沈玉21号玉米栽培要点与制种技术

沈玉21号(原沈试3005)组配于2000年,亲本自交系为自选系沈3336和自选系沈3265。2004年通过辽宁省农作物品种审定委员会审定,现已获国家植物新品种保护。该品种为黄色、半硬粒型、晚熟玉米单交种,具有产量高、适应范围广、抗倒性强、活秆成熟、籽粒赖氨酸含量较高等特点,深受广大农户欢迎。     

播期对洛麦21生长发育的影响

对洛麦21的生育规律进行了研究。结果表明:洛麦21的幼穗分化总体表现为前期慢、后期快,随着播期的推迟,幼穗分化的时间缩短。平均灌浆速率在1.453~1.697之间,在10月4日播种下平均灌浆速率最大,灌浆时间35~38 d,千粒重稳定在45 g以上。针对灌浆速率而言,以10月上旬适期播种对洛麦21最为有利。洛麦21主要依靠主茎和第1、2个一级分蘖成穗。随着播期的推迟,洛麦21的总分蘖数明显降低,成穗率提高。以10月14日播种的产量最高。     

禽传染性喉气管炎病毒体内外感染模型中鸡Toll样受体21mRNA转录水平的研究

为研究病毒与机体的相互作用,本研究参考GenBank中鸡Toll样受体21 (ChTLR21)的基因序列设计实时定量PCR特异性引物,以鸡核糖体蛋白L4 (RPL4)为内参基因,建立检测ChTLR21 mRNA相对转录水平的实时定量PCR方法,分析ChTLR21在禽传染性喉气管炎病毒(ILTV)感染的鸡胚成纤维细胞(CEF)中和感染SPF雏鸡免疫器官脾脏、法氏囊和胸腺组织中的转录水平.结果显示:ILTV感染CEF后2h、4h、8h和18h时间点ChTLR21 mRNA转录水平分别为未感染对照细胞的1 540.53、0.98、1.19和3.70倍.但仅在ILTV感染2h时引起ChTLR21转录水平升高,与对照组相比差异显著(P＜0.05).ILTV感染SPF雏鸡后6h、24 h和30 h脾脏中ChTLR21 mRNA转录水平分别为未感染对照的56.34、59.85和3.61倍;法氏囊中分别为0.03、25.98和3.08倍;胸腺中分别为2.52、50和7.32倍.在感染初期,脾脏中ChTLR21转录量显著升高,随后有所降低,但均高于未感染对照(p＜0.05);法氏囊中仅在感染6h时呈显著抑制(p＜0.01);胸腺中呈波动性转录水平升高,但与对照组无统计学差异(p＞0.05).本研究证明ChTLR21参与了鸡体对ILTV感染的应答,并在体内外感染模型中呈现不同的表达规律.     

生物反应器培养BHK-21悬浮细胞增殖伪狂犬病病毒工艺优化

旨在筛选伪狂犬病病毒（PRV）敏感的BHK-21细胞并分析其生长和病毒增殖特性,优化反应器中BHK-21悬浮细胞的培养和病毒增殖条件,建立生物反应器培养BHK-21悬浮细胞增殖PRV工艺。本研究利用响应面和单因素优化法,以细胞生长动力学特性、TCID₅₀病毒滴度等参数为指标,优化1.2 L生物反应器中BHK-21悬浮细胞的最佳培养和增殖病毒条件,在5 L生物反应器中进一步批培养验证。结果显示,筛选获得PRV高敏感的BHK-21-02贴壁细胞和BHK-21-XF02悬浮细胞各1株,BHK-21-XF02悬浮细胞在含3%血清的SLM-BHK低血清培养基和SFM-BHK无血清培养基中均能实现良好的生长和病毒增殖。利用响应面法优化得到1.2 L反应器最佳培养条件为接种密度1.20×10⁶cells·mL^-1、搅拌转速120 r·min^-1、DO值40%,5 L反应器批培养72 h细胞密度可达（7.61±0.18）×10⁶ cells·mL^-1、细胞活率为（96.93±1.18）%。利用单因素法优化得到1.2 L反应器最佳病毒增殖条件为MOI 0.001、培养温度37℃、细胞密度2.0×10⁶cells·mL^-1、搅拌转速80 r·min^-1,5 L反应器批培养接毒后48 h病毒滴度达到最大值（7.13±0.11） lgTCID₅₀·mL^-1。本研究可为PRV疫苗相关研究和规模化生产提供参考。     

miR-21:生成、表达调控及对其细胞凋亡的影响

凋亡是细胞的一种基本生物学现象,在机体生命过程中发挥着重要作用。miRNAs是在真核生物内发现的一类长度约22个核苷酸的内源性非编码单链小RNA,通过调控靶基因的表达参与调控细胞的凋亡、分化、增殖、侵袭和代谢。大量的研究表明,miR-21作为miRNAs重要成员之一,与细胞凋亡有重要联系。本文将从miR-21的生成、表达调控及对细胞凋亡的影响方面进行综述。     

小麦-簇毛麦6VS/6AL易位染色体在不同小麦背景中的遗传稳定性及其在配子中的传递

为研究小麦-簇毛麦6VS/6AL易位染色体在不同小麦背景中的遗传稳定性及其在配子中的传递,利用高抗白粉病的普通小麦-簇毛麦6VS/ 6AL易位系92R137与不抗白粉病的栽培品种百农64、百农9310、邯5310、小偃54、淮麦20、徐麦856进行杂交,并用这些品种分别与杂种F1进行正反回交.对杂种F1花粉母细胞减数分裂中期Ⅰ的细胞学观察表明,在减数分裂中期Ⅰ易位染色体6VS/ 6AL通常以6AL与6A 染色体配对形成棒状二价体.各杂种F1高抗白粉病,位于6VS上的抗白粉病基因Pm21呈显性,在F2中有69.0%～74.0%的植株抗白粉病,接近1对显性基因遗传的理论值.由于6VS与6AS在通常情况下不发生配对交换,因此可通过白粉病抗性鉴定并结合利用6VS上的分子标记来跟踪6VS/ 6AL易位染色体的传递.测交结果表明,以F1作母本6VS/ 6AL易位染色体通过雌配子的传递率为49.2%(45.8%～54.9%);以F1作父本6VS/ 6AL易位染色体通过雄配子的传递率为44.7% (43.1%～46.8%),均接近50%的理论值.但在各组合中,6VS/ 6AL易位染色体通过雄配子的传递率均低于通过雌配子的传递率.     

2种不同方法纯化乳酸杆菌重组S-层融合蛋白的比较试验

将重组菌E.coliBL21（含重组质粒pGEX-SLP）经IPTG诱导获得重组融合蛋白GST-SLP,分别用非特异性的氯化锂沉淀方法和特异性的Pierce（R）GST Spin Purification Kit纯化方法,将该融合蛋白进行纯化,纯化结果采用SDS-PAGE方法进行鉴定.结果显示,2种方法均能够获得大小约为71 ku的目的融合蛋白,但氯化锂沉淀方法纯化效果不及Pierce（R）GST Spin Purification Kit方法.该研究结果为进一步研究乳酸杆菌S-层蛋白生物学特性提供了参考.     

Effect of miR-21 over-expression on proliferation,migration and diffe-rentiation of c-Kit+ cardiac stem cells

AIM: To explore the effect of microRNA (miR)-21 on proliferation, migration and differentiation abilities of c-Kit⁺ cardiac stem cells (CSCs). METHODS: c-Kit⁺ CSCs were cultured and selected by the methods of enzyme digestion and magnetic bead separation. miR-21 mimics (50 nmol/L) and mimics negative control (MNC) were transfected into c-Kit⁺ CSCs with Lipofectamine^® 2000. The cells was divided into 3 groups:control group:c-Kit⁺ CSCs without any pretreatment; MNC group:the cells were transfected with MNC for 48 h; mimics group:the cells were transfected with miR-21 mimics for 48 h. qPCR was used to assess the expression of miR-21 in each group. CCK-8 and EdU assays were used to determine the cell proliferation. qPCR and immunofluorescence were used to detect the differentiation in each group. Scratch assay was adopted to explore the migration ability of the cells. RESULTS: The expression of c-Kit in the c-Kit⁺ CSCs were 90.8%, with 0.6% of CD45 and 0.5% of CD34. A significant increase in miR-21 expression was observed when the cells were transfected with miR-21 mimics for 48 h (P<0.05). CCK-8 and EdU assays showed that miR-21 significantly increased cell proliferation as compared with MNC group and control group (P<0.05). No difference in the expression of Nkx2.5, CD31 and α-SMA at mRNA and protein levels was observed, and no difference of the migration ability in 3 groups of the c-Kit⁺ CSCs was found. CONCLUSION: Over-expression of miR-21 significantly promotes the proliferation of c-Kit⁺ CSCs, without any effect on the cell migration and differentiation.     

MicroRNA-21 up-regulates expression of occludin by targeting ROCK1 in NCM460 cells

AIM: To investigate the role of microRNA-21 (miR-21) in regulation of tight junction (TJ)-associated protein occludin in human normal colon mucosal epithelial cell line NCM460, and to analyze related target genes. METHODS: Using miR-21 over-expression lentivirus, the NCM460 cells with miR-21 overexpression were established. The expression level of miR-21 was detected by qPCR. The expression of occludin was determined by Western blot. The target genes of miR-21 were predict by bioinformatic method. According to the scores and the appropriate literature search, a target gene was selected for further study. The miR-21 mimic and inhibitor were transfected into NCM460 cells, and the expression levels of miR-21 and ROCK1 were measured. Dual-luciferase reporter assay was also performed to verify ROCK1 as the target gene of miR-21. RESULTS: In miR-21-overexpressing NCM460 cells, the protein expression level of occludin was upregulated. Bioinformatics and literature analysis predicted that the target gene of miR-21 was ROCK1. The mRNA and protein levels of ROCK1 were down-regulated in the NCM460 cells transfected with miR-21 mimic. Accordingly, the mRNA and protein levels of ROCK1 were up-regulated in the NCM460 cells transfected with miR-21 inhibitor. Luciferase assay confirmed that miR-21-binding sequence was on the 3'UTR of ROCK1, indicating that ROCK1 is the target gene of miR-21. CONCLUSION: In NCM460 cells, miR-21 up-regulates the expression of occludin, which functions in the maintenance of intestinal epithelial mechanical barrier. ROCK1 is a target gene of miR-21, and is involved in the miR-21 regulation of occludin.