12.1  Constitutive overexpression of cDNA or cDNA with tags

Tomomichi Fujita


We found that 7133 promoter we has been used has a sequence of 7113 promoter. The original plasmid we obtained from Drs. Mitsuhara and Ohashi has the 7113 plasmid with a label of 7133 on the tube. Therefore all plasmids we used in my laboratory as 7133 should be 7113.


CaMV 35S promoter is a weak promoter in P. patens, although it gives enough resistance for drug selection with resistant genes. The rice actin promoter and the CaMV35S derived 7113 promoter are stronger promoters than CaMV 35S. As platform sites for targeting an overexpression construct, PpMADS2, Pphb7, and BS213 loci are used (Pphb7, Sakakibara et al. 2003; BS213, Schaefer et al. 1997).



(1) When Pphb7 is disrupted, development of rhizoid becomes abnormal. Development of protonemata is not distinguished from that in wild type (Sakakibara et al. 2003). We do not find any phenotypic effects by disruptions of PpMADS2 and BS213.


(2) Gene silencing is sometimes observed ccurs in some lines with overexpression constructs. The gene silencing is transmitted from generation to generation, although gene expression is recovered in the mucilage hairs of a gametophore and the foot of a sporophyte. If you select a line with a single insertion, gene silencing is rare. On the other hand, gene silencing usually happens for multi copy insertion lines. Northern or western analyses are recommended to confirm whether the targeted gene is effectively overexpressed. It is a good idea to fuse the targeted gene with GFP, although it is necessary to confirm the fusion protein is functional.


(3) Actin promoter works in protonemata well but much weaker in gametophores. The 7113 promoter is recommended to overdrive in gametophores.


(4) 7113 promoter was kindly provided from Dr. I. Mitsuhara and Dr. Ohashi (Mitsuhara et al. (1996), Plant Cell Physiol. 37: 49-59).  Actin promoter from rice was kindly provided from Dr. Wu (Zhang et al. (1991), Plant Cell, 3:1155-1165). Rice actin promoter worked >10 times stronger than 7113 promoter in P. patens protoplasts (Fujita et al. unpublished). 


Available plasmids (I)



- E. coli harboring a plasmid with the 7113 promoter should be cultured at 30ºC rather than 37ºC.  Truncation of 7113 promoter sequence is sometimes found at 37ºC.


- NotI is usually used to linearize the plasmid for transformation.


- To screen stable transformants, we perform green PCR (See a section of Green PCR for PCR strategy).


Available plasmids (II)


(1)  PpMADS2 target, rice Actin promoter

NotI site is usually used to linearize the plasmid for transformation.


(2) Pphb7 target, rice actin promoter


NotI site is usually used to linearize the plasmid for transformation.


(3) BS213 target, 7113 promoter, gateway construction, with tag sequence (His, mRFP, citrine)





*As LR reaction with pENTR Directional TOPO brings NotI site into the resultant product, NotI cannot be used for excision of a target fragment.