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:
Family | Construct | Type | Gene |
---|---|---|---|
4CL | a12 | IV | 4CL3 |
a13 | IV | 4CL5 | |
i15 | I | 4CL3 | |
4CL5 | |||
AldOMT | i30 | I | COMT2 |
C3H | i20 | I | C3H3 |
i69 | II | C3H3 | |
C4H | a9 | IV | C4H2 |
a10 | IV | C4H1 | |
i11 | I | C4H1 | |
C4H2 | |||
i69 | II | C4H1 | |
C4H2 | |||
CAD | i33 | I | CAD1 |
i34 | I | CAD2 | |
i35 | II | CAD1 | |
CAD2 | |||
o65 | I | CAD1 | |
o65 | V | CAD2 | |
o66 | I | CAD1 | |
o66 | V | CAD2 | |
o67 | V | CAD2 | |
CAld5H | a27 | IV | CAld5H1 |
a28 | IV | CAld5H2 | |
i29 | II | CAld5H1 | |
CAld5H2 | |||
CCR | i26 | I | CCR2 |
CCoAOMT | a22 | IV | CCoAOMT1 |
a23 | IV | CCoAOMT2 | |
i21 | IV | CCoAOMT3 | |
i24 | I | CCoAOMT1 | |
CCoAOMT2 | |||
i25 | II | CCoAOMT1 | |
CCoAOMT2 | |||
CCoAOMT3 | |||
HB | i50 | I | KNOX1 |
HCT | a17 | IV | HCT1 |
a18 | IV | HCT6 | |
i19 | I | HCT1 | |
HCT6 | |||
LIM | a40 | IV | LIM1 |
a41 | IV | LIM2 | |
i42 | I | LIM1 | |
LIM2 | |||
MYB | a43 | IV | MYB167 |
a44 | IV | MYB090 | |
a46 | IV | MYB156 | |
a47 | IV | MYB221 | |
a51 | IV | MYB002 | |
a52 | IV | MYB021 | |
a54 | IV | MYB003 | |
a55 | IV | MYB020 | |
i45 | II | MYB090 | |
MYB167 | |||
i48 | II | MYB156 | |
MYB221 | |||
i53 | II | MYB002 | |
MYB021 | |||
i56 | II | MYB003 | |
MYB020 | |||
PAL | a1 | IV | PAL1 |
a2 | IV | PAL3 | |
a3 | IV | PAL4 | |
a4 | IV | PAL5 | |
a5 | IV | PAL2 | |
i6 | II | PAL1 | |
PAL3 | |||
i7 | II | PAL2 | |
PAL4 | |||
PAL5 | |||
i8 | III | PAL1 | |
PAL2 | |||
PAL3 | |||
PAL4 | |||
PAL5 | |||
PO | i36 | I | PO1 |
i37 | I | PO2 | |
i38 | I | PO3 | |
i39 | I | PO8 |