果显示表达得到的可溶性蛋白产物占其总蛋白量为 10%,细胞粗提物具有活力为 30 nmol/min/mg 的吡哆醛激酶活性,结果证实了克隆到的 cDNA 编码家蚕中的吡哆醛激酶。