Transgenics

The transformation task of this project has been completed. Four types of constructs were used for P. trichocarpa transformation.
Many of our target genes are phylogenetically paired and may have redundant functions. Functional redundancy were evaluated as an integral part of the perturbation using RNAi and amiRNA knockdown transgenics. Gene knockdowns were carried out in a xylem-specific manner at three levels: (1) the individual gene level(2) the phylogenetic gene-pair level, and (3) the gene family level.
We designed three types of RNAi constructs (Types I to III in Figure below) and one type of amiRNA construct (Type IV in Figure below) for these knock-downs.amiRNA constructs were used to suppress gene separately. A P. trichocarpa miRNA gene, ptr-MIR408 was used as the transgene backbone to deliver the 21-nt mature amiRNA silencing sequence complementary only to a specific target gene region for cleavage (knockdown). RNAi constructs with either one (Type I), two (Type II) or three (Type III) cDNA silencing fragments suppressed the single genes, paired genes or whole gene families.

All knock-downs were controlled by a xylem-specific promoter (Ptr4CLXP) of 4CL gene we cloned from P. trichocarpa. 

Click on construct ID to see the target sequence(s) of the constructs.


Construct types I,II,III,IV:

FamilyConstructTypeGene
4CLa12IV4CL3
a13IV4CL5
i15I4CL3
4CL5
AldOMTi30ICOMT2
C3Hi20IC3H3
i69IIC3H3
C4Ha9IVC4H2
a10IVC4H1
i11IC4H1
C4H2
i69IIC4H1
C4H2
CADi33ICAD1
i34ICAD2
i35IICAD1
CAD2
o65ICAD1
o65VCAD2
o66ICAD1
o66VCAD2
o67VCAD2
CAld5Ha27IVCAld5H1
a28IVCAld5H2
i29IICAld5H1
CAld5H2
CCRi26ICCR2
CCoAOMTa22IVCCoAOMT1
a23IVCCoAOMT2
i21IVCCoAOMT3
i24ICCoAOMT1
CCoAOMT2
i25IICCoAOMT1
CCoAOMT2
CCoAOMT3
HBi50IKNOX1
HCTa17IVHCT1
a18IVHCT6
i19IHCT1
HCT6
LIMa40IVLIM1
a41IVLIM2
i42ILIM1
LIM2
MYBa43IVMYB167
a44IVMYB090
a46IVMYB156
a47IVMYB221
a51IVMYB002
a52IVMYB021
a54IVMYB003
a55IVMYB020
i45IIMYB090
MYB167
i48IIMYB156
MYB221
i53IIMYB002
MYB021
i56IIMYB003
MYB020
PALa1IVPAL1
a2IVPAL3
a3IVPAL4
a4IVPAL5
a5IVPAL2
i6IIPAL1
PAL3
i7IIPAL2
PAL4
PAL5
i8IIIPAL1
PAL2
PAL3
PAL4
PAL5
POi36IPO1
i37IPO2
i38IPO3
i39IPO8