柞蚕热休克蛋白70基因的克隆和序列分析

HSP70蛋白是受热等因素刺激后而诱导产生的蛋白质,是热休克蛋白家族中最重要的一员。采用RT-PCR方法克隆了柞蚕(Antheraea pernyi)热休克蛋白70基因(HSP70)的ORF序列(GenBank登录号:GU945199),该片段的序列长度为1905bp。生物信息学分析表明,该序列共编码634个氨基酸,预测蛋白的等电点和分子量大小分别为5.62kD和69.5kD。具有HSP70的保守性结构特征,与天蚕(Antheraea yamamai)、家蚕(Bombyx mor)、甘蓝夜蛾(Mamestra brassicae)、棉铃虫(Heliothis viriplaca)、甜菜夜蛾(Spodoptera exigua)、烟草夜蛾(Manduca sexta1)、膜翅目寄生蜂(Cotesia rubecula)的同源性分别为95.7%、78.5%、76.1%、77.3%、76.6%、74.7%、65.9%。根据它们的一级结构构建了系统进化树,进一步确立了它们之间的亲缘关系。     

禾谷缢管蚜阳离子通道基因RSC1的克隆、分子特性及诱导表达分析

【目的】SC1通道(sodium channel 1)是昆虫体内一种重要的离子通道,被认为是一种开发新型杀虫剂的神经靶标。本研究拟克隆禾谷缢管蚜Rhopalosiphum padi的SC1通道基因,并初步分析其生理功能及其与SC1类通道、电压门控钠离子通道、电压门控钙离子通道的进化关系。【方法】采用RT-PCR技术,克隆了禾谷缢管蚜SC1基因完整的开放阅读框;利用实时荧光定量PCR技术,分析禾谷缢管蚜成蚜在不同浓度的高效氯氟氰菊酯诱导下SC1基因表达变化。【结果】获得了禾谷缢管蚜SC1基因(命名为RSC1)完整的开放阅读框(Gen Bank登录号为KU640190),其长度6 687bp,编码2 228个氨基酸。RSC1具有SC1通道的结构特征,有一个不同于电压门控钠离子通道和电压门控钙离子通道的特殊DEEA模体(motif)。系统进化分析结果显示,RSC1与电压门控钠离子通道组成一个进化枝,电压门控钙离子通道组成另外一个进化枝,SC1与电压门控钠离子通道在进化上有更近的起源关系。实时荧光定量PCR分析结果表明,LC15,LC35和LC503种剂量的高效氯氟氰菊酯处理6 h后,禾谷缢管蚜RSC1基因表达量相对于清水对照显著下调,表达量分别为对照的0.57,0.82和0.78倍;3种剂量的高效氯氟氰菊酯处理24 h后,禾谷缢管蚜RSC1基因表达量分别为对照的2.19,1.33和1.19倍,其中LC15(0.1484 mg/L)胁迫下RSC1基因的表达量显著上调。【结论】SC1类通道与电压门控钠离子通道在进化起源上有更近的关系。RSC1通道可能是高效氯氟氰菊酯的次级靶标。由于RSC1和其同源基因只存在于节肢动物中,脊椎动物尚未发现该类基因,因此这类通道可能作为开发新型杀虫剂的神经靶标。     

大鼠胎脑神经干细胞HPRT基因的敲除

根据已知大鼠次黄嘌呤鸟嘌呤磷酸核糖转移酶 (HypoxanthineGuaninePhosphoribosylTransferase ,HPRT)基因的外显子序列 ,从大鼠HPRT基因组DNA序列的细菌人工染色体 (BacterialArtificialChromosome ,BAC)中用酶切和PCR方法分别分离得到用于构建基因敲除载体的 3 0kb的 5′长臂 (LongArm ,LA)和 1 7kb的 3′短臂 (ShortArm ,SA) ,并分别克隆到pSL1180和pCR2 1中。进一步构建大鼠HPRT基因打靶载体———pKO HPRT ,经酶切鉴定后的大鼠HPRT基因敲除载体用NotⅠ酶切使其线性化 ,经溴乙锭、正丁醇、酚、酚 /氯仿提纯后 ,将终浓度调至 1μg μl。在FuGene 6转染试剂的作用下转染培养 2 4h的第二代大鼠胎脑神经干细胞 (RatFetalNeuralStemCells,rFNSCs)。转染后的细胞用 80 μg mlG4 18和 0 2 μmol L的Ganc全培养液筛选 ,2w后将存活细胞进行悬浮培养 ,使细胞形成球形物 ,挑选单个的球形物进行单克隆增殖 ,其中一部分细胞 (约 2～ 3× 10 3)用裂解液处理 ,取上清用于PCR检测 ,大部分细胞 (5× 10 7)用于DNA和RNA的提取 ,进行Southernbolt和RT PCR检测 ,剩余细胞冷冻保存。最后 1次实验共分离培养了 32个rFNSCs单克隆 ,其中 3个单克隆 (9 3% )经PCR、Southernbolt和RT PCR证实HPRT基因已被敲除     

不同性别类型黄瓜ACC合酶基因的单核苷酸多态性标记和酶切扩增长度多态性标记

黄瓜的性别分化与乙烯密切相关,1-氨基环丙烷-1-羧酸(ACC)合酶是乙烯生物合成过程中的关键酶.根据ACC合酶基因家族的保守序列设计PCR引物,从8个不同性别类型(雌雄同株、强雌性和全雌性)黄瓜品种中克隆了长度为1188bp的ACC合酶基因(CS-ACS2)片段(GenBank登记号为:DQ115884～DQ115886和DQ115875～DQ115879).经测序分析,3个雌雄同株性别类型品种的序列完全相同.与之相比,5个强雌性和全雌性品种中存在8个单核苷酸多态性(SNPs)标记,SNPs标记为4个A←→G和4个T←→C之间的转换.在8个SNPs中,有1个SNP位于内含子区域,其余7个SNPs都位于外显子区域.在7个位于外显子区域的SNPs中,有3个为非编码区的SNPs,4个为cSNPs.而在4个cSNPs中,有3个导致了编码的氨基酸序列改变.研究结果表明,与雌雄同株性别类型相比,雌性系中均存在单核苷酸的变异,这提示ACC合酶基因CS-ACS2的单核苷酸变异可能与黄瓜雌性系的发生形成有关.另一方面,根据SNP多态性还发展了一个酶切扩增长度多态性(CAPS)标记C-MT700.利用CAPS标记C-MT700能将强雌性优良品种MT-705与其他黄瓜品种相区别,该标记在黄瓜育种生产上具有一定的应用价值.此外,研究获得的SNPs标记和CAPS标记丰富了黄瓜的分子标记种类.     

新疆绵羊种布鲁氏菌HtrA基因的克隆与序列分析

根据已发表的牛流产型布鲁氏菌(B.abrotus)HtrA(High temperature requirment A)基因的核酸序列设计引物,从新疆绵羊种布鲁氏菌(B.ovis)基因组中扩增到了大约1600bp的片段。将该片段纯化后克隆到PBS-T载体上,对所得到的重组质粒进行PCR鉴定、酶切分析后,对克隆的片段进行测序表明,新疆绵羊种布鲁氏菌HtrA基因与发表的牛流产型布鲁氏菌、马耳他布鲁氏菌(B.melitensis)、猪种布鲁氏菌(B.suis)的HtrA基因核苷酸序列的同源性分别为99.68%、99.81%、99.55%,推导的氨基酸序列也存在很高的同源性。     

Photosynthesis and Chlorophyll Fluorescence in Two Hybrids of Sorghum under Different Nitrogen and Water Regimes

In two hybrids of sorghum (Sorghum bicolor Moench.), C51 and C42, high nitrogen concentration (HN) increased net photosynthetic rate (PN), stomatal conductance (gs), and transpiration rate (E) of well watered (HW) plants. Water stressing (LW plants) resulted in low PN, gs, and E in both hybrids, but the values were still higher in HN plants as compared to low nitrogen-grown (LN) plants. Intercellular CO2 concentration (Ci) increased in droughted plants. This increase was much higher in LN plants as compared to HN plants. Instantaneous water use efficiency was lower in LN plants as a consequence of a greater effect of water stress on photosynthesis. Leaf water potential was reduced by water stress in all treatments. Analysis of chlorophyll a fluorescence at room temperature showed that photosystem 2 (PS2) was rather tolerant to the water stress imposed. Water stress caused a slight decrease in the efficiency of excitation capture by open PS2 reaction centres (Fv/Fm). The in vivo quantum yield of PS2 photochemistry (PS2) and the photochemical quenching coefficient (qP) were slightly reduced, while the nonphotochemical quenching coefficient (qN) was increased under the water stress. However, in hybrid C42 these characters were little or not affected by the water stress.     

Industrial enzymes--developments in production and application

The utilization of gene technology and of new production technologies have made industrial enzymes with improved properties or better cost performance available. This has in turn opened important new areas of enzyme applications. The benefits to the customers are considerable: cost savings in the application process, improved product quality, and in most cases also a significantly reduced impact on the environment.

Gene technology offers several benefits to the enzyme industry. This technology enables the use of safe, well-documented host organisms easy to cultivate, the microbial production of enzymes of animal and plant origin, the realization of enhanced efficiency and high product purity, and also the production of enzymes with improved stability and activity.

Developments in production technology include advanced control methods, the use of expert systems, and the application of large-scale crystallization.

As case stories the development of a lipase and of a cellulase is described. The effect on environment of enzyme application and production is discussed.     

The plantibody approach: expression of antibody genes in plants to modulate plant metabolism or to obtain pathogen resistance

Immunomodulation is a molecular technique that allows the interference with cellular metabolism or pathogen infectivity by the ectopic expression of genes encoding antibodies or antibody fragments. In recent years, several reports have proven the value of this tool in plant research for modulation of phytohormone activity and for blocking plant-pathogen infection. Efficient application of the plantibody approach requires different levels of investigation. First of all, methods have to be available to clone efficiently the genes coding for antibodies or antibody fragments that bind the target antigen. Secondly, conditions to obtain high accumulation of antigen-binding antibodies and antibody fragments in plants are being investigated and optimized. Thirdly, different strategies are being evaluated to interfere with the function of the target molecule, thus enabling immunomodulation of metabolism or pathogen infectivity. In the near future, optimized antibody gene isolation and expression, especially in reducing subcellular environments, such as the cytosol and nucleus, should turn immunomodulation into a powerful and attractive tool for gene inactivation, complementary to the classical antisense and co-suppression approaches.     

RNAi介导的OsBTF3基因沉默及其对水稻籽粒相关性状的影响

为了创制OsBTF3基因沉默的水稻植株、验证该基因在水稻籽粒相关性状中的功能、评价其在水稻遗传改良中潜在的应用价值,设计和合成OsBTF3基因序列的引物、扩增部分基因片段,构建RNAi基因沉默载体、通过农杆菌介导转化愈伤组织、植株再生、潮霉素抗性筛选和PCR验证、定量分析OsBTF3基因表达量,测定转基因水稻籽粒相关性状。结果表明,成功地获得了20个T1代OsBTF3-RNAi转基因株系,OsBTF3基因表达量得到显著的抑制和干扰,抑制效果平均达到85%;与野生型对照株相比,5个所测定RNAi转基因株系的穗长、穗粒数、千粒重和穗粒重等籽粒相关性状明显地减小或降低。因此,RNAi介导的基因沉默导致了OsBTF3基因表达水平抑制以及在籽粒性状中的功能缺失;OsBTF3可能是一个调控水稻籽粒相关性状重要的功能基因。