Revealing frequent alternative polyadenylation and widespread low-level transcription read-through of novel plant transcription terminators.

Plant genetic engineering can create transgenic crops with improved characteristics by introducing trait genes through transformation. Appropriate regulatory elements such as promoters and terminators have to be present in certain configurations for the transgenes to be properly expressed. Five terminators native to soybean genes-encoding a MYB family transcription factor (MYB2), a Kunitz trypsin inhibitor (KTI1), a plasma membrane intrinsic protein (PIP1), a translation elongation factor (EF1A2) and a metallothionein protein (MTH1) were cloned and tested for their ability to enable transgene expression, mRNA polyadenylation and transcription termination. The terminators are as good as a control terminator of the potato proteinase inhibitor II gene (PINII) in conferring proper transgene expression, leading to mRNAs with various polyadenylation sites and terminating mRNA transcripts. RNA transcription read-through was detected in all transgenic plants and was quantified by qRT-PCR to be <1% at positions approximately 1 kb downstream of the 5' ends of different terminators. The detection of read-through RNA transcripts of the corresponding endogenous genes up to approximately 1 kb beyond the polyadenylation sites suggests that limited RNA transcription read-through is a normal phenomenon of gene expression. The study also provided more choices of terminators for plant genetic engineering when constructing DNA constructs containing multiple gene expression cassettes.

A Cre/loxP-mediated self-activating gene excision system to produce marker gene free transgenic soybean plants.

Marker-gene-free transgenic soybean plants were produced by isolating a developmentally regulated embryo-specific gene promoter, app1, from Arabidopsis and developing a self-activating gene excision system using the P1 bacteriophage Cre/loxP recombination system. To accomplish this, the Cre recombinase gene was placed under control of the app1 promoter and, together with a selectable marker gene (hygromycin phosphotransferase), were cloned between two loxP recombination sites. This entire sequence was then placed between a constitutive promoter and a coding region for either beta-glucuronidase (Gus) or glyphosate acetyltransferase (Gat). Gene excision would remove the entire sequence between the two loxP sites and bring the coding region to the constitutive promoter for expression. Using this system marker gene excision occurred in over 30% of the stable transgenic events as indicated by the activation of the gus reporter gene or the gat gene in separate experiments. Transgenic plants with 1 or 2 copies of a functional excision-activated gat transgene and without any marker gene were obtained in T0 or T1 generation. This demonstrates the feasibility of using developmentally controlled promoters to mediate marker excision in soybean.