Recipient Organization
MICHIGAN TECHNOLOGICAL UNIV
1400 Townsend Drive
HOUGHTON,MI 49931
Performing Department
College of Forest Resources and Environmental Science
Non Technical Summary
One of the most important traits of woody tree species is the branch angle, which can influence photosynthesis efficiency, tree canopy sizes, tree trunch height and wood quality. Despite its importance, the molecular mechanism underlying the branch angle regulation remains to be elusive and hard to comprehend. At the time being, the completion of poplar genome sequencing and the availability of gene transformation methods as well as high-throughput gene expression profiling methods enable us to initiate a project to investigate this. Here, we propose asystemic approach to scrutinize the underlying molecular mechanism. We will first get started from a set of regulatory genes that are known to regulate the shoot initiation and angle in Arabidopsis and fruit trees. Using an in-vitro system called DAP-seq, we will first identify the target genes of the regulatory gene set and then evaluate their possible functions based on their target genes' types and functions. We will select some regulatory genes from the set and then develop transgenic lines to verify their functions in regulating shoot angle.
Animal Health Component
30%
Research Effort Categories
Basic
50%
Applied
30%
Developmental
20%
Goals / Objectives
Objectives:1. Cloning the above mentioned regulatory genes that include TAC1, LAZY1, ESR1, ESR2 and WIND1 from Populus trichocarpa.2. Identification of target genes of the above mentioned regulatory genes including TAC1, LAZY1, ESR1, ESR2 and WIND1. We will predict the functions of these regulatory genes based on their target genes.3. Generation of transgenic lines of the above regulatory genes to test their effects on shoot number and angle.
Project Methods
Gene cloning: Cloning of TAC1, LAZY1, ESR1, ESR2 and WIND1 from poplar trees. Dr. Yinan Yuan will be responsibe for cloning these genes into gateway plasmidvectors. We have cloned all genes except TAC1. We will run 5'RACE assay to acquire the upstream sequences of TAC1 genes. After that, we will re-design the primers to clone the TAC1 genes using PCR. All cloned genes will be verified by DNA sequencing before they are used for further studying.Identification of target genes of TAC1, LAZY1, ESR1, ESR2 and WIND1 using DAP-seq. We will identify the target genes of TAC1, LAZY1, ESR1, ESR2 and WIND1 using DAP-seq technology [6]. First, DNA libraries will be constructed using genomic DNA from shoots of Populus trichocarpa. Secondly, the individual open reading frames (ORFs) of each of above TFs will be transferred to the Gateway-compatible pIX-HALO expression vector. The pIX-HALO-TF construct is then expressed with a plant (wheat germ)-based in vitro transcription/translation coupled system to yield HaloTag-fused protein which can bind to magnetic HaloTag ligand and used for fishing the specific DNA fragment in library. The proteins are purified and then combined with the adaptor-ligated genomic DNA library. The fused protein can bind to genomic fragments containing the promoter sequences of target genes of this TFs. Bound genomic DNA is eluted from the TF and are sequenced using next-generation sequencing. Sequence reads will be mapped to Populus trichocarpa genome to identifying genome-wide binding locations for each TF assayed, from which target genes will be identified. This method may not be appliable to TAC1 and LAZY1 because we still do not know if they have DNA-binding domain, but other three genes are transcription factors.Identification of target genes of TAC1, LAZY1, ESR1, ESR2 and WIND1 through constructing a multilayered hierarchical network. We will develop a transient expression system to perturb the expression levels of TAC1, LAZY1, ESR1, ESR2 and WIND1 genes transiently, and then collect a time series of shoot samples for RNA-seq experiment. The resulting data will be used for building a multilayered hierarchical network, from which we can infer the target genes of these regulators. This is an alternative approach to infer the functions of these regulatory genes if DAP-seq fails to work.Generation of transgenic lines: To generate overexpression transgenic lines of these regulatory genes, we will amplify the full-length of these regulatory genes, and clone it into pCAMBIA1300 vectors, where they will be driven by 35S promoter. To generate suppression lines via RNAi technology, we will use a PK7GWIWG2(II) (http://www.vib.be/VIB/EN/) expression binary vector that was modified from a small binary vector called pPZP222 for generating efficient RNAi constructs. Gene-specific fragments of 200~300 bp can be easily cloned into PK7GWIWG2(II) vector at two different loci and in opposite orientation to facilitate the formation of double-strand RNAs that are extraordinarily efficient in triggering gene silencing. The constructs will be transferred into Agrobacterium tumefaciens AGL1 strain using a freeze-thaw protocol [7]. To generate transgenic lines, we will use A. tumefaciens containing different constructs to infect the leaves of poplar clone INRA 717-IB4 (P. tremula x P. alba) with our standard transformation procedure used routinely. We will generate at least 5 independent transgenic lines per gene construct for selecting up/down-regulated lines with various efficiencies.Characterization of transgenic plants: All transgenic plants will be propagated in vitro and transferred to the greenhouse. Their growth will be monitored and compared to wild-type plants. Transgenics will be subjected to molecular analyses to characterize the integration of transgenes and their expression levels using RT-PCR in order to select the desirable lines for further characterization. Branch angle will be measured and imaged. The transgenic lines with significantly altered shoot number and angle will be subjected to more molecular characterization towards producing a publication.