Transgenic Paulownia

Received 21 Feb. 2005 Accepted 24 Mar. 2005Supported by the National Natural Science Foundation of China (39770626) and the President Foundation of the Chinese Academy ofSciences.*Author for correspondence. Tel: +86 (0)27 8719 7513; E-mail: <[email protected]>.

Journal of Integrative Plant BiologyFormerly Acta Botanica Sinica 2005, 47 (12): 1500−1506

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Transgenic Paulownia Expressing shiva-1 Gene Has Increased Resistance toPaulownia Witches’ Broom Disease

Tao DU, Yao WANG, Qin-Xue HU, Jie CHEN, Sheng LIU, Wen-Jin HUANG and Mu-Lan LIN*

(Wuhan Institute of Virology, the Chinese Academy of Sciences, Wuhan 430071, China)

Abstract: Stem segments from diseased Paulownia tomentosa×P. fortunei and leaves from healthycontrol were transformed with the expression vector p438PRSI via Agrobacterium tumefaciens. The p438PRSIvector contained shiva-1 gene, which encodes an antibacterial peptide under the control of a CaMV35Spromoter. The regenerated plants from transformed explants were planted in a greenhouse and nursery. PCRand Southern blotting analysis showed that the shiva-1 gene was successfully integrated into the Pau-lownia genome. Transcription of the integrated shiva-1 gene was confirmed by RT-PCR. Bioassay in thegreen house and phytoplasma DNA-dot blotting demonstrated that resistance to Paulownia witch’s broomdisease (PWB) increased significantly in shiva-1-transgenic Paulownia. Further investigations indicatedthat higher Shiva-1 expression correlated with fewer phytoplasma and less symptoms in diseased transgenicPaulownia. Together, our findings strongly suggest that breeding shiva-1-Paulownia is an effective strat-egy to control PWB disease.Key words: Agrobacterium tumefaciens; Paulownia; Paulownia witch’s broom; phytoplasma; shiva-1.

Paulownia witch’s broom (PWB), caused by PWBphytoplasma, is the most important disease inPaulownia. PWB seriously slows down tree growthand can even cause seedling death. It is difficult totreat infected Paulownia with bactericides becausethere is no good method to deliver the chemicals to thephloem, where the pathogen parasitizes. Conventionalphytoplasma-free seedlings are one option, but rein-fection in the field usually makes this strategyineffective. Success in developing a phytopathogen-resistant crop through genetic engineering has led usto investigate the possibility of breeding phytoplasma-resistant Paulownia.

Cecropins are a series of peptides segregated fromHyalophora cecropia that have broad antibacterialactivities. Cecropins-transgenic plants have been gen-erated and show pathogen resistance in several cropsand vegetables, including pepper, rice (Huang et al.

1997; Sharma et al. 2000), Chinese cabbage (Wang etal. 2002), potato (Yi et al. 2004), tobacco (Wang et al.2001), and grape. Previous studies have demonstratedthat mycoplasma, which belongs to mollcutes, is verysensitive to Shiva-1 at low concentrations. In the presentstudy, we report on our investigation in protectingPaulownia against PWB phytoplasma by geneticengineering.

1 Materials and Methods

1.1 Plants, plasmids, and bacteriaStem segments of diseased Paulownia tomentosa ×

P. fortunei were collected from the Wuhan Institute ofVirology. Healthy seeds from the same variety weresterilized and cultured on 1/2 MS medium as the start-ing material. A4, a plasmid containing PWB-phytoplasmaDNA fragments (Zhang et al. 1994) was used to makea probe to detect phytoplasma. The p438PRSI vector

Tao DU et al.: Transgenic Paulownia Expressing shiva-1 Gene has Increased Resistance to Paulownia Witch’s BroomDisease 1501

was as constructed by Huang et al. (1994) and is shownin Fig. 1. The vector contains neomycin phosphotrans-ferase (NPTII) gene (conferring kanamycin resistance),a pathogenesis-related protein 1b signal peptide sequence(PR-1b), and the shiva-1 gene under the control of theCaMV35S promoter. The p438PRSI vector was trans-formed to Agrobacterium tumefaciens LBA4404 (kindlyprovided by Professor Xue-Xian PENG, Institute ofMicrobiology, the Chinese Academy of Sciences,Beijing, China) by electric pulse.1.2 Transformation of Paulownia

Paulownia was transformed via A. tumefaciens asdescribed by Wang et al. (2002). Briefly, stem seg-ments from PWB phytoplasma-infected Paulowniaseedlings were precultured at 28 °C for 3 d on MSnutr ient medium supplemented wi th 4 mg/L6-benzyladenine (BA) and 0.2 mg/L α-naphthaleneaceticacid (NAA). After coculture with A. tumefaciens in thedark at 28 °C for 3 d, the explants were rinsed oncewith 500 mg/L carbenicillin for 5 min and subsequentlyplaced on MS medium supplemented with 300 mg/Lcarbenicillin and 50 mg/L kanamycin. Explants weresubcultured every 2 weeks.

The transformation procedure for healthy tissue wassimilar to that used for diseased Paulownia. Intact leaveswere used as explants in this procedure. The MS nutri-ent medium used for healthy tissue was supplementedwith 4.0 mg/L BA, 0.4 mg/L NAA, and 300 mg/Lcarbenicillin. Kanamycin (30 mg/L) was used in thefirst 4 weeks, with the concentration being increasedto 50 mg/L in the following weeks. In the present study,

line was designated as plants derived from an indepen-dent transformation event.

After growing to 2–3 cm long, the Kanamycin re-sistance (KanR) buds (Fig. 2) were transferred to root-ing medium, and then planted in a greenhouse in March.The healthy transgenic Paulownia was transferred toa nursery.1.3 Molecular analysis of transformed Paulownia

S a m p l e D N A w a s e x t r a c t e d u s i n g t h ecetyltrimethylammonium bromide (CTAB) method(Lodhi et al. 1994). Primers (5'-CCGGGGATCCTCT-AGA-3'; 5'-GCTAGCGAATTCTCAACC-3') were de-signed to amplify the PRSI which constituted of PR1-B and shiva-1 gene. PCR was performed in a 50-µLvolume containing 0.8 pmol/L of each primer, 0.3 mmol/LdNTP, 0.1 µg sample genomic DNA, and 2.5 U Taqpolymerase for 30 cycles (94 °C for 5 min; 94 °C for1 min, 52 °C for 1 min, 72 °C for 1 min for 30 cycles;and extenstion at 72 °C for 5 min).

For Southern hybridization, 10–18 µg DNA was di-gested with HindIII at 37 ºC overnight and resolved onan 0.8% agarose gel. The DNA was transferred to anylon membrane after electrophoresis. The PCR prod-uct amplified from the PRSI gene was 32P-labeled as aprobe using a DNA labeling Kit (TaKaRa, China). Sub-sequent hybridization was performed at 42 ºC understringent conditions, as described by Sambrook et al.

Fig. 1. Schematic diagram of plant expression vectorp438PRSI. NPTII, neomycin phosphoryl transferase gene;35SP, CaMV35S promoter with double enhancers; Ω, “Ω”fragment of TMV cDNA; PRSI, PR-1b signal sequenceand shiva-1 gene; NOS 3', transcriptional terminator se-quence of nopaline synthase gene. Fig. 2. The regeneration of KanR shoots.

Journal of Integrative Plant Biology (Formerly Acta Botanica Sinica) Vol. 47 No. 12 20051502

(1989).For RT-PCR, total RNA was extracted from Pau-

lownia using a Total RNAultra; Extraction Kit (BeijingTianwei, China) according to the manufacturer’sinstructions. A 4.0-µL aliquot of template RNA was re-verse transcribed in a final volume of 20 µL reactionbuffer (1 × RT buffer, 0.2 mmol/L dNTP, 0.1 µgoligodT, 10 U AMV RTXL (TaKaRa, China) and 20 URnasin Ribonuclease inhibitor (TaKaRa, China)) at 42ºC for 1 h. A 1-µL aliquot of the cDNA reaction wasused as a PCR template and the PCR was performedas described above.1.4 Protein extraction and ELISA

One gram of tender leaves was boiled in 100 ºCwater for 45 s and immediately ground in liquid nitro-gen (Hu et al. 1999; Zhi et al. 2001). The powder washomogenized with 1 mL buffer (10 mmol/L Na2CO3,35 mmol/L NaHCO3, 1 mmol/L phenylmethylsulphonylfluoride (PMSF), and 1.5 mmol/L NaN3, pH 9.6). Theresulting homogenate was then centrifuged at 13 500gat 4 ºC for 15 min. A 100-µL aliquot of each superna-tant was used for ELISA. Synthesized Shiva-1 wasused as a positive control. Briefly, samples and con-trols were loaded and incubated at 37 ºC for 1 h. Afterthree washes with phosphate-buffered saline (PBS),plates were incubated with blocking buffer (PBSsupplemented with 1% (w/v) bovine serum albumin(BSA)) at 37 ºC for 1 h. Bound Shiva-1 was detectedwith a polyclonal antibody against Shiva-1 (diluted1:1 000 in PBS supplemented with 0.1% (w/v) BSA).After 1 h incubation at 37 ºC and three washes withPBS, plates were conjugated with secondary antibody(goat anti-rabbit IgG coupled to horseradish peroxidase)and incubated at 37 ºC for 1 h. After repeated washing,o-phenylenediamine was added and the reaction wasallowed to incubate for 30 min. This was followed theaddition of stop buffer (0.5 mol/L H2SO4) and absor-bance values were then read at 490 nm on a Sunriseabsorbance reader (Tecan , Austria).1.5 Evaluation of PWB resistance

The PWB disease was monitored in shiva-1-t r a n s g e n i c P a u l o w n i a d e r i v e d f r o m b o t h

phytoplasma-infected and healthy Paulowinia. Non-transgenic Paulownia from infected and healthy Pau-lownia were used as controls. Symptoms were classi-fied into one of four groups as follows: class I, one ortwo branches have PWB; class II, a few branches havePWB; class III, the majority of branches have PWB;and class IV, the plant is dead because of PWB. Symp-toms were recorded every month. Healthy transgenicPaulownia was kept in a nursery for natural infectionof phytoplasma.1.6 Detection of phytoplasma in transgenic Pau-lownia

Sample DNA was extracted using a modified CTABmethod. Phytoplasma-DNA fragments were recoveredfrom EcoRI-digested A4 plasmids and were labeled with32P as a probe using 6-mer random primers. Hybridiza-tion was performed at 65 ºC as described previously(Zhang et al. 1994).

2 Results

2.1 Transformation of PaulowniaFive days after being transferred to selective MS

medium, stems from phytoplasma-infected Paulowniabegan to expand. After 1–1.5 months, some explantsstarted to regenerate green buds. The KanR buds werecultured in selective medium for another 1–1.5 monthsto allow them to multiply.

Healthy leaf explants were placed on selective MSmedium with petioles in the medium. The petiole wasthe first place to expand. Regeneration from healthyPaulownia occurred within 4–5 weeks.

After shoots had reached 2–3 cm in length, the pu-tative transgenic shoots from phytoplasma-infectedPaulownia were transferred onto rooting inducingmedium. The 1/2 MS medium supplemented with 0.5mg/L kinetin and 0.3 mg/L NAA was suitable for healthyPaulownia to root, whereas phytoplasma-infected Pau-lownia failed to root in this medium. In contrast,phytoplasma-infected Paulownia rooted in 1/2 MS supple-mented with 0.5 mg/L NAA and 3 g/L carbon powder.2.2 Molecular analysis of transgenic Paulownia

To investigate whether the shiva-1 gene was


successfully integrated into the genome of Paulownia,DNA was extracted from KanR seedlings and subse-quently examined by PCR. As shown in Fig. 3, theresults showed that an expected 250-bp fragment wasamplified from the DNA extracted from KanR seedlings,but not from the untransformed Paulownia.

The integration of the shiva-1 gene was further con-firmed by Southern hybridization. As shown in Fig. 4,two ramets of line 6, designated as 6-1 and 6-2, andtwo ramets of line 3, designated as 3-2, 3-3, and 3-3,all had a single band. Taken together, the results ofPCR and Southern hybridization indicate that the shiva-1 gene was successfully integrated into the Paulownia

genome.The results of RT-PCR (Fig. 5) demonstrated that

all transgenic Paulownia tested had a 250-bp band,indicating that the shvia-1 gene could be transcribed inramets of transgenic Paulownia lines 3 and 6.2.3 Detection of phytoplasma in transgenic Pau-lownia engineered from infected stems

Dot-blot hybridization was performed to detectphytoplasma-DNA in shiva-1-transgenic Paulowniashoots derived from infected stems, whereas DNAextracted from healthy and diseased Paulownia wasused as a negative and positive control, respectively.As shown in Fig. 6, phytoplasma-DNA was detectedin all transgenic Paulownia engineered from infectedstems, although the amount of phytoplasma-DNAvaried.2.4 Bioassay of transgenic Paulownia

The symptoms of each Paulownia were recordedaccording to the four-point scale described earlier.Three ramets of line 3 (designated as 3-1, 3-2, and 3-3) and line 6 (designated as 6-1, 6-2, and 6-3) wereused for bioassays. In May, 6-3 and two of the dis-eased untransformed Paulownia that has the same ori-gin with the shiva-1 Paulownia (CK) died of PWB.During year 1, the symptoms of transgenic Paulowniawere alleviated, whereas those of the CK became moreserious during the bioassay period (Fig. 7a). When thebioassay was finished, lines 3 and 6-2 had no PWBsymptoms; the symptoms of line 6-1 were alleviated toclass II, whereas the controls continued to exhibit aserious PWB syndrome (Fig. 7b–g).

Fig. 3. PCR result of KanR seedlings. Lane 1, negativecontrol (untransformed Paulownia); lane 2, positive con-trol (plasmid p438PRSI); lanes 3–11, KanR seedlings; lane12, molecular marker.

Fig. 4. Southern hybridization of Paulownia genomicDNA. Lane 1, 6-2; lane 2, 6-1; lane 3, 3-2; lane 4, 3-3; lane 5,negative control; lane 6, DL15000 molecular marker.

Fig. 5. RT-PCR result of transgenic Paulownia. Lane 1,molecular marker; lane 2, positive control; lane 3, negativecontrol (untransformed Paulownia); lanes 4–8, 3-1, 3-2, 3-3, 6-1 and 6-2, respectively.

Fig. 6. Dot blot detection of phytoplasma in infectedPaulownia. b1, plasmid A4 (positive); b2, Paulownia in-fected with phytoplasma (positive); A2, healthy paulownia(negative); a3–a7, transgenic plant 15, 14, 11, 5 and 10,respectively; b3–b7, transgenic plant 1, 3, 4, 6, 7 lines,respectively.


After observation for two years in the nursery, nosymptoms of PWB were found in either healthytransgenic Paulownia or healthy non-transgeniccontrol.2.5 Dot blot analysis of phytoplasma-DNA in dis-eased Paulownia

Dot blot analysis showed that all samples containedphytoplasma-DNA in May of year 1. As shown in Fig.8a, lines 3-2 and 3-3 have lighter hybridization signalsof phytoplasma that correspond to their less serioussymptoms. The content of phytoplasma in the lowerpart of the plant was much higher than that in the

upper part of the plant in line 6-1. These results are inagreement with the symptoms observed: the lower partof Paulownia was more seriously affected by PWBthan the upper part of the plant. In October of year 1,the same trees were sampled and analyzed again. Asshown in Fig. 8b, the phytoplasma-DNA hybirdizationsignal in controls was stronger, whereas the signal intransgenic Paulownia was weaker. In summary, dotblot results from two time points clearly indicate thatphytoplasma in transgenic Paulownia decreased,whereas that in controls increased.2.6 Detection of shiva-1 expression

The concentration of Shiva-1 was related to absor-bance value over the 0–50 ng/mL (data not shown).When absorbance values were converted toconcentration, the concentration of Shiva-1 differedbetween the transgenic Paulownia lines, as shown inFig. 9. The results also indicated that the higher theconcentration of Shiv-1 was the less phytoplasma andthe milder the symptoms would be.

Fig. 7. Disease grade and symptom of Paulownia witch’sbroom disease (PWB). a. The change of average symptomgrade of PWB during a year-long period; black bar, aver-age symptom of CK; white bar, average symptom oftransgenic Paulownia. b, e. The symptom of CK on May,Oct., respectively. c, f. The symptom of 3-2 on May, Oct.,respectively. d, g. The symptom of 6-1 on May, Oct.,respectively.

Fig. 8. Dot blot analysis of phytoplasma in diseasedPaulownia. a. Result of hybridization on May. b. Result ofhybridization on Oct. II-1, V-4, plasmid A4 (positive). I-2,III-2, healthy Paulownia (negative); II-3–II-8, DNA quan-tification of Paulownia infected with phytoplasma(phytoplasma quantity control) 4 µg, 1 µg, 250 ng, 65 ng,16 ng, 4 ng, respectively; I-1, III-1, Paulownia infectedwith phytoplasma (positive); I-3–I-8, 6-1 uper part, 6-1 lowerpart, 6-2, 3-1, 3-2, 3-3, respectively on May; III-3, III-4, 6-2and 6-1 respectively on Oct.; IV-1–IV-3, 3-3, 3-2 and 3-1,respectively on Oct.


3 Discussion

PWB is a systemic disease caused by phytoplasma,a pathogen unable to be cultured in vitro. In the presentstudy, we developed transgenic Paulownia by intro-ducing the shiva-1 gene into Paulownia, and moni-tored its antiphytoplasma activities using molecular andbiological approaches.

In general, there are three methods to test for PWBresistance following challenge of Paulownia withphytoplasma: grafting, dodder bridging, and transfer-ring by insects. Grafting is widely used, but needs tobe conducted in the proper season because season mayaffect the content of phytoplasma. Affinity betweenscion and stock is another important factor that mayinfluence the outcome of grafting (Tian et al. 1999). Adodder bridge between healthy and diseased Paulowniacan transfer phytoplasma. However, it usually takes along time and consumes nutrients from the host tree.Certain sucking insects can also transfer phytoplasmafrom diseased Paulownia to healthy Paulownia.Halyomorpha mista is the most important insect vec-tor for transferring PWB phytoplasma. Sun (1999)undertook a systemic investigation using H. mista asthe vector and showed that 53% of tested Paulowniaseedlings was lost as a result of insect sucking. In thepresent study, we used PWB-diseased Paulowniastems to generated Shiva-1-transgenic Paulownia. Thetransformed materials were selected in medium supple-mented with kanamycin and carbenicillin. Phytoplasma

is sensitive to members of the tetracycline family, butnot to kanamycin and carbenicillin. This was confirmedby Dot blot analysis showing that phytoplasma propa-gation was not seriously affected. Our findings sug-gest that using diseased plant materials to transformand obtain phytoplasma-resistant plants may be a reli-able and efficient way to evaluate antiphytoplasmaeffects, avoiding the difficulty of challenging plants withphytoplasma.

In the current study, the shiva-1-transgenic Pau-lownia showed reasonable resistance to PWB in thegreenhouse, although symptoms varied. This may bedue to differences in the phytoplasma content beingdirectly affected by Shiva-1 expression. The CaMV35Spromoter is a constitutive promoter and can drive theexpression of genes from the cecropin family (Wangand Jia 1996; Yi et al. 2004). However, genes underthe control of the CaMV35S promoter are not neces-sarily expressed at similar levels in different tissues andorgans. In Cry1A-transgenic cotton, which usedCaMV35S as a promoter, the highest expression ofCry1A was in fresh leaves (381 ng/g) compared with35 ng/g in the pith (Liu and Wang 2003). In rice andtomato, the results are similar: genes under the controlof the CaMV35S promoter were better expressed ingreen tissue than in non-green tissue (Battrawet andHall 1990). In β-glucuronidase gene (gus)-transgenicscots pine (Pinus sylvestris L.) with the CaMV35Spromoter, it was demonstrated that the expression ofGUS varied from season to season (Aronen et al. 1995).Such an expression pattern controlled by the CaMV35Spromoter may account for the symptom relapse ob-served in some transgenic Paulownia in the presentstudy. Phytoplasma could be inhibited in green tissuewhere Shiva-1 is expressed in high concentrations.However, phytoplasma in non-green tissue (e.g. root,cortex) with a poor expression of Shiva-1, can stillexist and may survive as a pathogen source, migratingalong the phloem and infecting other parts of the plant.The expression pattern of shiva-1 in transgenic Pau-lownia needs further investigation.

Taken together, the results of the present study

Fig. 9. Quantification of Shiva-1 in tender leaves ex-tracted from transgenic lines by ELISA. The Shiva-1 aver-age level of 3, 6, 15 and CK were 10.8, 3.8, 2.3, 1.7 ng/ 100mg tender leaves (fresh weight), respectively.


indicate that shiva-1 gene-transgenic Paulownia de-rived from diseased materials show good PWB resis-tance in the greenhouse. The PWB resistance oftransgenic Paulownia derived from healthy materialsneeds further examination.

References

Aronen T, Hohtola A, Laukkanen H, Haggman H (1995). Seasonal

changes in the transient expression of a 35S CaMV-gus gene con-

struct introduced into Scots pine buds. Tree Physiol 15, 65–70.

Battraw MJ, Hall TC (1990). Histochemical analysis of CaMV

35S promoter beta-glucuronidase gene expression in

transgenic rice plants. Plant Mol Biol 15, 527–538.

Chen WX, Xiao GF, Zhun Z (2002). Obtaining high pest-resis-

tant transgenic upland cotton cultivars carrying Cry1Ac gene

driven by chimeric OM promoter. Acta Bot Sin 44, 963–970.

Guo HN, Wu JH, Tian YC et al. (2003). Cotton plants trans-

formed with the activated chimeric CrylAc and API-B genes.

Acta Bot Sin 45, 108–113.

Hu TS, Wei WG, Liu JF et al. (1999). Preparation and applica-

tion of monoclonal antibodies against anti-bacterial peptide

magainin II. Chin J Cell Mol Immunol 15, 302–304 (in Chi-

nese with an English abstract).

Huang DN, Zhu B, Yang W et al. (1997). Cecropin B gene trans-

ferring to rice and the detection of transgenic plants. Chin Sci

(Ser. C) 27, 55–62 (in Chinese).

Huang WJ, Cui XJ, Tian YC, Lin ML, Peng XX (1994). Cloning

of T7 lysozyme gene and construction of the vector for

transgenic plants resistant to bacterial infection. Acta Microbiol

Sin 34, 261–265 (in Chinese with an English abstract).

Lin ML, Zhang CL, Yang JH, Shi XY (1994). Detection of my-

coplasma-like organism of Paulownia witches’ broom using

nuclear acid hybridization. Chin Sci Bull 39, 376–379 (in

Chinese).

Liu YJ, Wang GY (2003). Inheritance and expression of Cry1A

gene in the transgenic maize. Acta Bot Sin 45, 253–256.

Lodhi MA, Ye GN, Weeden NF, Reisch BI (1994). A simple and

efficient method for DNA extraction from grapevine cultivars,

Vitis species and Ampelopsis. Plant Mol Biol Rep 12, 6–13.

Sambrook J, Fritsh EF, Maniatis T (1989). Molecular Cloning:

A Laboratory Manual, 2nd edn. Cold Spring Harbor Labora-

tory Press, New York.

Sharma A, Sharma R, Imamura M, Yamakawa M, Machii H

(2000). Transgenic expression of cecropin B, an antibacterial

peptide from Bombyx mori, confers enhanced resistance to

bacterial leaf blight in rice. FEBS Lett 484, 7–11

Sun ZQ, Fu JM, Qiao J, Yan ZS, Du KY, Dong SQ (1999).

Capacity of Halyomorpha picus transmitting MLO associ-

ated with Paulownia Witches’ Broom. J For Res 12, 606–

611 (in Chinese with an English abstract).

Tian GZ, Zhu SF, Luo F, Li HF, Qiu WF (1999). Response of

resistance and susceptivity of in vitro cultured Paulownia to

the graft inoculation with phytoplasma. Sci Silv Sin 35, 31–

39 (in Chinese with an English abstract).

Wang GL, Fang HJ, Wang HX, Li HY, Wei YT (2002). Pathogen-

resistant transgenic plant of Brassica pekinensis by trans-

ferring antibacterial peptide gene and its genetic stability.

Acta Bot Sin 44, 951–955.

Wang H, Sun C, Peng XX (2001). Construction of plant expres-

sion vectors harboring a peptide antibiotic-apidaecin gene

and resistance analysis of the transgenic tobacco. Chin J

Biotechnol 17, 423–427 (in Chinese with an English abstract).

Wang Y, Liu S, Du T, Lin ML (2001). Transformation of

Agrobacteria tumefaciens on healthy and infectious

Paulownia. Acta Bot Boreali-Occident Sin 21, 406–412 (in

Chinese with an English abstract).

Wang ZX, Jia SR (1996). Construction of vectors harboring a

barley G-amylase signal sequence and a Cecropin B or Shiva

A gene that resulted in the extracelluar protein secretion in

potato transgenic plants. J Agr Biotechnol Sin 4, 277–286 (in

Chinese with an English abstract).

Yi JY, Seo HW, Yang MS, Robb EJ, Nazar RN, Lee SW (2004).

Plant defense gene promoter enhances the reliability of shiva-

1 gene-induced resistance to soft rot disease in potato. Planta

220, 165–71.

Zhang CL, Lin ML, Hu QX, Huang WJ (1994). Molecular clon-

ing and sequence analysis of DNA in mycoplasma-like or-

ganism of the Paulownia Witches’ Broom. Acta Bot Sin 36,

278–282 (in Chinese with an English abstract).

Zhi JL, Ubramanian J, Gray DJ (2001). An improved enzyme-

linked immunoabsorbent assay protocol for the detection of

small lytic peptides in transgenic grapevines (Vitis vinifera).

Plant Mol Biol Rep 19, 341–351.

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