Because of the concern about escape of antibiotic- or herbicide-resistant transgenes from transgenic crops, selectable marker genes from plant origin would be an alternative choice for plant transformation. In this study, a feedback-insensitive anthranilate synthase gene ( ASA2 ) cloned from a tobacco cell line was tested for Agrobacterium-mediated transformation of axis tissue of soybean mature embryo, with a tryptophan analogue 5-methyltryptophan (5-MT) as the selective agent. Southern blot analysis of the To transgenic lines confirmed the integration of the ASA2 gene into the soybean genome. Northern blot analysis showed the ASA2 gene was also expressed in the leave tissue, and the free tryptophan content in the leaf tissue of transgenic soybean was about 59% to 123% more than that in the wild type. PCR analysis of the T1 progeny showed that the transgene was inherited in a Mendelian fashion. All these results indicate that this feedback-insensitive ASA2 gene can be used as a selectable marker gene for plant transformation. This work also demonstrated that the ASA2 gene coding for the a-subunits from one plant (tobacco) can interact with the n-subunits of a heterologous plant (soybean) to form an active anthranilate synthase enzyme. The use of this feedback-insensitive gene as a novel selectable marker for plant transformation is also discussed.
Anthranilate Synthase ; genetics ; Feedback, Physiological ; Plants, Genetically Modified ; genetics ; Soybeans ; genetics ; Transformation, Genetic ; Tryptophan ; analogs & derivatives ; metabolism

OBJECTIVETo investigate the effects of nitrogen forms on the camptothecin (CPT) content, tryptophan synthase (TSB) and tryptophan decarboxylase (TDC) activities in Camptotheca acuminata seedlings.

METHODThe seedlings of C. acuminata with 6 pairs of leaves were subjected to 5 different NH4(+) -N/NO3(-) -N ratio (0 : 100, 75 : 25, 50 : 50, 25 : 75, 100 : 0) treatments by sand culture in a greenhouse. The CPT content, TSB activity in the young leaves and TDC in the stem barks of the seedlings were determined by HPLC on the 15th, 30th, 45th, 60th and 75th day, respectively.

RESULTThe obvious relationship between CPT content and nitrogen forms was observed. When NH4(+) - N /NO3(-) -N ratio was 25 : 75, CPT accumulation in young leaves displayed the best advantages (the highest value is 5.69 per thousand) and increased in the early 30 days of treatment and then declined. There was no obvious relationship between TSB activity in the young leaves and nitrogen forms. TDC activity in the stem bark was the highest when NH4(+) -N /NO3(-) -N ratio was 25 : 75, and the change of TDC activity paralleled to CPT content in the young leaves.

CONCLUSIONA short-term treatment that NH4(+) -N /NO3(-) -N ratio was 25:75 may gain high CPT content in the young leaves through enhancing the TDC activity in the stem bark of C. acuminata seedlings.

Aromatic-L-Amino-Acid Decarboxylases ; metabolism ; Camptotheca ; drug effects ; enzymology ; metabolism ; Camptothecin ; metabolism ; Drugs, Chinese Herbal ; metabolism ; Nitrogen ; chemistry ; pharmacology ; Plant Leaves ; drug effects ; enzymology ; metabolism ; Seedlings ; drug effects ; enzymology ; metabolism ; Tryptophan Synthase ; metabolism

To improve tryptophan production in Escherichia coli, key genes in the tryptophan biosynthesis pathway -aroG, trpED, trpR and tnaA were manipulated. TrpR gene was knocked out to eliminate the repression on the key genes controlling tryptophan biosynthesis and transportation on bacteria chromosome, and the tryptophan degradation was blocked by tnaA gene knockout. Then the bottleneck in tryptophan biosynthesis pathway was removed by co-expressing aroGfbr gene and trpEDfbr gene. Compared with the MG1655, the tryptophan production of trpR knockout and double-genes knockout strains was improved 10-folds and about 20-folds, respectively. After the trpEDfbr was expressed, the tryptophan production increased to 168 mg/L, and when the aroGfbr and trpEDfbr were co-expressed, the tryptophan production increased to 820 mg/L. This work laid the foundation for further construction of higher-efficient engineered strain for tryptophan production.
3-Deoxy-7-Phosphoheptulonate Synthase ; metabolism ; Amino Acid Transport Systems ; genetics ; Bacterial Proteins ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; Gene Knockout Techniques ; Genetic Engineering ; Repressor Proteins ; genetics ; Tryptophan ; biosynthesis