11.6 Overexpression

Minoru Kubo, Tomomichi Fujita, and Mitsuyasu Hasebe

Introduction

Gain-of-function experiments using over expression and ectopic expression are indispensable to analyze gene function. While the cauliflower mosaic virus (CaMV) 35S promoter is a strong promoter in flowering plants, in Physcomitrella patens, CaMV35S promoter is strong enough to induce antibiotic resistance gene for selection but is too weak to see the effects of over expression and ectopic expression for genes involved in development. For stronger induction, the rice actin promoter (Zhang et al. 1991), the 7113 promoter modified from CaMV35S (Mitsuyara et al. 1996), and the PpEF1-α promoter (Kubo et al unpublished) are useful. The actin promoter works well in protonemata but is much weaker in gametophores. On the other hand, the 7113 and PpEF1-αpromoters work in almost tissue including gametophores, although it seems that not all of the cells in gametophores show overexpression of genes. In this way, you have to select the promoter for overexpression in accordance with your purpose.

To introduce the construct of overexpression to P. patens genome, some targeting sites, the PpMADS2, Pphb7, BS213 have been reported (Pphb7, Sakakibara et al. 2003; BS213, Schaefer et al. 1997). Additionally, we established new targeting sites, PIG1 and PTA1, by using informatics and genome resource of P. patens (Kubo et al. unpublished). Here we display the list and schematic representations of available vectors for overexpression using various promoters and targeting sites.

Materials and Methods

Construction of a targeting vector

Conventional methods

The PCR fragment with blunt ends, which is amplified with proof-reading DNA polymerase such as KOD plus DNA polymerase (TOYOBO), can be directly cloned in the EcoRV and SmaI sites of these vectors.

Gateway compatible

Your gene and DNA fragment, which is amplified with proof-reading DNA polymerase such as KOD plus DNA polymerase (TOYOBO), are subcloned into pENTR/D/TOPO vector. Your gene or DNA fragments in these pENTR/D/TOPO vectors (entry clones) are integrated into destination vectors by the LR reaction. For details of the GATEWAY system (Invitrogen), you can refer to websites as follows: http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html

Transformation and selection of candidate lines

See Chapter 11.3

Overexpression vector list

vector	promoter	targeting site	selection	Vector map
pPpMADS2 7113	7113	PpMADS2	G418	(1)
pPpMADS2 7113 rev	7113	PpMADS2	G418	(1)
pPphb7 7113	7113	Pphb7	G418	(1)
pPphb7 7113 rev	7113	Pphb7	G418	(1)
pPpMADS2 Actin	ACT	PpMADS2	G418	(2)
pPpMADS2 Actin rev	ACT	PpMADS2	G418	(2)
pTFH15.3	ACT	Pphb7	G418	(3)
pBS213-7113-mRFP-G	7113	BS213	Zeo	(4)
pBS213-7113-G-mRFP	7113	BS213	Zeo	(4)
pBS213-7113-citrine-G	7113	BS213	Zeo	(4)
pBS213-7113-G-citrine	7113	BS213	Zeo	(4)
pBS213-7113-HA-G	7113	BS213	Zeo	(4)
pBS213-7113-G-HA	7113	BS213	Zeo	(4)
pPOG1	PpEF1-α	PIG1	Hyg	(5)
pPOYG1	PpEF1-α	PIG1	Hyg	(5)
pPOCG1	PpEF1-α	PIG1	Hyg	(5)
pT1OG	PpEF1-α	PTA1	Zeo	(6)
pT1OGY	PpEF1-α	PTA1	Zeo	(6)
pT1OGC	PpEF1-α	PTA1	Zeo	(6)

ACT: rice actin promoter, Zeo: zeocin, Hyg: hygromycin

Remarks

1. By Pphb7 disruption, its transformants show abnormal development of rhizoids. Development of other tissue such as protonemata is not distinguishable from that in wild type (Sakakibara et al. 2003). We have not found any phenotypic change in the transformants introducing the harmless transgene to PpMADS2, BS213, PIG1 and PTA1.

2. Gene silencing is sometimes observed in some lines with overexpression constructs. The gene silencing is hereditable, although gene expression is occasionally recovered in the mucilage hairs of gametophores and the foot of sporophytes. You should select the transformants with a single insertion, because multi copy insertion lines have a greater tendency to induce gene silencing than single ones. To confirm whether the gene is effectively overexpressed, expression analyses of the transcript and protein are recommended. It might be useful for monitoring gene expression to construct GFP-fusion protein which maintains native function in P. patens.

3. The 7113 promoter was kindly provided by Dr. I. Mitushara and Dr. Ohashi (Mitsuhara et al. (1996), Plant Cell Physiol. 37: 49-59). The actin promoter from rice was kindly provided by Dr. Wu (Zhang et al. (1991), Plant Cell, 3:1155-1165). The rice actin promoter was >10 times stronger than 7113 promoter in P. patens protoplasts (Fujita et al. unpublished). The PpEF1-αpromoter was isolated from P. patens genome (Kubo et al. unpublished).

Vector map

(1) PpMADS2 target, 7113 promoter

Note: Please substitute “p7113” for “pE7133” in the figures.

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

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

(2) PpMADS2 target, rice actin promoter

NotI is usually used to linearize the plasmid for transformation.

(3) Pphb7 target, rice actin promoter

NotI is usually used to linearize the plasmid for transformation.

(4) BS213 target, 7113 promoter, gateway compatible, with tag sequence (His, mRFP, citrine)

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

(5) PIG1 target, PpEF1-α promoter, gateway compatible, with tag sequence (citrine as YFP), cerulean as CFP)

- PmeI and Sse8387I are usually used to linearize the plasmid for transformation.

(6) PTA1 target, PpEF1-α promoter, gateway compatible, with tag sequence (citrine as YFP), cerulean as CFP)

- PmeI and Sse8387I are usually used to linearize the plasmid for transformation.