以山羊捻转血矛线虫第三期幼虫作为诱饵线虫,利用撒布分离法对新疆石河子地区土壤中的捕食线虫性真菌进行分离,并对所分离的捕食线虫性真菌进行了形态学及分子生物学鉴定.结果成功分离出了9株捕食线虫性真菌,其形态学符合少孢节丛孢菌.将分离株5.8S rDNA和ITS2基因序列与国内外已知少孢节丛孢菌菌株的对比分析,证明少孢节丛孢菌新疆分离株具有区域性特征,其捕食活性高于报道的菌株.分离出适合本地区生态环境的当地菌株对以后家畜线虫病的生物防控尤为必要.

This experiment was conducted to understand the role of the σB factor in regulating the environmental stress of Listeria monocytogenes(LM).We constructed SigmaB gene deletion LM strain by using gene overlap extension PCR(SOE-PCR) and homologous recombination,and the environmental stress response of the strain on the different temperature,different pH condition and high osmotic pressure conditions were detected,and the transcription level of the genes that related with environment stress were tested by using qRT-PCR.The results showed that the environmental stress abilities of the SigmaB gene deletion mutant at the non-suitable conditions weakened and the transcriptional level of genes corresponding to environmental stress(rsbV,rsbW,hpt,clpP,ctsR) decreased significantly comparing with the standard strain.The results indicated that σB factor plays an important role in responding to environmental stress(temperature,pH and osmotic pressure) of LM.The study provides an important theoretical basis for further studies on its molecular mechanisms of environmental stress response and new target for drug action.

The nematophagous fungus Arthrobotrys oligospora is a potential biological agent against parasitic gastrointestinal nematodes. Its subtilisin-like serine proteases play an important role in nematode cuticle breach. In this study, the cDNA of the mature serine protease XAoz1 from A. oligospora XJ-XAo1 was expressed in Pichia pastoris to assess the in vitro nematicidal activity of recombinant XAoz1 (reXAoz1) on Caenorhabditis elegans and Haemonchus contortus. The cDNA sequence of the protease XAoz1 was amplified by reverse transcription polymerase chain reaction (RT-PCR) and inserted into the vector pPIC9K for expression in P.pastoris GS115. Our results show that the reXAoz1 had a molecular mass of 50 kDa after 3 days of 1.5%-methanol induction at 28 °C. The highest specific protease activity was achieved at 12 168 U mg(-1) protein. The reXAoz1 had the highest hydrolytic activity at pH 6.5-9.5 with an optimal pH at 8.5. Moreover, the purified reXAoz1 displayed a highly toxic and biological activity to immobilize C. elegans and H. contortus by degrading their cuticles and inducing death.

To understand the tick-borne anaplasmataceae in the south edge of Gurbantunggut desert,ticks specimens from 11 sites in three different habitats areas were collected and identified.Nested PCR was carried to detect positive ticks carring anaplasmataceae,and then 5′end variable region of 16S rRNA gene of anaplasmataceae was amplified by semi-nested PCR for cloning and sequencing.The obtained DNA sequences were compared with the GenBank sequence and phylogenetic tree was constructed with the Mega 5.0 software.The 708 ticks collected from 11 sites were identified as 4 genera and 8 species.25 were detected positive in the 236 tick samples,with a positive rate of 10.59%.By sequence analyses,Ehrlichia chaf feensis,Ehrlichia canis,Anaplasma marginale and Anaplasma ovis were positive in Hyalomma asiaticum,Rhipicephalus sanguineeus,Hyalomma detritum and Haemaphysalis longicornis,respectively.The results showed that there exists anaplasmataceae in ticks parasited in livestock from the south edge of Gurbantunggut desert,suggesting that the region may be one of natural foci of anaplasmosis.

Caenorhabditis elegans is an important model nematode for modern developmental biology,genetics and genomics etc.It was found that C.elegans was able to grow at 16 ℃ to 25 ℃ in NGM medium.However,it was better to grow at 20 ℃ from the morphology,culture condition and preservation of C.elegans.And under the condition of-80 ℃ or liquid nitrogen,stocks were survived for at least 2 years in 30% glycerol solution or SA frozen solution.Meanwhile,the induction ability of different larvae of C.elegans to A.oligospora XJ-XA was explored with the detection of trapping rate of predation of the same fungus to different larvae,and the relationship was analyzed between the production of trapping device,predation ability and juvenile stages of nematode.The results demonstrated that there was a significant difference in the induced amount of the capturing device of the predating strain,and there were also great differences in the predating ability of the same strain to different larvae.

为研究新疆地区捕食线虫性真菌的生物活性,本实验采用玉米粉琼脂培养基(cMA)培养分离自新疆不同地区的捕食线虫性真菌,通过对分离株纯培养物的形态学观察和18S rDNA基因序列分析,鉴定4株少孢节丛孢菌,分别命名为XA3、XA6、XD1和XD4.18S rDNA基因遗传进化分析表明,XA3、XA6、XD1和XD4新疆分离株之间同源性较高,与GenBank登录的少孢节丛孢菌CBS 289.82株同源性最高,分别为99.6%、99.5%、99.1%和98.9%.与形态学鉴定结果一致.捕食线虫活性测定试验表明,4个分离株在CMA中的捕食率为92.8%～97.6%;体外对绵羊粪便中线虫幼虫的捕食率为90.7%～95.4%,均具有很强的捕食活性.本研究为研究绵羊消化道线虫生物防控制剂奠定基础.

根据已经报道的少孢节丛孢菌丝氨酸蛋白酶基因序列,设计特异性引物,通过PCR技术对少孢节丛孢菌新疆分离株(XJ-XA)丝氨酸蛋白酶(命名为Aoz1)基因序列进行了扩增,并与国内外少孢节丛孢菌不同地域分离株相应序列进行了同源性分析,构建该基因系统进化树.结果显示,XJ-XA Aoz1基因全长为1344 bp,包含2个外显子和1个63 bp的内含子(第517～579位核苷酸),编码426个氨基酸.Aoz1的信号肽位于氨基酸序列的第1～21位氨基酸；氨基酸序列的第160～171位、200～210位和351～361位分别是天冬氨酸(D)、组氨酸(H)和丝氨酸(S)活性位点所在区域,表明该酶属于Subtilase家族；该酶还含有2个潜在的N末端糖基化位点及2个与底物结合的S1区.系统发生分析发现,少孢节丛孢菌不同地域分离株Aoz1基因序列具有较高的亲缘性,与其他捕食线虫性真菌Aoz1基因亲缘性较远.本研究为研发家畜线虫病生物防控制剂奠定了前期基础.