Effects of Ppc Gene Construction of Monocotyledon on Seedling Growth of Transgenic Nicotiana tabacum

Guifang ZHANG,Zaisong DING,Ming ZHAO*

1.College of Life Sciences/Editorial Department of Journal of Beijing Normal University(Natural Science),Beijing Normal University,Beijing 100875,China;2.Institute of Crop Sciences,CAAS/Key Laboratory of Crop Physiology and Ecology,Ministry of Agriculture,Beijing 100081,China

Phosphoenolpyruvate carboxy lase (PEPC)is a irreversible reaction enzyme for catalytic reaction of phosphoenolpyruvic acid(PEP)with HCO3-to form oxaloacetic acid.It plays a key role in CO2fixation in C4plant photosynthesis.C4plant has extremely high net photosynthetic rate under drought,high temperature,highlight,and low concentration of CO2,which enlightens the scholars use key enzyme of C4pathway to optimize the photosynthesis of C3plant to enhance the net photosynthetic rate of C3plant.Most C4plants belong to monocotyledonous Gramineae or Cyperaceae[1].ButNicotiana tabacumin C3plant belongs to dicotyledonous Solanaceae.More than90% dry matters ofN.tabacumcome from the photosynthesis of leaves,andN.tabacumleaves are the most impor tant organ to form dry matters.Therefore,it is of great significance to cultivateN.tabacumwith high photosynthetic efficiency.At present,most researches are focused on the trans formation of C4PEPC genes ofZea mays[2-3],Sorghum[4]andS.officinarum[5]into monocotyledonous Poaceae C3plant(such asOryza sativa),in order to improve the photosynthetic efficiency of rice.There are also reports on the transformation of C4photosyntheticenzyme genesinZea maysandS.officinarum[6-9].However,no reports are found on the transformation ofphotosynthetic enzyme genes in wild C4plantEchinochloa crusgallitoNicotiana tabacum.The genetic distance of phylogenetic origin affects the transformation effects of photosynthetic enzyme genes from monocotyledonous C4to dicotyledon[6,10].Based on these,the cDNA ofPpcgenes in Echinochloa crusgalli and the complete DNA sequence ofPpcgenes inZea mayswere transferred into dicotyledonN.tabacumbased on Agrobacterium-mediated transformation. Their physiological performance was compared,so as to obtain transgenicN.tabacumstrain with high PEPC expression in C4plant,to improve the photosynthetic carbon assimilation ofN.tabacum,and to enhance the yield and quality ofN.tabacumleaves.

Materials and Methods

Tested materials

Flue-cured tobacco variety G140 was provided by Institute of Genetics and Developmental Biology,Chinese Academy of Science.It was stored in College of Life Sciences,Beijing Normal University.Restriction enzyme,ligase and polymerase were all purchased from Takara;antibiotics were purchased from Sigma Cooperation;and other reagents were all analytically pure.Expression vectorPUbi-Eppccontaining cDNA ofPpcgenes inE.crusgalliwas constructed[11]and stored in Institute of Crop Sciences,Chinese Academy of Agriculture Sciences.Expression vectorpCB-ZM Ppccontaining complete DNA sequence ofPpcgenes inZ.mayswas provided by Matsuoka.AgrobacteriumtumefactionsLBA4404,Escherichia coliDH5α andE.colicontaining power-assist plasmid pRK2013 were all cultivated and stored in Institute of Crop Sciences,Chinese Academy of Agriculture Sciences.

Transformation of A.tumefactions LBA4404

Plant expression vectors containing target genes were transformed intoA.tumefactionsLBA4404 by triparental crossing method,

N.tabacum transformation

Sterilization and pre-cultivation of leavesHealthy and unfolded young leaves ofN.tabacumwere sterilized by 70% ethanol and 0.5% sodium hypochlorite solution.After prepared into leaf plates,they were pre-cultivated on MS plate with 0.1 mg/L 6-BA 1mg/L and NAA 0.1 mg/L for 2 d.

Transformation and co-cultivationDisk edge was wetted by LBA4404 solution for infection for several minutes.Then,it was co-cultivated on MS＋1 mg/L 6-BA＋0.1 mg/L IAA covering by a sterile filter paper in dark under 25℃for 2-3 d.

Subculture under lightWhen there were visible small colonies on co-culture medium,leaf plates were transformed into MS subculture medium with 1 mg/L 6-BA,0.1 mg/L IAA and 500 mg/L Cef,and cultivated under light at 28℃for 3 d.

Screening and differentiationLeaf plates were transformed into MS screening medium with 1 mg/L 6-BA,0.1 mg/L IAA,500 mg/L Cef and 50 mg/L Hyg.They were cultivated for 7-10 d and transformed to MS regeneration medium with 1 mg/L 6-BA,500 mg/L Cef and 50 mg/L Hyg.After 15-20 d,callus formed in disk edge.On about one month,calluses were transformed to new culture medium.Formation of differentiated seedlings was observed.When several young leaves were differentiated,seedlings were transformed to MS rooting medium with 50 mg/L Hyg.Seedlings after rooting were put in water for one week and then transformed to pot for cultivation.

Identification of transgenic N.tabacum

Histochemical Staining of GUS(βglucuronidase gene)The undifferentiated leaf plates with wound and the differentiated young leaves were cut into small pieces of 0.5 cm2,and put in GUS straining fluid [0.1 mol/L phosphate buffer(pH 7.0),50 mmol/L K3Fe(CN)6,50 mmol/L K4Fe(CN)6,0.5 mol/L Na2EDTA,1% Triton and 20 mmol/L X-Gluc].After processed at 37℃for 1-2 h or incubated over night,they were decolorized by 70% ethanol until the histohaematin was completely removed.Straining situation was observed under microscope.

Detection of PCR and RT-PCRTotal DNA was extracted from the leaves of transgenicN.tabacum.Target gene Ppc was amplified by primers P1C(5’-ATCCGC AGA ACC CCT CCC ACT CCT CAA G-3’)and P2C(5’-GGCGTTTCTCCTCCGACC ACTCAG CAT A-3’)[11].Total RNA was extracted from the leaves of transgenicN.tabacum.Reverse transcription was carried out with Oligo-T17as the primer.Then,Ppc gene was amplified with reverse transcription products as the template and P1Cand P2Cas the primers.After amplification,the target gene fragments were recycled and the sequences were detected.

Cultivation of transgenic positive plants

Rooting and strong seedling culture were of transgenic positive plants were detected by histochemistry,PCR and RT-PCR tests.Then,they were transferred to flowerpots in greenhouse.

Detection ofleafphotosynthetic rate(Pn)

Photosynthetic rate(Pn)was detected by Licor-6400 photosystem.Light intensity and environmental temperature were designed to be 1 200 μmol/(m2·s)and 28 ℃,respectively.

Results

Transformation, cultivation and differentiation of transgenic N.tabacum

Transgenic leaf plates(Fig.1a)were cocultured in dark and subcultured under night for one week.During screening and differentiation, leaf plates gradually became thick and large;its color turned to light;and callus appeared in disk edge.After several weeks,predifferentiated yellowgreen granules were observed on callus.The green tissues were transferred to differentiation medium,and cultivated under light at 28℃for 3-4 weeks.Greenseedlingswereobserved (Fig.1b,1c).When there were 4-5 leaves,the seedlings were transformed to rooting medium(Fig.1d).After rooting,seedlings were put in water for one week(Fig.1e)and cultivated in flower pot(Fig.1f).

Identification of transgenic N.tabacum

After transformation,several leaf plates with wounds were observed by microscope based on GUS staining.And scattered blue spots(Fig.2)were found.During seedling exercising,seedling leaves were cut off for GUS staining and alcohol decolorization.Results showed that all the transgenic leaves of seedlings showed blue precipitation,which were positive plants.

Detection of PCR and RT-PCR

Fig.3a illustrated the PCR electrophoresis results of leaf DNA sample,showing that transgenicN.tabacumcould all amplify 0.8 kbPpcgene fragments (Lanes 1-9)fromE.crusgalli;and amplification signals of corresponding non-transgenic plants were very weak (lanes control 1 and 2).Fig.3b illustrated the PCR electrophoresis results, showing that transgenicN.tabacumcould all amplify 0.8 kbPpcgene fragments(Lanes 1-7)fromE.crusgalli;and there were almost no amplification signals of corresponding non-transgenic plants.Bands with relatively strong amplification signals were cut off and their sequences were detected. Results showed that gene sequence of 0.8 kb gene fragment perfectly matched with the target genePpc,which proved that the target gene was expressed at tran-scriptional level.

Table 1 Pn of N.tabacum transformed with Ppc-cDNA of E.crusgalli

Comparison of the differentiation and growth of N.tabacum transformed with Ppc-cDNA of E.crusgalli and Ppc-DNA of Z.mays

Differentiation and growth ofN.tabacumwere observed,which were transformed withPpc-cDNA ofE.crusgalliandPpc-DNA ofZ.mays.After transformed withPpc-cDNA ofE.crusgalliandPpc-DNA ofZ.mays,seedling leaves differentiated from calluses were all fresh green.However,most leaves ofN.tabacumtransformed withPpc-DNA ofZ.maysgradually turned from dark green to light yellow and completely white during rooting.And the seedlings stopped growing due to whitening(Fig.4a,4c).Some leaves twisted into malformation,and finally died.At the same time,most leaves ofN.tabacumtransformed withPpc-cDNA ofE.crusgalliwere healthy green during the whole growth period (Fig.4b);only a small amount of leaves showed whitening.

Net photosynthetic rate of N.tabacum transformed with Ppc-cDNA of E.crusgalli

Pn ofN.tabacumtransformed withPpc-DNA ofZ.mayswas not obtained,since most seedlings died in potted period.Pn of sevenN.tabacumseedlings transformed withPpc-cDNA ofE.crusgalliwas detected.Table 1 reported thatPn values of all the seven seedlings were higher than that of control,exceptthose ofseedlings G140-Ecp4 and G140-Ecp7.Among them,Pn of G140-Ecp5 was the maximum[26.50 μmol/(m2·s)],which was 16.84% higher than that of control[22.68 μmol/(m2·s)].Pn values of G140-Ecp3 and G140-Ecp6 were also higher than that of control by more than 10%.These results preliminarily showed that PEPC ofE.crusgallihad certain regulatory effects on the photosynthesis ofN.tabacum.

Discussions

PpcDNA ofZ.maysandPpccDNA ofE.crusgalliin monocotyledonous C4plants were transformed toN.tabacumin dicotyledonous C3plant,which had relatively far genetic distance.Results showed thatPpccDNA had relatively strong growth and differentiation abilities after transformed toN.tabacum.Pnof plant leaves increased up to16.84% more compared with that in non-transgenic control.However,N.tabacumafter transformation of complete DNA sequence of Ppc genes had relatively poor ability of growth. The differentiated green seedlings had the phenomenon of yellowing,because the chloroplasts in leaf histocyte were seriously damaged during the growth period.Leaf photosynthesis ability was gradually reduced,the vegetative growth process could not be completed,and finally seedlings died.Since the transformedPpcDNA ofZ.maysincluded the promoter,introns and terminator ofPpcgene,it was concluded that promoter ofZ.maysPpc might not accurately identified the initiation site or termination site;and the incorrect m-RNA splicing led to the abnormal gene expression.However,transgenicO.sativawith high-level expression of PEPC was obtained after transformation of complete DNA sequence ofPpcgenes ofZ.mays[13],which proved that the complete gene introns played an important role in gene expression.Gene transformation between fargenetic distance might reduce the normal expression rate of complete gene DNA in heterologous plant cells.But there was also possibility of correct transformation.And transformation population should be increased in order to select the transgenic plants with normal expression.

This research preliminarily proved that during transformation of cDNA sequence between relatively far genetic distance,exogenous promoter with relatively wide subject range could avoid the misidentification and wrong stitching during transcription.In previous transgenic research,constitutive expression promoter CaMV 35S was selected for the construction of many plant expression vectors[9,14-15],which had effective expression in dicotyledon.In this research,the transformedPpccDNA was started by promoterpUbiof constitutive expression gene Ubiquitin1.The effects of promoter pUbiwas 10 times of CaMV 35S.And transgenicN.tabacumwas obtained with normal expression ofPpccDNA ofE.crusgalli,the photosynthetic efficiency of transgenic plants enhanced in different degrees.Furthermore,N.tabacumlines with high PEPC expression of C4plant would be screened,in order to improve the photosynthetic carbon assimilation ofN.tabacum,and to enhance the productivity ofN.tabacumleaves.

TransformingPpccDNAofE.crusgalliintoN.tabacumwas of certain significance to the changes of photosynthetic physiological characteristics ofN.tabacumunder natural conditions.PEPC isozyme also participated in other regulative processes of metabolism[16-17].Expression restriction of PEPC changed the metabolic pattern,and turned the flow of carbon atoms from saccharides synthesis to fatty acid synthesis[18-19].As a nonphotosynthetic isoenzyme,PEPC participated in many other functions during the plant growth.For instance,increase of PEPC isoenzyme activity enhanced the ammonia assimilation ability of tomato[20].Further research was needed on the growth status of transgenicN.tabacumunder various stress conditions,such as drought,high temperature,and high luminous intensity,so as to determine the concrete functions PEPC isoenzyme inN.tabacumafter expression.

Conclusions

PpccDNA ofE.crusgalliin monocotyledonous C4plant was regulated by the promoter of constitutive expression geneUbiquitin1,and transformed into dicotyledonN.tabacumwith relatively far genetic distance.Results showed that the transgenicN.tabacumhad relatively strong differentiation ability;itsPn was higher than that in control.However,N.tabacumafter transformation of complete DNA sequence ofPpcgenes inZ.mayshad relatively poor ability of growth.The differentiated green seedlings had the phenomenon of yellowing;and photosynthesis ability of leaves was poor.This might be caused by the misidentification and wrong splicing in transcription,so that the gene was abnormallyexpressed.Thisindicated that the expression rate of monocotyledonous complete DNA might be reduced in the monocotyledonous cells with relatively far genetic distances.

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