基于转录组测序分析象草木质素合成的研究

采用高通量测序技术Illumlna HiSeq 2000对高木质素的象草品系eg7和低木质素的象草品系eg87(对照)茎组织进行转录组比较测序。测序获得了169630902个序列读取片段(reads),包含13788439920nt碱基信息。对reads进行序列组装,获得87641个单基因簇(unigene),平均长度580nt。从长度分布、GC含量等方面对unigene进行评估,数据显示测序质量好,可信度高。将获得的unigene与Nr、Nt、Swiss-Prot、COG、GO和KEGG数据库进行序列同源性比较和功能分析,62557个unigene与其他生物的已知基因具有不同程度的同源性,象草与高粱序列同源性最高。共鉴定出33323个差异表达基因,其中上调基因9704个(29.12%),下调基因23619个(70.88%);GO分析显示39968个unigene归为54个功能类别,大量unigene与细胞进程、代谢过程、催化活性等相关;KEGG pathway分析富集得到127条代谢通路,包括光合作用、betalain生物合成、苯丙烷类代谢、苯丙氨酸代谢等,苯丙烷类代谢途径差异基因富集程度高、差异基因数目最多,达285条,该途径中64条木质素单体合成酶基因表达上调,79条ClassⅢ型植物过氧化物酶基因表达下调、22条上调。挑选9个差异基因进行qRT-PCR验证,9个基因的表达趋势与高通量测序结果一致。为象草的分子生物学研究提供了宝贵的基因组数据,对于了解象草茎生物合成与木质素调控基因挖掘和多用途定向育种具有指导意义。     

玉米赤霉烯酮对趋化因子诱导的小鼠T细胞迁移效应和黏附与迁移相关蛋白表达的影响

为揭示玉米赤霉烯酮(ZEA)免疫毒性机理,通过体外试验研究了ZEA对趋化因子诱导的小鼠T细胞迁移效应的影响,以及这一过程中与细胞黏附与迁移相关蛋白的变化。以ConA作为T细胞活化剂,以不同浓度ZEA(0、10、20、40μmol/L)染毒处理细胞后,Transwell法检测T细胞分别在CCL19、CCL21作用下的迁移效应,以及利用流式细胞术检测迁移后CD4、CD8阳性T细胞所占比例。激光共聚焦显微镜拍摄细胞穿膜形态,Western blot检测T细胞迁移及细胞黏附相关蛋白的表达。结果显示,ZEA可以分别降低CCL19和CCL21趋化的T细胞的迁移指数并扰乱迁移后CD4、CD8阳性T细胞之间的比例。此外,ZEA可以不同程度地抑制CCL19和CCL21介导的T细胞穿膜效应。Western blot结果显示,20、40μmol/L的ZEA作用可下调T细胞迁移及细胞黏附相关蛋白的表达。结果表明,ZEA可以抑制趋化因子介导的T细胞迁移效应,在这一过程中,黏附蛋白及迁移蛋白的表达均受到抑制,提示ZEA可能通过抑制T细胞运动过程中黏附及迁移这两个环节,进而影响T细胞的免疫应答,从而发挥免疫抑制作用。     

非典型猪瘟病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立

参照GenBank上登录的非典型猪瘟病毒(atypical porcine pestivirus virus,APPV)全基因组序列设计特异性引物成功从感染APPV阳性样品中扩增出270 bp的APPV基因片段,并将其克隆到pEASY-Bluent Zero Cloning Kit载体,以纯化的重组质粒为模板优化反应条件,建立了检测APPV的特异性荧光定量PCR方法。结果显示,建立的荧光定量PCR方法C_t值与标准品在1.49×10~1～1.49×10¹⁰ copies/μL呈现良好的线性关系,相关系数为R^20.999,斜率为-3.442,扩增效率为95.207%。该方法与PCV2、PRV、PRRSV、PPV及CSFV基因组均无交叉反应,敏感性比常规PCR高出1 000倍,组内和组间重复试验变异系数均小于2%。对临床采集的20份疑似感染APPV的样品进行检测,结果显示应用所建立的荧光定量PCR成功检测到3份APPV阳性样品,而常规PCR仅检测到1份,表明所建立的荧光定量PCR比常规PCR灵敏度更高。本试验所建立的APPV SYBR GreenⅠ实时荧光定量PCR检测方法,可实现对APPV早期诊断及感染进行定量分析检测。     

高寒干旱地区新引桃树良种抗寒耐旱性研究

通过抗寒耐旱生理指标测试分析、露地越冬表现调查、不同栽培措施分析,选择出抗寒耐旱性强、特别是抗寒性强,适宜于张掖川灌区气候条件的桃树品种与品种群,以及抗寒栽培模式与措施,为张掖川灌区及同类地区桃树良种推广栽培提供科学依据。     

赤霉素和温度对贵州特有观赏植物习水报春种子萌发的影响

以习水报春种子为试验材料,采用不同温度梯度和20℃恒温下不同浓度赤霉素处理,研究了各种处理对习水报春种子的发芽率、发芽势和发芽指数的影响,以期找到最适萌发条件。结果表明:15℃处理发芽率(44.45%)最高;20℃处理发芽势(32.22%)最高和发芽指数(4.64)最高;25℃处理各项指标最低,发芽率1.11%,与10、15、20℃处理差异显著(P0.05),发芽势1.11%,与20℃处理差异显著(P0.05),发芽指数0.27,与20℃处理差异显著(P0.05)。最适萌发温度范围为15~20℃。在20℃恒温条件下,200mg·L~(-1) GA3溶液处理发芽率(41.11%)最高;100、200mg·L~(-1) GA3溶液处理发芽势(34.44%)最高,100mg·L~(-1) GA3溶液处理发芽指数最高(5.53)。400、500mg·L~(-1) GA3溶液处理与CK相比发芽率、发芽势差异均显著(P0.05),200、300、400、500mg·L~(-1) GA3溶液处理与CK相比发芽指数差异显著(P0.05),GA3溶液的最适萌发浓度范围为100~200mg·L~(-1)。     

Effects of Dietary Ginger Root,Star Anise and Dan-shen Root on Antioxidant Status and Reproductive Performance of Sows during Late Gestation and Lactation

The experiment was conducted to study the effects of diatary ginger root, star anise and dan-shen root on antioxidant status and reproductive performance of sows during late gestation and lactation.A total of 36 Laiwu Black pregnant sows with average weight of 164.28 kg±1.30 kg and similar parity were randomly divided into 4 treatments with 9 replicates per treatment.The control group was fed a basal diet, and the experimental groups were fed the basal diet supplemented with 10 g/kg ginger root, 5 g/kg star anise and 10 g/kg dan-shen root, respectively(ie Cont., GIN, SA, DS groups).The results showed that the concentrations of glutathione in serum and milk protein in colostrum, activity of total antioxidant capacity in colostrum of GIN, SA and DS groups were significantly higher than that of Cont.group(P<0.05).The concentration of milk fat in colostrum of GIN and DS groups were higher than that of Cont.group(P<0.05).Comparing to Cont.group, the concentration of lactose in colostrum was significantly higher(P<0.05) of SA and DS groups.All groups had similar number of born live, 21 d survival rate, birth weight of born and 21 d weight of born(P>0.05).In conclusion, dietary supplementation of 10 g/kg ginger root, 5 g/kg star anise and 10 g/kg dan-shen root could increase antioxidant status of serum and colostrum of sows, improve the nutritional value of colostrum, but had no effect on reproductive performance.     

Analysis of Ribosome Genotyping and Antimicrobial Resistance in Enterococcus faecalis Isolated from Pigs and Chickens

This study was to investigate the ribosome genotyping and antimicrobial resistance in Enterococcus faecalis strains isolated from 2 large-scale farms in Shanghai.40 isolates were evaluated for their sensitivity to 12 antimicrobial agents by broth microdilution,and the ribosome genotyping (ribotype) was characterized by the Riboprinter^® Microbial Characterization System.The resistance rates of most antimicrobial drug were relatively high and multidrug resistant strains were detected with more than 80%.4 strains of Enterococcus faecalis isolated from pigs were resistant to vancomycin,including 2 strains of vancomycin highly resistant (MIC>64 mg/L) with the same time highly resistant to gentamicin and streptomycin (MIC>2 048 mg/L).The results showed that multidrug resistance of Enterococcus faecalis was a serious issue,and the resistant phenotypes of the same type of ribotype was not entirely consistent.     

Substitution between Virulent and Avirulent PPRSV Vaccine Strains with Reverse Genetics System

The Nsp9 gene of porcine reproductive and respiratory syndrome virus（PRRSV）coded the RNA polymerase,which played an important role in the virus replication.In order to determine the interaction between the Nsp9 gene and virulence,replication capacity and cell tropism,the Nsp9 gene sequence of PPRSV vaccine strain CH-1R was cloned,and ligated to a virulent strain XH-GD infectious clone after restriction enzyme digestion,and transformed the virus under the basis of the reverse genetics.The results showed that the virus was successfully saved.And the results of biological characteristics test showed that titers and growth rate of saved virus were obviously higher than that of parental strain rXH-GD,but were lower than vaccine strain CH-1R.While plaques of two strains were similar in size.The polymerase of vaccine strain CH-1R could help to improve the drop degree of the virus,and this test could lay the foundation for further study of the effect of Nsp9 gene on virus virulence.     

小鼠胚胎胃肠道细菌的分离与初步鉴定

对常规饲养、正常妊娠的小白鼠胚胎后期胃肠道细菌进行分离鉴定,通过细菌培养特性、形态特征和生化试验鉴定,初步确定小鼠胚胎后期胃肠道中存在的微生物菌群有微球菌属（Micrococcus）、葡萄球菌属（Staphylococcus）、变形杆菌属（Proteus）和双岐杆菌属（Bifidobacterium）等细菌,其中G^＋细菌占据了绝对优势,小鼠胚胎胃肠道中细菌的平均数量达到1．2&#215;10^2CFU／g-3．2&#215;10^2CFU／g。本研究为哺乳动物胚胎时期存在于胃肠道中细菌的深入研究奠定了基础。     

犬瘟热病毒疫苗株H蛋白基因片段的克隆与表达及表达产物抗原性的初步鉴定

根据发表的犬瘟热病毒（CDV）参考株Ondetstepoort的序列设计一对引物,以CDV感染的Vero细胞总RNA为模板,经RT-PCR扩增得到549bpDNA片段,将该片段以正确的阅读框架定向克隆于pGEX-4T-1中,然后将重组质粒转化宿主菌Rosetta^TM,在37℃、1．0mmol／LIPTG诱导下外源基因获得良好表达。经SDS．PAGE鉴定,表达的融合蛋白约46ku,与预期值一致。Westernblot试验显示,CDV免疫的小鼠血清可特异的识别该重组蛋白,表明该重组蛋白具有一定的免疫原性。