猪圆环病毒2型/3型双重荧光定量PCR检测方法

为了能够同时快速地检测和鉴别猪圆环病毒2型和3型(PCV2和PCV3),参考GenBank中已发表的PCV2和PCV3基因组序列,针对其保守区分别设计了2对特异性引物,经优化反应体系和条件,建立了能快速检测和鉴别PCV2/PCV3双重荧光定量PCR方法.结果 表明,PCV2和PCV3的R2分别为0.999、0.9993,E值分别为3.5731、3.3734.该方法能同时特异地检测PCV2和PCV3,而对其他5种猪病原均未检测到荧光信号;PCV2和PCV3的最低检测值分别为41.1 copies·μL-1、27.0 copies·μL-1;批内和批间变异系数均小于1％.临床样本检测结果显示,PCV2和PCV3的阳性率分别为62.12％(41/66)、48.48％(32/66),二者混合感染的阳性率为46.96％(31/66).表明该方法具有敏感、特异和可靠等特点,该方法为PCV2和PCV3单独或者混合感染的早期诊断、定量检测及其流行病学调查提供了可行的技术支持.     

反义AcInv基因转化马铃薯方法的研究

马铃薯反义AcInv基因,采用农杆菌介导法,选用生产上推广的9个普通四倍体栽培种,对影响转化的各个因素进行了优化研究。结果表明:在愈伤组织诱导过程中,卡那霉素(Km)的适宜浓度为100 mg/L;生根过程中为50 mg/L。外植体的预培养为:茎段2 d,叶盘3 d效果较好;农杆菌浓度当OD600值为0.5时对茎段和叶盘的转化效果均较佳;茎段和叶盘侵染时间分别达8 min和10 min时转化率较高;外植体与农杆菌共培养时间分别为2 d和3 d时,茎段和叶盘的转化率较高;各个品种的外植体在卡那霉素抗性培养基上均能形成愈伤组织,但只有1号(“大西洋”)最后从愈伤组织上分化成苗,形成正常植株,植株再生率为11.2 %,PCR扩增检测表明大部分转基因植株为阳性。     

DNA分子标记技术的研究与应用

DNA分子标记是继传统的形态学标记、细胞学标记、生化标记之后发展起来的以DNA为基础的遗传标记技术,因其独有的技术特点,迅速在各领域得到广泛的应用与发展。本研究总结概括了基于m RNA的表达序列标签(ESTs)、差异显示逆转录PCR (DDRT-PCR)、特征性差异分析(RDA)、基因表达系列分析(SAGE);基于目的基因的保守DNA衍生多态性(CDDP)、目标起始密码子多态性(SCoT)、保守区域扩增多态性(Co-RAP)和基于反转录转座子的反转录转座子位点间扩增多态性(IRAP)、反转录转座子-微卫星扩增多态性(REMAP)等几种新型DNA分子标记技术的原理、特点、优缺点,以及常见DNA分子标记技术的特点比较及其应用,以期在其基础上推动分子标记技术进一步融合、创新与发展。     

层出镰刀菌处理对食用百合保护酶相关基因表达的影响

以4种食用百合为试材,利用实时荧光定量PCR(RT-qPCR)方法,研究了层出镰刀菌处理对食用百合保护酶相关基因(POD、CAT)相对表达量的影响,以期为研究食用百合抵抗逆境的分子机制提供参考依据。结果表明:POD在“龙牙×兰州”(R6-2、6)杂交百合、“铁炮×兰州”杂交百合、“兰州”百合被处理后的第1天相对表达量达到峰值且显著高于处理后的其它时间段,分别约为对照组的18.57、63.81、51.41、37.42倍,然而“铁炮”百合则是被处理后的第3天相对表达量达到峰值且显著高于处理后的其它时间段,约为对照组的113.19倍;CAT在“龙牙×兰州”(6)杂交百合、“铁炮×兰州”杂交百合均是被处理后的第1天相对表达量达到峰值且其显著高于处理后的其它时间段,分别约为对照组的68.02、19.77倍,在“龙牙×兰州”(R6-2)杂交百合、“兰州”百合则是被处理后的第3天相对表达量达到峰值,且显著高于处理后的其它时间段,分别约为对照组的30.69、25.19倍,铁炮百合是被处理后的第5天基因相对表达量达到峰值且显著高于其它时间段,约为对照组的71.83倍。     

小麦籽粒淀粉合成酶基因表达与酶活性特征的研究

为研究小麦籽粒淀粉合成酶基因表达与酶活性的特征,以普通小麦品种秦麦11(粒重36.942 mg/粒,淀粉含量61.02%)和中优9507(粒重50.636 mg/粒,淀粉含量68.36%)为材料,采用实时荧光定量RT-PCR方法,对灌浆期籽粒淀粉合成酶相关基因的表达进行了研究,并对其表达量与相应酶活性的相关性做了分析.结果表明,腺苷二磷酸葡萄糖焦磷酸化酶基因(AGPase)、束缚态淀粉合成酶基因(GBSSI)、可溶性淀粉合成酶基因(SSS)、淀粉分支酶基因(SBE)表达均呈单峰曲线变化,在花后3 d 内检测不到其表达,花后4～6 d 这些基因开始表达.AGPase和SSS在花后12 d左右表达量达到高峰,GBSSI和SBE在花后15 d左右的表达量最大.自花后18 d开始,各基因的表达量均显著下降.中优9507在表达高峰期(即花后第12、15、18 d)的酶基因相对表达量均显著高于秦麦11,且差异均达显著水平.整个灌浆期间中优9507的四种淀粉合成酶活性高于秦麦11,尤其在灌浆中期差异更显著.相关分析表明,AGPase、SSS、SBE基因在花后15 d的相对表达量与相应酶活性间呈极显著正相关,而GBSS基因的相对表达量与GBSS酶活性间相关不显著,暗示AGPase、SSS、SBE基因在籽粒淀粉合成过程中属于转录水平调控,而GBSSI基因属于转录后调控.     

Comparative proteomics analysis of maize (Zea mays) leaves infected by small brown planthopper (Laodelphax striatellus)

Maize rough dwarf disease (MRDD) is a viral disease caused by brown planthopper infestation, and leads to great yield loss, especially in China. Comparative proteomics was performed using maize inbred line Zheng 58 and LN 287. MRDD pathogen was detected as rice black-streaked dwarf virus (RBSDV) by quantitative real time PCR (qRT-PCR) in Shandong Province, China. The modified trichloroacetic acid (TCA)/acetone method was used for soluble protein extraction from leaves. Two-dimensional electrophoresis (2-DE) analysis was performed on 24-cm long, pH 4–7 linear immobilized pH gradient (IPG) strips, and gels were stained with silver and coomassie brilliant blue. We identified 944 proteins expressed in RBSDV infected maize leaves by proteomics approaches. Among these, 44 protein spots that revealed a 1.5-fold difference in intensity were identified by mass spectrometry between mock-inoculated and RBSDV infected samples. Among these, 17 and 26 spots were up-regulated, and 27 and 18 spots were down-regulated in the virus infected samples of Zheng 58 and LN 287, respectively. Differential protein spots were analyzed by mass spectrometry identification, which could be divided into six categories. Furthermore, the expression of stress-related proteins was detected and confirmed by qRT-PCR. This study lays the foundation for further investigations, enabling the enhancement of MRDD resistance in maize.     

木薯遗传图谱16号连锁群与其细胞学图谱的整合

利用荧光原位PCR技术体系整合木薯的分子遗传图谱,将16号连锁群上的NS376和SSRY86标记定位到华南6号木薯的染色体上,结果表明:这2个标记均能在华南6号不同时期的细胞上检测到1个信号.结合核型分析,将NS376标记定位在华南6号的第4号染色体的短臂上,扩增位点到着丝粒的百分距离是31.25,将SSRY86标记定位在第4号染色体的长臂上,扩增位点到着丝粒的百分距离是75.86.NS376和SSRY86 2个标记位于木薯的同一对染色体上,并且分别位于该染色体的两端,进一步揭示了16号连锁群对应的是木薯的4号染色体.     

半滑舌鳎StAR基因克隆及其在不同组织的时空表达分析

利用半滑舌鳎性腺转录组测序获得的StAR基因部分序列,设计RACE引物,克隆了半滑舌鳎StAR基因的cDNA序列,全长为1 294 bp,5'端UTR为132 bp,3'端UTR为310 bp,开放阅读框(ORF)为852 bp,共编码283个氨基酸。将半滑舌鳎StAR基因与其他物种StAR基因进行氨基酸同源性分析,结果显示,半滑舌鳎StAR与塞内加尔鳎、大口黑鲈、花鲈、金头鲷的同源性都达到了85%,与虹鳟、斜带石斑鱼及日本鳗鲡的同源性分别为81%、83%和76%。雌、雄鱼不同组织StAR基因的表达分析表明,StAR基因在雄鱼性腺中高表达,在雄鱼的肝脏、脑及心脏中表达量较低,而在雄鱼的其他组织中不表达;在雌鱼肠中不表达,在其他组织(卵巢、肝脏、脾脏、脑、垂体、肌肉、心脏、肾脏)中微量表达。荧光定量PCR分析不同组织与不同时期性腺表达谱表明,雄鱼性腺中StAR基因的表达量显著高于雌、雄鱼其他各组织(P0.05),提示StAR基因对雄鱼精巢发育起重要作用。雄鱼不同时期表达谱分析结果显示,StAR基因在66天前的精巢中不表达,在150天时表达量急剧增加,至2龄时表达量最高,3龄时表达量下降,说明该基因在精巢发育成熟过程中起重要作用。原位杂交结果显示,StAR基因主要在雄鱼精巢的精子细胞中表达,而在雌鱼的卵巢中不表达。研究表明,StAR基因在半滑舌鳎精巢发育中发挥作用,且可能在精子形成中发挥重要作用。     

采用通用引物PCR配合SSCP和RFLP技术检测鱼病病原菌

采用通用引物PCR（UPPCR）、PCR－RFLP、PCR－SSCP技术,研究快速鉴别鱼病病原菌的分子生物学诊断技术。结果发现,采用细菌16S rRNA基因保守区特异性引物,以嗜水气单胞菌、鲁克氏耶尔森菌、鳗弧菌、柱状曲挠杆菌、乙型链球菌、荧光假单胞菌等部分常见鱼病病原菌为对象,可以建立一种UPPCR技术。该技术能在保证实验条件不变的基础上,检出上述所有细菌,并还可检出大肠杆菌和双歧杆菌等非鱼病病原菌。并且认为,该法与SSCP配合即采用UPPCR－SSCP技术能较好地鉴别被检菌而用于鱼病病原菌的快速诊断。     

Cloning, tissue distribution and hormonal regulation of stearoyl-CoA desaturase in tilapia, Oreochromis mossambicus

The stearoyl-CoA desaturase cDNA in tilapia (Oreochromis mossambicus) was cloned by RT-PCR and RACE, and it was compared with those in grass carp, common carp and milkfish. Nucleotide sequence analysis revealed that the full length of cDNA (1172 bp) clone encompasses 1008 bp open reading frame (ORF) encoding 336 amino acid residues. The deduced amino acid sequence shares 78–82% identity with the teleosts and 64–66% with mammals compared, and like these fish, the cloned tilapia stearoyl-CoA desaturase amino acid sequence conserves three histidine cluster motifs (one HXXXXH and two HXXHH), which functioned as non-heme iron binding sites, essential for stearoyl-CoA desaturase activity. RT-PCR and Northern blot analysis reveal that tilapia stearoyl-CoA desaturase is expressed only in liver, but the stearoyl-CoA desaturase expression in multiple tissues was observed in milkfish, grass carp and carp. Further, the hormonal regulation of stearoyl-CoA desaturase gene expression was investigated by a single injection of 17β-estradiol and testosterone. The results showed that the administration of 17β-estradiol to tilapia led to a greater increase in desaturase activity than testosterone, and higher doses of steroids produced greater increases in enzyme activity. The comparative RT-PCR analysis showed that the stearoyl-CoA desaturase mRNA level increased significantly in 17β-estradiol treated animals, especially in the groups receiving a single injection of 50 mg 17β-estradiol. This was reflected in the decrease in the saturated fatty acids and the increase in the monounsaturated fatty acids. The proportion of the polyunsaturated fatty acids was not affected.