牛支原体新疆分离株P48基因的克隆、原核表达及重组蛋白免疫原性研究

试验旨在确定牛支原体P48基因的免疫原性，为进一步筛选牛支原体免疫保护性基因奠定基础。本研究以牛支原体新疆分离株为研究对象，运用Overlap PCR方法扩增得到点突变后的牛支原体新疆分离株P48基因，构建原核表达载体pET-32a （+）-P48，转化大肠杆菌BL21（DE3）感受态细胞，在诱导剂ITPG的诱导下获得重组蛋白P48，纯化后的重组P48蛋白免疫BALB/c小鼠制备多克隆抗体，运用Western blotting和ELISA方法验证其反应原性和免疫原性。结果表明，试验成功构建原核表达载体pET-32a （+）-P48，重组蛋白P48大小约为66 ku，纯化后的牛支原体P48重组蛋白免疫小鼠后可产生良好的免疫反应，血清抗体滴度达到较高水平（D_{450 nm}值为1.126）。Western blotting结果显示，抗牛支原体P48重组蛋白的鼠血清与牛支原体P48重组蛋白及牛支原体全菌蛋白抗原均能产生明显的抗原抗体反应，表明P48重组蛋白具有良好的免疫原性与反应原性，可作为牛支原体新型疫苗的候选基因，且牛支原体新疆分离株P48基因与国内外5株牛支原体P48基因的同源性很高，亲缘关系较近。     

葡萄WRKY54基因克隆及表达特性分析

为了探究葡萄WRKY54基因的功能,以抗盐葡萄品种Vidal Blanc为材料,采用同源克隆法克隆得到Vv WRKY54基因,并对其进行生物信息学和表达特性分析。结果表明,Vv WRKY54基因c DNA序列为942 bp,编码313个氨基酸。生物信息学分析结果表明,Vv WRKY54蛋白分子量约为35.3091 k Da,等电点为5.45,不稳定系数为55.03,推测其为不稳定蛋白,与已知毛果杨及拟南芥WRKY54蛋白高度同源;亚细胞定位预测结果显示其主要存在于细胞核中。实时荧光定量PCR分析表明,Vv WRKY54在葡萄不同组织中均有表达,其中在梢尖中表达量最高;盐和低温等逆境胁迫因子均能诱导Vv WRKY54上调表达;此外,Vv WRKY54受水杨酸和一氧化氮诱导上调表达,其中水杨酸诱导Vv WRKY54相对表达量在12 h达到最大值,约为对照的50倍。推测Vv WRKY54在植物发育和抵御逆境胁迫中起着重要作用,这为进一步阐明Vv WRKY54的功能及作用机制奠定了分子基础。     

Effects of dietary protein and lipid levels on growth performance,fatty acid composition and antioxidant‐related gene expressions in juvenile loach Misgurnus anguillicaudatus

Dojo loach (Misgurnus anguillicaudatus) is one of the most important cultured freshwater fish in several East Asian countries. However, a little information is available in its nutritional requirements. Thus, this study was conducted to evaluate the effects of feeding varying levels of dietary protein and lipid on growth, fatty acid composition and antioxidant‐related gene expressions in juvenile loach. Six practical diets at three levels of protein (30%, 40% and 50%) and two levels of lipid (6% and 12%) were fed to loach juveniles (initial weight 0.40 g) in triplicated groups (20 fish per replicated) for a period of 8 weeks. Results showed that regardless of lipid level, body weight gain of fish was significantly increased with incremental dietary protein level. Meanwhile, feed conversion ratio was significantly decreased by dietary protein levels, and the lowest value was observed in fish fed dietary protein levels of 50%, regardless of dietary lipid level. Moreover, the percentage of 22:6n‐3 in viscera was significantly increased by different protein levels. The expression level of catalase was significantly increased with incremental dietary protein level with both lipid levels. Meanwhile, the expression level of hepatic nuclear factor erythroid 2‐related factor 2 (Nrf2) was downregulated with incremental dietary protein level with 6% of lipid level, but the expression was upregulated with incremental dietary protein level with 12% of lipid level. In conclusion, these data suggested that 6% lipid and 50% protein in diet was optimal for loach during early development stage.     

RNA干扰对大鲵蛙病毒主要功能基因表达及其增殖的影响

采用q PCR与病毒滴度测定观察RNA干扰(RNA interference,RNAi)对大鲵蛙病毒(Chinese giant salamander ranavirus,CGSRV)主要衣壳蛋白(major capsid protein,MCP)、甲基转移酶(methyltransferases,MTases)、DNA多聚酶(DNA polymerase)基因表达与病毒增殖的影响。结果表明,小干扰RNA(small interfering RNA,siRNA)能推迟鲤鱼上皮瘤细胞(epithelioma papillosum cyprini,EPC)出现病变,且病变程度也较对照组轻。在各功能基因表达量上,NC-FAM对照组与阴性对照组差异不显著(P0.05);而干扰组的干扰率极显著高于阴性对照组,其中siR-DP-1组siRNA对MCP基因的干扰率为79%,极显著高于其余组(P0.01);siR-MT-1、siR-MT-2组siRNA对MTases基因的干扰率为77%,极显著高于其余组(P0.01);siR-DP-1组siRNA对DNA polymerase基因的干扰率为79%,极显著高于其余组(P0.01)。干扰实验组病毒滴度与NC-FAM对照组和阴性对照组相比也有不同程度的降低。阴性对照组lg TCID50为8.362,NC-FAM对照组lg TCID50为7.848,siR-MCP、siR-MT、siR-DP实验组最低lg TCID50分别为5.764、5.317、5.362。证实RNAi能够抑制CGSRV主要功能基因的表达并影响病毒的复制。     

茶树几丁质酶基因的克隆及其在干旱胁迫下的表达分析在干旱胁迫下的表达分析

以铁观音茶树叶片为材料,利用逆转录PCR及RACE法,克隆了茶树几丁质酶基因CsChi(GenBank登录号为KR078345).CsChi基因的cDNA全长为1 192 bp,包含972 bp的开放阅读框(ORF),编码323个氨基酸.生物信息学分析结果表明,CsChi蛋白的分子量为34.33 ku;理论等电点pI为8.44;原子组成为C1519H2285N413O464S18,总原子数为4 699;蛋白质结构分析显示该蛋白有6个蛋白的跨膜区域,属于跨膜蛋白;存在于细胞外;没有卷曲螺旋结构存在;CsChi基因编码的蛋白属于糖苷水解酶19家族,含有保守的ChtBD1结构域,与溶菌酶的保守结构域类似,可能兼具几丁质酶活性和溶菌酶活性,qPCR定量分析结果显示在不同干旱胁迫处理下茶树的CsChi基因的表达量,与对照组相比有所增加.推测CsChi基因在茶树干旱等逆境胁迫中起重要作用.     

Effect of substitution of dietary fish meal by soybean meal on different sizes of gibel carp (Carassius auratus gibelio): digestive enzyme gene expressions and activities,and intestinal and hepatic histology

A 7‐week growth trial was conducted to evaluate the effects of dietary soybean meal (SBM) on digestive enzyme activity of intestinal mucosa, mRNA levels of digestive enzymes in hepatopancreas, and the mid‐intestinal and hepatopancreas histology of gibel carp CAS III (Carassius auratus gibelio). Four different growth phases of gibel carp (initial body weight: fry, 0.8 g; juvenile, 5.0 g; 1‐year‐old, 62.7 g; and broodstock, 135.6 g) were tested. Seven isonitrogenous and iso‐energetic diets were formulated to contain different SBM replacement levels (0%, 20%, 40%, 60%, 80% and 100% of dietary fish meal protein), and another diet (SBMAA) contained all SBM protein and supplied crystalline amino acids. The results showed that the activities of mid‐intestine trypsin, α‐amylase and gamma glutamyl transpeptidase reduced with increased dietary SBM, while the chymotrypsin activity increased first and then decreased. The ultrastructures of intestinal epithelial cells and hepatopancreas cells in fry and broodstock fish were distinctly affected by 200 g kg^‐1 dietary SBM. Supplementation of dietary amino acid to the highest replacement groups was not sufficient to improve digestive and absorptive capacities and growth performance. Gibel carp may be adapted to dietary SBM through increase in gene expression of hepatopancreas digestive enzymes and has potential to utilize proceeded SBM as feedstuffs.     

茶树MADS-box转录因子基因的克隆与非生物胁迫响应分析

本研究基于茶树转录组数据库,以茶树龙井43为试验材料,通过RT-PCR方法从该茶树的cDNA中克隆得到1个CsMADS1基因。序列分析表明:茶树CsMADS1基因开放阅读框长度为657 bp,编码218个氨基酸,是典型的植物MADS-box家族转录因子。序列多重比对显示,该序列与多个相关物种的MADS-box序列一致性为65.65%,含有高度保守的MADS结构域和半保守的K结构域。氨基酸理化性质、亲疏水性、亚细胞定位预测、无序化分析,以及二级和三级结构分析显示,CsMADS1转录因子是亲水性蛋白,可能定位于细胞核中,无序化程度明显,以α-螺旋结构为主,并与人MEF2蛋白具有相似的三级结构。利用实时荧光定量PCR方法分析了茶树龙井43中CsMADS1基因在非生物胁迫下的表达。结果表明,茶树中CsMADS1基因对高温、低温、干旱和高盐等不同非生物胁迫有响应,且表达存在差异。     

Detect unique molecular structure associated with physiochemical properties in CDC varieties of oat grain with unique nutrient traits [Feed Type vs. Milling Type] in comparison with barley grain using advanced molecular spectroscopy as a non-destructive biological tool

The objective of this study was to determinate grain unique protein inherent molecular structure that are related physiochemical and nutrient profiles in CDC developed oat varieties [CDC Nasser (Feed Type) and CDC Seabiscuit (Milling Type)] grown in cool climate condition in western Canada in comparison with conventional barley variety of CDC Meredith as a control using advanced molecular spectroscopy. Multivariate analyses, including an agglomerative hierarchical cluster analysis (CLA) and principal component analysis (PCA), were performed to identify protein molecular structural differences among the grains. The results revealed that CDC Seabiscuit contained greater (P < 0.05) protein structural Amide I and II than CDC Nasser and CDC Meredith, while the greater (P < 0.005) structural Amide I to II area and height ratios was detected in CDC Meredith. New oat grains had greater (P < 0.05) β-sheet height than barley grains, however, there was no difference in α-helix to β-sheet ratio values among the varieties. In conclusion, CDC Nasser and CDC Meredith had no difference in protein molecular structural features, while CDC Seabiscuit contains different protein structural characteristics as compared to CDC Meredith grain. The molecular structure features are highly associated with physiochemical and nutrient profiles in grains, which indicate that it also affect nutrient utilization and availability.