Cloning and characterization of a sigma 70 homologue gene, sigA from Corynebacterium ammoniagenes

A sigma 70-like gene, sigA, has been identified from Corynebacterium ammoniagenes. The sigA gene encodes a polypeptide of 467 amino acids with a calculated molecular mass of 52036 Da. The deduced amino acid sequence preserves the common motifs of the primary sigma factors and shows very high similarity to those of SigA (sigmaA) homologues from high G+C Gram-positive bacteria, which suggest that the sigA gene encodes the primary sigma factor. The sigA gene is transcribed as a monocistronic mRNA of 2 kb and its mRNA occurs during the exponential growth phase and decays rapidly on entry into the stationary phase. The open reading frame encoding polyphosphate glucokinase-like protein is closely linked to the sigA gene.     

烟草磷转运蛋白基因NtPHT2;1的克隆和表达分析

植物对磷酸盐的吸收与利用主要依靠磷转运蛋白,其中PHT2家族编码的低亲和磷转运蛋白主要负责植物在正常供磷条件下磷酸盐的吸收、转运与再利用。为了探究低亲和磷转运蛋白基因NtPHT2;1在烟草转运磷酸盐中的作用和表达模式,本研究以普通烟草K326的cDNA为模板,克隆得到NtPHT2;1,对该基因进行生物信息学分析和蛋白质的亚细胞定位,并通过荧光定量PCR技术对该基因在低磷等非生物胁迫下的基因表达模式进行分析。结果表明：（1）NtPHT2;1基因的全长为1 764 bp,编码587个氨基酸。（2）亚细胞定位结果表明,NtPHT2;1蛋白定位于叶绿体上。（3）同源性比对发现,NtPHT2;1蛋白与辣椒CaPHT2;1蛋白的同源性最高达到91.00%。（4）启动子分析表明,NtPHT2;1启动子含有参与调控植物衰老、逆境胁迫相关的顺式作用元件。（5）组织表达模式分析表明,NtPHT2;1在叶片中的表达量最高,新叶中的表达量比老叶中的高;在低磷诱导条件下,该基因的表达量与正常条件相比差异不显著。（6）不同非生物胁迫下的表达模式表明,在盐胁迫和干旱胁迫下,该基因的表达量显著降低。研究认为,NtPHT2;1基因主要是负责烟株正常生长发育条件下磷酸盐的转运与利用。     

High expression of foot-and-mouth disease virus structural protein VP1 in tobacco chloroplasts

A tobacco chloroplast expression vector, pTRVP1, containing the foot-and-mouth disease virus (FMDV) VP1 gene and the selective marker aadA gene, was constructed and transferred to tobacco by biolistic method. Three resistant lines were obtained through spectinomycin selection, and each transgenic line was subjected to a second round of spectinomycin selection. PCR and PCR southern blot analysis revealed that the VP1 gene had integrated into the chloroplast genome. Western blot and quantification ELISA assays indicated that the VP1 gene was expressed in tobacco chloroplasts and accounted for 2–3% of total soluble protein. This suggested that plant chloroplasts were an efficient expression system for the potential production of recombinant antigens in plants.     

Overexpression of the Arabidopsis thaliana MinD1 gene alters chloroplast size and number in transgenic tobacco plants

The Arabidopsis thaliana (L.) Heynh. minD gene (AtMinD1) was isolated and constitutively expressed in tobacco (Nicotiana tabacum L.) plants using the CaMV 35S promoter. Confocal and electron-microscopic analysis of the AtMinD1 transgenic tobacco lines revealed that the chloroplasts were abnormally large and fewer in number compared with wild-type tobacco plants. The abnormal chloroplasts were less prevalent in guard cells than in mesophyll cells. Chloroplast and nuclear gene expression was not significantly different in AtMinD1-overexpressing plants relative to wild-type tobacco plants. Chloroplast DNA copy number was not affected, based on the relative level of the rbcL gene in transgenic plants. Transgenic tobacco plants constitutively overexpressing AtMinD1 were completely normal phenotypically with respect to growth and development, and also displayed normal photosynthetic electron transport rates. These results show that the Arabidopsis MinD1 gene also functions in a heterologous system and confirm the role of the MinD protein in regulation of chloroplast division.