Cloning and expression analysis of ScBAK1 gene and its alternative spliceosome in sugarcane北大核心CSCD

Alternative splicing (AS) is an important part of regulation of eukaryotic gene expression. BAK1 (Brassinosteroid insensitive1-associated receptor kinase 1) is a specific type of plant serine/threonine protein kinases, and can regulate growth and development and natural immunization. To reveal the responses of sugarcane BAK1 gene to the adverse environment, a ScBAK1 gene and its alternative spliceosome, termed ScBAK1 (GenBank accession number: KP032226) and ScBAK1 S1 (GenBank accession number: KP032227), were cloned from leaf cDNA of Yacheng 05-179 utilizing the methods of electronic cloning and RT-PCR. The open reading frame (ORF) length of ScBAK1/ScBAK1 S1 gene was 1 860bp/1 770bp, encoding 619/589 amino acids residues. The predicted molecular weight of the protein was 69.28 kDa/ 65.76 kDa. Both proteins were located in plasma membrane, estimated as acid, hydrophikic and secretive proteins. Random coil and alpha helix gave priority to extended strand in their secondary structure without beta turn. The most important protein function was cell envelope, secondly biosynthesis of amino acids and cofactors. Real-time quantitative PCR analysis revealed that the expression of sugarcane ScBAK1 S1 gene exhibited the reduced expression trend under smut fungus stress and various abiotic exogenous stresses, including SA, CuCl2, PEG, ABA, NaCl and JA, while the expression of ScBAK1 gene was induced by SA, CuCl2, PEG, NaCl and smut fungus stresses. The phenomenon showed that the absent sequences or amounts of ScBAK1 S1 gene plays a key role in the response of ScBAK1 to the stress of sugarcane smut fungus, osmotic stress and cell growth. The differential expression of ScBAK1 and ScBAK1 S1 lays a foundation for further research on the function of ScBAK1 gene under biotic and abiotic stress.     

伊乐藻-氮循环菌联用对太湖梅梁湾水体脱氮的研究

从太湖梅梁湾采集无扰动泥芯样,分别添加伊乐藻、固定化氮循环菌,模拟生态修复并探讨其机制.采用同位素配对技术测定了伊乐藻-氮循环菌技术对反硝化速率的影响.结果表明,伊乐藻与氮循环菌联合作用的试验柱的反硝化速率(以N计)最高,为104.64μmol·(m2·h)-1,与裸泥试验柱相比增加了150%.采用实时荧光定量PCR技术(RT-qPCR)对沉积物中反硝化菌功能基因nirS、nirK和nosZ进行定量研究,结果显示,反硝化菌的功能基因nirS和nosZ比对照裸泥组高出1～2个数量级,表明较高的微生物量促进了反硝化脱氮的能力.室内模拟实验还表明,沉水植物提高了耦合硝化反硝化的作用,氮循环菌提高了非耦合硝化反硝化的作用,沉水植物与微生物的联合作用提高了沉积物的总反硝化速率,促进了湖泊水体氮素的脱除,起到了净化作用.     

污泥厌氧消化和后续处理中沙门氏菌杀灭及VBNC发生

应用最大或然数(MPN)培养法和反转录定量PCR(RT-qPCR)技术研究了4种污泥厌氧消化方式以及后续处理过程中沙门氏菌杀灭及有活性但不可培养(VBNC)状态的发生情况.中温厌氧消化(MAD)和高温预处理后中温消化(65℃+MAD)的沙门氏菌分别减少了2.8和5.6个数量级;高温厌氧消化(TAD)和高温-中温两相厌氧消化(TPAD)的沙门氏菌含量均低于检测限.沙门氏菌的杀灭速率常数排序为65℃+MAD>TAD>TPAD> MAD.结果显示高温对病原菌的灭活效果显著增加,高温短时预处理使得杀灭速率明显加快.TAD和TPAD的污泥VBNC沙门氏菌数量高于MAD和65℃+MAD 2个数量级,表明长时间的高温厌氧消化易导致沙门氏菌进入VBNC状态.消化污泥经过离心脱水后会出现沙门氏菌数量增加的现象,MAD、65℃+MAD、TAD和TPAD的消化污泥中沙门氏菌分别增加了1.1、2.4、3.7和2.5个数量级.但其中VBNC状态的沙门氏菌数量明显降低了1~3个数量级,表明消化后污泥中部分VBNC沙门氏菌在离心脱水过程中复活并生长,使得MPN检测得到的可培养沙门氏菌数量增加,初步解释了消化污泥经离心脱水后的病原菌再生长现象.消化污泥中沙门氏菌数量在室温放置过程中会进一步下降.     

Diaphorobacter sp.PDB3菌好氧反硝化脱氮特性

研究了Diaphorobactersp.PDB3菌在发酵罐内好氧反硝化特性.经过分析比较确定柠檬酸钠是好氧反硝化最佳碳源,采用响应面法对影响好氧反硝化的条件（温度、pH值、发酵罐转速、碳氮比）进行优化,构建出好氧反硝化数学模型.本模型预测值与实测值相关性非常好,预测PDB3菌发酵罐内好氧反硝化最佳条件：pH值、碳氮比、温度、转速分别为7.3、8.1、30.1℃、299.9r/min,最佳好氧反硝化比速率为2.25h^-1.验证实验表明,PDB3菌在发酵罐内好氧反硝化比速率为2.12h^-1,误差仅为5.8%.通过氮平衡分析表明,29.0%的总氮去除量转化为胞内氮,氮损失占总氮去除量的67.3%,细胞主要通过好氧反硝化作用和细胞同化作用脱氮.荧光定量PCR分析测定好氧反硝化过程中脱氮基因（napA,nirS,cnorB,nosZ）的表达水平以进一步解释脱氮特征,为Diaphorobacter sp.PDB3菌的实际应用提供一定的理论依据.     

栉孔扇贝内参基因稳定性研究

合适的内参基因对准确定量目标基因表达水平非常重要。利用实时定量聚合酶链式反应(RT-qPCR)分析了不同发育阶段和雌激素暴露条件下栉孔扇贝性腺组织中β-actin、β-TUB、EF-lα、18SrRNA和GAPDH5个内参基因的表达水平,并利用RefFinder软件对其表达稳定性进行了分析。结果表明:在所考察的内参基因中,EF-lα在栉孔扇贝不同发育阶段和雌激素暴露下的表达均最为稳定,可作为定量目标基因表达水平的内参基因之一。