Transgenic root clones gene confirmation - 根毛農桿菌對黃豆之基因轉形研究

Chapter 3 Results

3.5 Transgenic root clones gene confirmation

The 90 root clones underwent 42 days of liquid cultivation were ground in liquid nitrogen and genomic DNA were extracted, DNA confirmation was then carried out by PCR amplification. For all candidate root clones, the first target in DNA confirmation is the 543 base pairs rolC sequence. In WT construction, rolC was not observed on gel for all thirty root clones (data not shown). For 2BV and 2AR construction, rolC was not observed either. Even the two root clones with the hairy roots like morphology did not

show rolC band as well (Figure 13). Because of the inexistence of rolC in all candidate root clones, we could not verify hairy roots and adventitious roots at this point.

The other two targets for 2BV and 2AR construction in DNA confirmation was gfp and gus, each sized 833 and 638 base pairs. For gfp confirmation, two of the root clones, 2BV-C30-2, and 2AR-C31-1 showed gfp band on gel (Figure 13). All root clones from these two constructions were then examined under fluorescent microscopy for the confirmation of functional GFP. For the two root clones that gfp was observed, GFP was functionally expressed and observed. In addition, root clone 2AR-C16-2 also showed GFP expressing (Figure 14).

For gus confirmation, all sixty candidate root clones from 2BV and 2AR construction did not show gus band on gel. With the experience of false estimating gene transcription of gfp, all root clones underwent examination of GUS activity. We found no root clones functionally expressing GUS.

The two fast growing root clones were the only ones that had sufficient material for protein extraction, and the 2BV-C16-2 clone expressing GFP was also extracted. We extracted total protein from the three root clones as mentioned above. After quantification, the protein extracts were further concentrated 5 fold because of the low protein content. SDS-PAGE was first carried out (Figure 15). According to the result on gel, both roots 2AR-C16-2 and 2AR-C31-1 had a 27 kD GFP band as expected. Root

2BV-C16-2 had protein content too low for observing GFP band on gel. For GUS (68 kD), a light band of around 70 kD which might be GUS was observed for root 2AR-C31-1 and 2BV-C16-2. We then further examined the three protein extracts with Western blotting, using the 6x His tag on both GFP and GUS as target. We did not observe any band on the membrane, which might be due to the low protein content of root protein extracts (data not shown).

All of the gene confirmation results are summarized in Table 5.

Chapter 4 Conclusions and Discussions

4.1 A. rhizogenes transformation

In this study, we attempted to construct an A. rhizogenes-mediated multiple gene transformation system in soybeans. The strategies could be divided into two parts concerning the binary vector number being used. The first strategy is transforming two genes in separate binary vectors simultaneously, either harbored by one A. rhizogenes transformant or by two separate transformants. The second strategy is transforming two genes in a single binary vector, either in a single T-DNA region or in separate T-DNA regions. We have only managed to test the first strategy so far in this study.

Using the strategy of transforming genes in two separate binary vectors, we first have to understand the efficiency of constructing A. rhizogenes transformants.

Transforming a single binary vector into wild-type A. rhizogenes had a transformation efficiency averaged at 6.88 x 10⁵, which had little difference depending on the vector transformed. This protocol is considered quite efficient of gaining A. rhizogenes transformants. Further transforming the second binary vector into transformed A.

rhizogenes had a efficiency averaged at 9.5 x 10². The protocol was still capable of gaining sufficient transformants but the transformation efficiency was a thousandth of transforming the first vector into A. rhizogenes. The transformation efficiency transforming two vectors simultaneously into wild-type A. rhizogenes was even lower at 2.75 x 10². This decrease in the acceptability of A. rhizogenes might be related to

plasmid incompatibility due to the considerable similarity between the two binary vectors being transformed.

Plasmid incompatibility is due to the sharing of one or more element of the plasmid replication or partitioning systems (Novick, 1987). In this case, pCAMBIA 1201 and pCAMBIA 1302 share the backbone sequence which contains the replication origin and a hygromycin resistance gene, a total of 8737 base pairs (pCAMBIA 1201 and pCAMBIA 1302 are sized 12,001 and 10,549 base pairs, respectively). Vectorial plasmid incompatibility, in which case one plasmid is lost exclusively or with higher probability, might happened in this study. When transforming the second binary vector into a pre-transformed A. rhizogenes, most cells retained only the original vector harbored. Only 1 x 10^-9 of the transformed cells lost the original vector and gained the newly transformed vector. Although plasmid incompatibility seems to played an important role affecting the transformation process, it is considered not to have its affect after the cells are cultured under selection stress, which is antibiotics in this case (Novick et al., 1976).

4.2 Soybean and transgenic soybean roots

We examined six genotypes of soybean for selecting the most suitable material for further soybean researches, and we found soybean Tainan #2 showed the highest

germination rate and a high utilization factor compared to the other five genotypes of soybean tested. The selection strategy was based on the germination rate and also considering the growth rate, plant height and shoot thickness at an early period of germination as the index of selecting the suitable soybean material. With this strategy, it seemed we have overlooked the difference of soybean genotype characteristics between the six soybean genotypes tested.

Using soybean Tainan #2 as the material for A. rhizogenes mediated gene transformation, we gained plenty candidate root clones which showed fast growth while still attached to the A. rhizogenes infected soybean tissue. However, from these candidate root clones we only gained two root clones which maintained fast growth on solid plate and in liquid medium after detaching the original tissue. Based on the morphology and the growth, these two clones had a high possibility being transgenic hairy roots, and the rest of the candidate root clones might be adventitious roots.

The PCR results for gene confirmation were not quite as we expected. For the poor growing root clones which were not expected to be hairy roots, rolC was not detected as expected. But for the fast growing clones which were expected to be hairy roots based on the morphology and growth, rolC was not found, either. Secondly, there was a root clone which had GFP functionally expressed but gfp not detected in the PCR confirmation. The two confusing results gave us a conclusion that the problem might

lies in PCR. We then tried many modifications with the PCR reaction mixture and the PCR sequence, but still gained the same results. As the PCR problem still not solved, for now we will make our judgments based mainly on the morphology and on the protein level. As a conclusion, we had gained two transgenic hairy root clones, 2AR-C16-2 and 2AR-C31-1, both expressing GFP but not GUS. We also gained one adventitious root 2BV-C30-2 expressing GFP, and we gained no wild-type hairy roots.

As with the PCR problem, because of the massive numbers of gene within soybean genomic DNA pool and the unknown site of T-DNA insertion (Zambryski, 1988), it is somehow difficult to amplify some certain genes. To avoid this problem, reverse-transcription PCR can be carried out. Because not all genes in soybean genomic DNA are being transcript in hairy roots, RT-PCR can enhance the chance of finding the desired genes.

The two hairy root clones we gained is considered that both gained two of the three T-DNA we transformed, which in this case was a T-DNA region containing rolC and a T-DNA region containing gfp. Furthermore, the adventitious root expressing GFP can be considered which gained only one of the three T-DNA being transformed. From the above results, we suspect that A. rhizogenes might have a transformation limit for T-DNA numbers being transformed simultaneously into soybean. On the other hand as we mentioned above, plasmid incompatibility has a high probability of happening when

A. rhizogenes is cultured under no selection stress. After A. rhizogenes was applied to

the wounded soybean tissue, A. rhizogenes and soybean were co-cultivated for a 14 to 21 days period on antibiotic free 1/2 MS plates. In this period, there was no selection stress for A. rhizogenes to retain the two nonessential extra-chromosomal binary vectors.

So, plasmid incompatibility might be another reason causing some of the genes not being transformed into soybean.

This low efficiency of gaining soybean transgenic hairy roots, compared to previous studies in our lab of inducing tobacco hairy roots, might be related to the soybean characteristics. Soybean Tainan #2 is a crossbred genotype bred by the Tainan District Agricultural Research and Extension Station (Tainan, Taiwan) in 1993. This soybean genotype has a strong disease-resistant ability to soybean downy mildew caused by Peronospora manshurica and soybean rust caused by Phakopsora pachyrhizi, it also has a medium disease-resistant to bacterial leaf spots disease caused by

Xanthomonas axonopodis and bacterial pustules caused by Xanthomonas campestris (連

大進等，1993). These disease-resistant abilities of soybean Tainan #2 might have an effect on the A. rhizogenes-mediated gene transformation. Agrobacterium, as mentioned above, is a soil born plant pathogen, so Tainan #2 might also have the ability to resist or partially-resist the infection of A. rhizogenes, which might be the reason that we were not able to gain sufficient transgenic hairy roots as expected.

Also, we suspected that the low efficiency of inducing soybean hairy roots might also have relation with the tissues used for induction. In this study, we used cotyledon and the lower shoot for induction of hairy roots. From our results, we suspected that many of the root clones inducted from lower shoot and cotyledon might just be soybean root which will grow even without A. rhizogenes induction. One member of our lab is currently studying A. rhizogenes-mediated soybean Tainan #5 gene transformation, most of the root clones induced from the upper shoot of Tainan #5 showed hairy roots morphology on plates and in liquid cultures. We then tested the induction rate of soybean Tainan #2 upper shoot with wild-type A. rhizogenes. The induction rate was 51.4%, and 32.6% of the induced roots showed fast growth while still attached to the upper shoot tissues. Roots induced from upper shoot generally showed more branching than those inducted from lower shoot and cotyledon (Figure 12 (C)), more likely to be hairy roots. Although this assumption is not yet confirmed, it is a point that should be considered in the following studies.

4.3 Perspectives

Our current strategy of transforming multiple genes into soybean is using multiple binary vectors (Figure 3 and Figure 4), this strategy is thought to be easier to construct.

But since plasmid incompatibility might have occurred and had a negative effect on the

transformation efficiency, other transformation strategies should be considered. The other strategy we constructed but not yet utilized in this study, transforming two genes in a single binary vector (Figure 5 and Figure 6), is thought to be more suitable for multiple genes transformation. Construction of two genes in a single binary vector, either constructing the two genes in a single T-DNA region or in separate ones, can avoid plasmid incompatibility happening during the co-cultivation period of

Agrobacterium and plant tissues. This will ensure the two genes both have its chance to

be transformed into plant cells. So to transform soybean with the two constructions that use only one binary vector is the first step for the follow ups of this study. And the binary vector with the two genes in separate T-DNA regions can also be a model for the study to find out if there’s a transform limit of T-DNA number for A. rhizogenes.

We had an assumption that soybean genotype characteristic and the tissue being induced might affect soybean susceptibility to A. rhizogenes-mediated transformation.

This assumption needs a broad scanning of different tissues of plenty soybean genotypes, testing sterile plant germination rate, wild-type A. rhizogenes induction rate, and the percentage of hairy roots over induced roots. This test may take long to finally select the most suitable soybean tissue, but it should be an important step to construct a complete soybean gene transformation system.

The A. rhizogenes-mediated soybean multiple genes transformation is not yet fully constructed. Although this study leaves a lot of unsolved questions behind, it still give us some knowledge about A. rhizogenes-mediated soybean hairy roots induction system:

1. the A. rhizogenes our lab uses can successfully induce soybean hairy roots and the hairy roots can propagate under solid and liquid medium; 2. the foreign gene transformed is successfully inserted into soybean hairy roots genome; 3. the foreign gene transformed can be functionally expressed in soybean hairy roots. These knowledge assure us that A. rhizogenes-mediated soybean gene transformation system can be used to become a foreign protein expression system, and this system can also be used for Genomics like promoter trapping and gene tagging.

Tables and Figures

52 Table 1 Proteins of medical relevance produced in plant cell cultures

Expressed protein Expression host Promoter Localization, yield

Human serum albumin N. tabacum suspension culture Modified 35S Secretion/apoplast targeting, 0.25 μg mg–1 protein in supernatant

scFv antibody fragment N. tabacum suspension culture 35S Secretion, up to 0.5 μg l–1 up to 0.5% of TSP Human erythropoietin N. tabacum cv BY-2 suspension culture 35S Secreted, 1 pg g–1 FW

Mouse monoclonal heavy-chain γ

N. tabacum cv NT-1 suspension culture

35 S Native heavy-chain secretion signal, ca. 10 μg l–1 extracellular without, 350 μg l–1 with PVP Mouse IgG2b/κ N. tabacum cv Petite Havana SR-1 Enhanced 35S 15 μg g–1 FW, ∼0.3% of TSP

Heavy chain mAb N. tabacum cv NT-1 suspension culture

35S Secreted (native signal peptides), 8–180 μg l–1 of culture broth

Recombinant ricin N. tabacum suspension culture 35S 25–37.5 μg l–1 scFv antibody fragment Oryza sativa cv Bengal (rice) callus

culture

Maize ubiquitin Apoplast targeting (optimized Ig leader peptides) and ER-retention, up to 3.8 μg g–1 callus FW

Full size IgG-2b/κ N. tabacum cv Petite Havana SR-1 Enhanced 35S 0.3% of TSP or 15 μg/g wet weight Human α1-antitrypsin O. sativa cv Taipei 309 suspension

culture

RAmy3D Secreted, 85 mg l–1 in shake flask, 25 mg l–1 in bioreactor

HBsAg N. tabacum NT-1 suspension culture A. thaliana ubq3 Secreted, up to 10 μg l–1 of particulate HBsAg (Hellwig et al., 2004)

Table 1 Proteins of medical relevance produced in plant cell cultures (Continue)

Expressed protein Expression host Promoter Localization, yield

biscFv antibody fragment N. tabacum cv BY-2 suspension culture Enhanced 35S Cytosolic, apoplast-targeted (up to 0.0064% of TSP), ER-retained (up to 0.064% of TSP)

hGM-CSF N. tabacum cv NT-1 suspension culture

35S Secreted/targeted to the apoplast ∼250 μg l–1 extracellular, ∼150 μg l–1 intracellular

scFv antibody fragment N. tabacum suspension culture 35S Apoplast targeting (sporamin secretion signal) 1 mg l–1 extracellular, 5 mg l–1 intracellular

Human α1-antitrypsin O. sativa suspension culture RAmy3D Up to 200 mg l–1 (calli suspended to 40% (vol/vol)) cell density in induction medium

HBsAg Glycine max cv Williams 82 & N.

tabacum NT-1 suspension cultures

(ocs)3mas Intracellular up to 22 mg l–1 in soybean ∼2 mg l–1 in tobacco

hGM-CSF N. tabacum 35S 1.6 to 6.6 μg ml–1 upon homogenizing the entire culture broth

Human lysozyme O. sativa cv Taipei 309 suspension culture

RAmy3D Intracellular (although RAmy3D signal peptide was used), up to 3%–4% of TSP

IL-12 N. tabacum cv Havana suspension culture

Enhanced 35 S Secreted, up to 800 μg l–1 of supernatant hGM-CSF Lycopersicum esculentum cv

Seokwang suspension culture

Enhanced 35S Secreted, up to 45 μg l–1 of supernatant

mAb against HBsAg N. tabacum cv BY-2 suspension culture 35S Secreted, ∼50/50 between supernatant and cells, total max ∼15 mg l–1

(Hellwig et al., 2004)

54 Table 2 Recent reports on valuable metabolites produced by hairy roots

Plant species Metabolites Properties Year

Camptotheca acuminate Camptothecin Anti-cancer, antiviral 2004

Gingko biloba Ginkgolides Aging disorders 2003

Gmelina arborea Verbascoside Stomach disorders, fevers, skin problems 2005

Gynostemma pentaphyllum Gypenoside Detergent 2005

Linum flavum Coniferin Anti-cancer 2003

Papaver somniferum Morphine, sanguinarine, codeine Sedative, analgesic 2004 Pueraria phaseoloides Puerarin Hypothermic, spasmolytic, hypotensive,

anti-arrhythmic 2003

Rauvolfia micrantha Ajmalicine, ajmaline Anti-hypertensive 2003

Saussurea medusa Jaceosidin Anti-tumor 2004

Solidago altissima Polyacetylene

(cis dehydromatricaria ester) Unknown 2003

(Guillon et al., 2006)

55 Table 3 A. rhizogenes transformation efficiency

Target A. rhizogenes (T-DNA)

Binary vector transforming

pCAMBIA1201 pCAMBIA1302 pCAMBIA1201 + pCAMBIA1302

WT (Ri) 5.5 x 10⁵ 8.25 x 10⁵ 2.75 x 10²

1201 (Ri + pCAMBIA1201) - 1.05 x 10³ -

1302 (Ri + pCAMBIA1302) 8.5 x 10² - -

Transformation efficiency (transformants/μg) = (number of colonies on plate / ng of DNA plated) x 1000 ng/μg

56 Table 4 Induction rate of soybean roots

Construction Root induced tissues / Total infected tissues (%)

Fast growing roots / Total roots (%)*

Root clones picked

WT Cotyledon 24/50 (48.0) 30/74 (40.5) 30

Lower shoot 10/65 (15.4) 1/10 (10.0) 0

Upper shoot 55/107 (51.4) 48/148 (32.6) -

2BV Cotyledon 40/101 (39.6) 41/93 (44.1) 29

Lower shoot 16/108 (14.8) 2/19 (10.5) 1

2AR Cotyledon 45/66 (68.2) 43/75 (57.5) 30

Lower shoot 21/65 (32.3) 0/21 (0) 0

* Root induced tissues / Total infected tissues

** Roots were still attached to the soybean tissue from which the roots were induced

Table 5 Gene confirmation of 2BV and 2AR root clones

rolC, gfp, and gus: PCR confirmation of three foreign genes. HR: Hairy roots phenotypes. GFP and GUS: functional protein confirmation.

2BV rolC HR* gfp GFP gus GUS 2AR rolC HR* gfp GFP gus GUS

(Tzfira and Citovsky, 2006)

Figure 1 Agrobacterium-mediated genetic transformation model.

○¹ Bacterium–plant attachment. ○² -○³ Transmembrane protein complex

induction by plant signals and expression of vir region. ○⁴ ssT-DNA production.

○⁵ -○⁶ -○⁷ ssT-DNA export into host cell, transport through cytoplasm and then into nucleus. ○⁸ Intranuclear transport. ○⁹ T-DNA uncoating. ○¹⁰ Integration.

(A)

(B)

Figure 2 Binary vectors (A) pCAMBIA 1201 (B) pCAMBIA 1302

60 Figure 3 The two A. rhizogenes (2AR) construction

Figure 4 The two binary vector (2BV) construction

Figure 5 Binary vector p-2-Reporter-Geens (p-2RG)

Figure 6 Binary vector p-2-T-DNA (p-2TD)

62 (A)

(B)

Figure 7 Calibration curve of A. rhizogenes OD600 vs. colony number (A) A. rhizogenes 1201 (B) A. rhizogenes 1302

(Utilization factor = total usable tissue fragments per seed planted)

Figure 8 Soybean germination tests. (A) Seeds of 6 genotypes of soybean. (B)

Germination rate, average plant height (from the bottom of lower shoot to the top of cotyledon or leaf), and utilization factor 7 days after sterile plant germination.

Figure 9 Growth of wild-type root clones. Left: growth from day 0 to 21 in 1/2 MS medium with cefotaxime; Center: growth from day 22 to 42 in 1/2 MS medium without cefotaxime; Right: Sugar consumption in the culture medium during cultivation from day 22 to 42.

Figure 10 Growth of 2BV root clones. Left: growth from day 0 to 21 in 1/2 MS medium with cefotaxime; Right: growth from day 22 to 42 in 1/2 MS medium without cefotaxime.

Figure 11 Growth of 2AR root clones. Left: growth from day 0 to 21 in 1/2 MS medium with cefotaxime; Right: growth from day 22 to 42 in 1/2 MS medium without cefotaxime.

*: 400 mg of 2AR-C31-1 was taken for experiments at day 22.

Figure 12 Morphology of transgenic roots (A) lower shoot 0 day after induction; (B) lower shoot 20 days after induction; (C) Upper shoot 20 days after

induction; (D) Root WT-C01-01 after 42 days liquid culture; (E) Root 2BV-C13-2 after 42 days liquid culture; (F) Root 2AR-C05-2 after 42 days liquid culture; (G) Root 2AR-C16-2 after 42 days liquid culture; (H) Root 2AR-C31-1 after 42 days liquid culture.

Figure 13 PCR confirmation of transgenic hairy roots

M: marker; 1: colony PCR of A. rhizogenes 1610 (rolC control ,543 bp); 2:

colony PCR of A. rhizogenes 2BV (gfp control, 833 bp); 3: colony PCR of A. rhizogenes 2BV (gus control, 638 bp); 4: PCR of rolC in genomic DNA from 2AR-C31-1; 5: PCR of gfp in genomic DNA from 2AR-C31-1; 6:

PCR of gus in genomic DNA from 2AR-C31-1

Figure 14 GFP confirmation under fluorescence microscope with Olympus C7070 WZ (aperture: 4.8, shutter: 30 sec, ISO: 80)

(A) wild-type root; (B) Root 2BV-C30-2;

Figure 15 SDS-PAGE analysis of protein extracts from three root clones M: marker; 1: 2AR-C16-2; 2: 2AR-C31-1; 3: 2BV-C16-2

Arrows indicate the putative position of GFP (27 kDa) and GUS (68 kDa)

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