hrpZ_(Psta)在转基因大豆中定量表达与疫霉根腐病和灰斑病抗性相关研究

以T5转hrpZPsta基因大豆JN29-705-15和JL30-187为材料,利用实时荧光定量PCR技术(Quantitative Real Time PCR,qRT-PCR)检测了目标基因在转基因大豆不同组织中的表达量,分别利用下胚轴侵染法和叶面喷施法鉴定疫霉根腐病抗性和灰斑病抗性,并分析目的基因表达量与疫霉根腐病和灰斑病抗性的相关性。结果表明:hrpZPsta基因在大豆的叶、茎、根、籽粒中均有表达,二个株系平均相对表达量分别为8.2/6.1、0.9/0.7、6.5/4.6和0.8/0.7;T5转hrpZPsta基因大豆抗疫霉根腐病和灰斑病能力与野生型相比均有所提高,JN29-705-15对疫霉根腐病抗性从感病提高到中抗,而JL30-187从中抗提高到抗病;hrpZPsta基因在叶中的表达量也与抗灰斑病能力呈正相关,与病情级别呈极显著负相关。试验结果初步证明了外源基因hrp ZPsta在大豆植株中的表达量与受体植株对疫霉根腐病和灰斑病抗性存在一定的相关性。     

Identification and expression analysis of 11 MADS-box genes in peach (Prunus persica var. nectarina ‘Luxing’)

MADS-box genes play a central role in the development of flowers in plants. In this study, 11 PpMADSs were isolated from ‘Luxing’ peach, and the expression levels were detected in different tissues and fruit development. Eleven PpMADSs, designated as PpMADS13, 14, 18, 23, 24, 25, 32, 33, 34, 35, and 39 were isolated. Phylogenetic analysis revealed that PpMADS13, 14, and 18 belonged to SVP, AGL15, and MIKC* group respectively; PpMADS23, 24, 25, and 39 were in the Mα group; PpMADS32, 33, 34, and 35 belonged to the Mγ group. Predictions from subcellular localization showed that 10 PpMADS were located in the nucleus. RT-PCR revealed that PpMADS13 was expressed in stems, leaves, and during fruit development (70d); PpMADS14 was expressed in sepals, stamens, petals, and during flower development; PpMADS18 was expressed in roots, stems, leaves, sepals, ovaries, stamens, petals, and during flower and fruit development; all MADS-box genes (expect for PpMADS33) in the Mα and Mγ group were expressed in roots, stems, leaves, sepals, ovaries, stamens, petals, and during flower development; few genes were expressed during fruit development. These results indicated that PpMADS may play a crucial regulatory role in vegetative growth and development processes of flowers and fruits in peaches.     

Identification and Molecular Characteristics Analysis of Porcine Epidemic Diarrhea Virus Variants

To ascertain a diarrhea case in a pig farm in Shandong province,the pathological changes of dead piglets were observed and nested RT-PCR test was carried out on 7 diarrhea samples for porcine epidemic diarrhea virus (PEDV).Pathological examination revealed that intestine was detected as enlargement,hyperemia and edema.After histological examination,the typical microscopic lesions of intestine were disappearance of epithelial cells,villus shrinkage and shortening.The result of nested RT-PCR showed that all of the 7 samples could amplify a specific target band of PEDV.Molecular characteristics of two field strains showed that they had an amino acid homology of 92.3% to 92.4% with vaccine CV777 and 96.6% to 98.6% with other previous field strains which sequences were downloaded from GenBank.Phylogenetic tree analysis further revealed that all of PEDV strains could be mainly divided into two clusters of G1 and G2. G2 consisted of the field strains of our study and other field strains from USA,China and so on,which had the same sequence characteristics of two insertions and one deletion,while G1 consisted of all vaccines of CV777 and several older field strains from China and Korea.These results indicated that our field strains were the dominant strains in recent epidemic diarrhea occurrence.Moreover,its molecular characteristics might be a characterization of differentiating the field and vaccine strains.     

巴西橡胶树乳管细胞HblMYC3互作蛋白筛选与功能分析

MYC是b HLH转录因子家族的亚家族成员,在植物茉莉酸信号转导过程中发挥着重要的调节作用。Hbl MYC3是从巴西橡胶树的乳管细胞中分离鉴定到的MYC类转录因子,其基因表达受割胶和茉莉酸上调。采用酵母双杂交方法初步筛选Hbl MYC3蛋白的互作蛋白,旨在进一步了解Hbl MYC3的功能。结果表明:Hbl MYC1、Hbl MYC2、DNAJ蛋白、谷氧还蛋白2、含A20和AN1锌指结构域的胁迫相关蛋白5、28 ku热和酸稳定的磷蛋白以及25 ku泛素连接酶E2等7种蛋白不同程度地与Hbl MYC3蛋白互作。基于这些互作蛋白的功能,推测Hbl MYC1或Hbl MYC2通过与Hbl MYC3形成二聚体对小橡胶粒子膜蛋白基因表达进行调控,其他蛋白参与胁迫条件下维持二聚体的稳定性和胁迫反应后降解该二聚体。     

猪胆囊中沙门菌L型的分离培养与invA基因检测

为了解猪胆囊中沙门菌L型携带情况,在贵阳市屠宰场采集970例健康生猪的胆囊组织与胆汁标本,用常规细菌学方法和非高渗分离培养法分离沙门菌及其细菌L型,用PCR和核酸序列分析方法对稳定L型纯培养物进行沙门菌的invA基因检测。结果显示,970例生猪胆囊标本未检出沙门菌细菌型,细菌L型检出率为8.25%;80例细菌L型分离物中有50例invA检测阳性,占5.15%;占细菌L型阳性分离物62.50%。研究结果为生猪胆囊沙门菌L型感染的流行病学及其检查提供了依据。     

m6A甲基化在鸡肌肉生长发育中的表达研究

试验旨在研究RNA m6A修饰相关基因去甲基化酶Alk B同源蛋白5（Alk B homologue 5，ALKBH5）、去甲基化酶肥胖相关蛋白（fat mass and obesity-associated protein，FTO）、甲基转移酶样蛋白3（methyltransferase like 3，METTL3）、甲基转移酶样蛋白14（methyltransferase like 14，METTL14）和成肾细胞瘤1-结合蛋白（Wilms’tumor 1-associating protein，WTAP）在鸡骨骼肌发育过程中的表达，分析其与骨骼肌m6A甲基化水平的相关性。首先，利用实时荧光定量PCR技术检测m6A甲基化相关基因在金茅花鸡12（E12）、14（E14）、16（E16）、18（E18）胚龄和1日龄腿肌和胸肌组织中mRNA表达水平，以及其在鸡成肌细胞50%、100%增殖期和1、2、3、4、5 d分化期的mRNA表达水平；随后，利用m6A甲基化试剂盒检测金茅花鸡E12和1日龄腿肌和胸肌组织中m6A甲基化修饰水平，与m6A甲基化相关基因表达水平进行相关性分析。结果显示，m6A去甲基化基因ALKBH5和FTO mRNA表达水平在骨骼肌发育过程中显著上调（P<0.05），即在E12、E14低表达，E16、E18逐渐上调，1日龄达到最高。m6A甲基化写入基因METTL14、METTL3和WTAP mRNA表达水平在E12、E14、E16逐渐上升，E18下降，随后至1日龄表达量回升。在细胞增殖过程中，ALKBH5、FTO、METTL14、METTL3和WTAP基因表达均上调；在细胞分化过程中ALKBH5和FTO基因表达水平显著上调（P<0.05），在分化第5天达到最高。METTL14、METTL3和WTAP基因mRNA表达水平在细胞诱导分化的1、2、3、4 d表达量呈下降趋势，而在诱导分化的第5天有所回升。甲基化水平检测结果显示，腿肌和胸肌m6A甲基化水平变化趋势一致，均在胚胎发育过程中显著下降（P<0.05），至1日龄达到最低。相关性分析结果显示，鸡骨骼肌RNA m6A甲基化水平与m6A去甲基化修饰基因ALKBH5、FTO mRNA表达水平呈显著负相关（P<0.05）。综合以上试验结果，推测m6A甲基化修饰与鸡骨骼肌发育相关，而去甲基化基因ALKBH5、FTO可能通过调控RNA m6A甲基化水平，影响鸡骨骼肌发育。本研究结果为进一步研究m6A甲基化修饰调控鸡骨骼肌生长发育的功能和分子机制提供理论依据。     

Enhanced muscle regeneration in freshwater prawn Macrobrachium rosenbergii achieved through in vivo silencing of the myostatin gene

Myostatin (MSTN) is an interesting negative growth‐regulating gene that has been well characterized in vertebrates but scantly described in invertebrates. The current study focuses on the downregulation of the MrMSTN gene and subsequently records any histological changes for giant freshwater prawn, Macrobrachium rosenbergii (Mr). In addition, the study also deals with the MrMSTN gene's influence on other growth‐related genes, which include myosin heavy chain, dystrophin‐dystroglycoprotein complex, tropomyosin, farnesoic acid o‐methyl transferase, arginine kinase, cyclophilin, and acyl CoA desaturase. The preliminary histological analysis following MrMSTN silencing favors muscle regeneration, which supports its functional role as a negative growth regulator and its significant effect on the expression of other growth‐related genes. Overall, our results show that the MrMSTN gene could therefore be a potential target for gene manipulation aimed at enhancing the growth and muscle development of M. rosenbergii, which could be beneficial in increasing the total mass production in the postlarva phase at the hatchery level.     

黄羽肉鸡ACSL1基因多态性与腹脂性状的相关性研究

长链酯酰辅酶A合成酶(ACSLs)是长链脂肪酸通过硫代酯化进而合成酰基辅酶A衍生物所必需的酶,也是脂肪酸代谢的第一步。哺乳动物ACSL家族由ACSL1、ACSL3、ACSL4、ACSL5和ACSL65个不同的成员组成,ACSL1是主要的异构体之一。为探讨黄羽肉鸡ACSL1基因作为腹脂性状分子标记的可行性,本实验采用PCR-直接测序技术对黄羽肉鸡ACSL1基因进行遗传多态性分析。结果显示:黄羽肉鸡ACSL1基因第17到第18外显子区域(1295 bp)SNPs位点较丰富,T32126C、C32013T、A31958G这3个位点的等位基因频率符合哈代-温伯格平衡,且A31958G突变位点、T32126C突变位点与腹脂重、腹脂率不相关,C32013T突变位点对鸡的腹脂重与腹脂率有显著影响,提示能够利用C32013T突变位点对黄羽肉鸡腹脂重进行分子标记辅助选择。     

中华卤虫家系构建几个关键技术探讨

本文就中华卤虫(Artemia.sinica)家系构建的几个关键技术进行了具体的讨论。交配产卵阶段基本参数研究结果表明,多数家系在抱对后24~60小时便开始产卵,平均间隔时间为55.68小时,具有较好的产卵及时性,同时有效地排除了假性交配现象,保证了在第20日龄时测定各性状值后建立家系、统计交配、产卵数据的准确性。各家系产卵情况统计结果表明,以生产2批卵的家系最多,46个,以产卵总量在151~200粒之间的家系最多,26个,产卵总量大于150粒的家系为70个,完全满足家系构建的需要。半同胞家系方面,在全同胞家系获得足够数量休眠卵的基础上,对仍在抱对的家系采用人工分离抱对雌雄个体的方法进行半同胞家系的组建,以便实现半同胞家系构建的同步性。本试验各生产阶段基本参数的获得,对指导中华卤虫构建家系进行遗传选育提供了理论依据。