Supplementary Material s15

Supplementary Material

Title: Root and Shoot Parts of Strawberry: Factories for Production of Functional Human Pro- Insulin

Authors: Ashkan Tavizia , Mokhtar Jalali Javaranb1, Ahmad Moienib , Manijeh Mohammadi- Dehcheshmehc , Mehdi Mohebodinid, Esmaeil Ebrahimiee1

aInstitute of Biotechnology, Shiraz University, Shiraz, Iran. bDepartment of Biotechnology, Tarbiat Modares University, Tehran, Iran. cSchool of Agriculture Food and Wine, The University of Adelaide, Adelaide, Australia. dDepartment of Horticulture, University of Mohaghegh Ardabili, Ardabil, Iran. eSchool of Molecular and Biomedical Science, The University of Adelaide, Adelaide, Australia.

Supplementary Materials and Methods

Agrobacterium Strains, Plant Material, Vector and Primers

The first step of human Pro-Insulin gene transformation to strawberry project is optimizing the tissue culture system of this plant for transforming the gene. We used cultivars Paros, Pajaro, 2

Selva, Gaviota, and Camarosa which were received from Tarbiat Modares University and two cultivars Alpine and Sarian Hybrid which had been bought from Australia and Netherlands.

In this research, the bacteria used for cloning pro-insulin gene were E. coli strain of DH5α. This strain was utilized for maintaining and producing the prepared structures. A. tumefaciens, which was used in this study, had the strains LBA4404, AGL1, EHA101, EHA105, C58, C58 (pGV2260), C58 (pGV3101) and A. Rhizogenes had the strains R1000, k599, A4, and MSU440. Apart from K599 which was obtained from Adelaide University, all the strains were received from Tarbiat Modares University. The expression plasmid pCAMBIA1304 with a length of 12.3 kb was used in this study. The expression vector of pCAMBIA1304 harbored the human pro- insulin gene. A resistance gene against kanamycin (NPT II) selected for expression in the bacterium. For inducing hygromycin resistance in plant, the hygromycin resistance gene was used. CaMV35S promoter, NOS terminator, the genes of GUS and GFP (encoding beta- glucuronidase and green florescent protein, respectively) were used in front of the 35S promoter and the cutting sequences for restriction enzymes of Bst EII and NcoI. Human pro-insulin was infused by the DNA sequence of the Immunoglobulin G binding protein A extracted from staphylococcus aureus and then it was cloned. The protein bonding with Immunoglobulin can trigger high expression of the recombinant pro-insulin gene and highly stabilizes the recombinant pro-insulin protein made in the Escherichia coli. Trapped chromatographically in purification processes, this protein can make easier purification of the human pro-insulin bound to itself. The sequence related to human pro-insulin and the one related to fusion protein A were placed at 3´ and 5´ ends respectively (Supplementary Fig. 7d).

Primers

Forward primer (Pro-F code):

5′-CATGCCATGGAAGCGGGATTCAACCAATTTAATAAGGCCATGGCATG-3′

Backward primer (Pro-R code):

5′-CCGGTCACCTCARRAGTTGCAGTAGTTTTCCAG-3′ 3

Human Pro-Insulin Gene Transformation

The Extraction of the Target Vector and Transfer of the Vector which Contains Pro- Insulin gene to Agrobaterium

Using the normal method for thawing and freezing, the pCAMINS structure was transformed into the strains of A. tumafasins and A. rhisogensis. After becoming transgenic, these bacteria were cultured on the medium containing antibiotics Streptomycin and Kanamycin. The grown colony was analyzed by the use of Colony PCR method and the specialized primers of human pro-insulin. Positive colonies containing gene were assigned for gene transformation to plants.

Gene Transformation of Human Pro-Insulin in Strawberry (Fragaria × ananassa Duch.) Via A. tumefaciens

Determining the concentration of antibiotics in the selection medium

Since the efficiency of the production of transgenic plants depends on the careful selection of cells and transgenic tissues, it is necessary to determine the threshold effectiveness of antibiotics. With this end in view, the explants of leaf, petiole, and terminal buds underwent different treatments with the concentrations of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg l-1 of hygromycin. The aim was to find the proper concentration of this antibiotic for the complete prevention of the regeneration of non-transgenic explants from these explants. In this way, the suitable concentration of hygromycin was determined.

The inoculation and transgenic of the explants via A. tumefaciens

A. tumefaciens was raised until reaching OD600 nm= 0.6 – 0.9 for the inoculation of leaf and

Petiole explant and OD600 nm= 1 for the terminal bud explant. In order to enhance its growth, power of pathogenicity, and the percentage of gene transformation, induction culture medium (MS containing 2 % glucose) were used. The explants were cultured in the solid regeneration medium for each explant (pre-culture). To do so, independent experiments were conducted on each of the cultivars; the explants were put in the bottle containing bacterium for 1, 5, 10, 15, 20, and 30 minutes in order to remain floated in the liquid medium containing bacterium. The control explants were only put in the liquid medium without bacterium. They were taken back to solid MS medium and lack any antibiotics (from the pre-culture medium used as a co-culture 4 medium for the simultaneous growth of bacteria and explant.). After two days, the plant explants were removed from the medium and transferred to the selection medium (the regeneration medium with 200 mg l-1 of cefotaxime and hygromycin is used as the selection index of vector pCAMBIA1304 as well as plants’ transgenic factor; also, cefotaxime is added to the medium in order to control the growth of Agrobacterium. Then, the explants were put in an isolated growth chamber applying the temperature 24± 2˚C with a light period of 16 hours of light and 8 hours of darkness. They were transformed into a new culture medium every 14 days. The transgenic direct shoots appear almost after 35 days of inoculation with Agrobaterium. Then the transgenic plants were transferred to flowerpots and put in an isolated greenhouse. In order to eliminate the possibility of gene escaping, each transgenic plant was covered by a bag.

Gene Transformation of Human Pro-insulin in Strawberry via A. rhizogenes

The strains of A. rhizogenes were grown OD600mm= 1.1_ 1.2 and also the MS containing 5 % glucose was used as the inoculation medium. For human pro-insulin gene transformation to hairy roots of strawberry, the petiole explant was used and this experiment was independently conducted for each cultivar. In order to prevent the infection of explants as a result of A. rhizogenes inoculation, every three days, they were washed under laminar air flow by the use of double sterile distilled water for a duration of one minute and were then subcultured. This action prevents the over-growth of bacterium and keeps the bacterium alive on the explant for the production of transgenic hairy roots. Afterwards, in order to have half-mass production as a bioreactor, the hairy roots which were the result of inoculation were cut from the petiole explant and were put inside the flask which contained hormone-free liquid MS medium. They were kept in darkness on the shaker. For preventing bacterium infection due to the existence of A. rhizogenes on the hairy roots, they were washed with sterile distilled water every two days and were subcultured to a new medium. The samples were studied over a period of 25 days. In order to remove the remains of A. rhizogenes 250 mg l-1 of cefotaxime antibiotic was used.

Re-generating the Complete plant of strawberry (Fragaria ×ananassa Duch.) Containing Human Pro-Insulin From Hairy Roots

After the transgenic of the hairy roots was confirmed, re-generation from the transgenic hairy roots was conducted in seven independent experiments. The transgenic root explants were put 5 under the condition of 24± 2 °C with a photo period of 16 hours of light and 8 hours of darkness. Every four weeks, they were sub-cultured to new mediums. Transgenic direct shoots appear approximately after 40 days. The direct shoots were separated from the explants and were transformed to the root-generating medium. Then, regenerated plants transferred to perlite for acclimatization. All the transgenic plants were put in an isolated greenhouse applying the temperature 24± 2˚C with a light period of 16 hours of light and 8 hours of darkness.

Analysis of the Strawberry (Fragaria × ananassa Duch.) Containing Human Pro-Insulin Gene

Analysis at DNA Level

Genomic DNA was extracted from the young leaves and hairy roots of strawberry by the use of CTAB method. To this end, each genome DNA sample extracted from plants and possibly transgenic hairy roots was tested in two concentrations (0.5 and 1) for PCR by the use of specific primers.

Analysis at the RNA Level

RNA was extracted from the young leaves and hairy roots of strawberry as well. RNXTM (-plus) (Sina-Gene Co.) was used to extract total RNA and RT-PCRkit (Fermentas Co.) used for cDNA synthesis. Respective primers mentioned above were also used for cDNA amplification.

Protein Analysis of the Transgenic Plants and Hairy Roots

The total protein must be extracted from plant tissue (leaf, fruit, and hairy root). 200 mg of strawberry plant tissue was used to extract total soluble protein (TSP) . In order to analyze the influence of time and the age of plant tissue on the amount of the human pro-insulin expression, two types of leaf (young and old) and two types of fruit (ripe and completely ripe) were used in four different hours of a day (8:00 AM, 12:00 PM, 6:00 PM, and 12:00 AM).

SDS-PAGE Assays

SDS-PAGE of proteins was done using 14% polyacrylamid gel followed by staining with Coomassie blue [2].

Dot-blot Immunoassay of Human Pro-Insulin 6

As described earlier [3], leaf and hairy root protein extracts were dotted on the nitrocellulose membrane and blocked for 1 hr. at room temperature, (25 °C) in 2 % BSA in PBS-T. To wash the membrane for three times, PBS-T was used and then it was incubated in a 1: 200 dilution of rabbit polyclonal primary antibody (Santa Cruz Biotechnology, Inc.) in PBS-T for 1 hr., washed three times and incubated with 1: 4000 dilutions of anti-rabbit IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Inc.) as a secondary antibody for 1 hr. DAB or 0.05 % 3, 3'-diaminobenzidine tetrahydrochloride and 0.01 % hydrogen peroxide in 50 mMTris (pH 7.5) were used as color development solution .In this method Sodium Azide was applied as a preservative for all buffers. Brown color was observed in dotted areas which were considered a transgenic line.

ELISA Assay

The double antibody sandwich ELISA (DAS ELISA) was carried out as explained by Clark and Adams [4]. ELISA plates (JET BIOFIL) were coated with 100 μl of the dilution of IgG in a carbonate coating buffer (15 mM Na2CO3, 35 mM NaHCO3 and 5 mM NaN3, pH: 9.6) and incubated overnight at 4 °C. The plates were, then, washed four times every five minutes with a washing buffer (PBS: 2.7 mMKCl, 3 mM NaN3, 8 mM Na2HPO4, 1 mM KH2PO4 and 0.13 M NaCl in addition 0.05 % Tween 20). One hundred μl (20 ng) of extracted plant protein was added to each well and incubated in the same fashion (Protein concentration was determined by the Bradford Protein Assay Reagent Kit (Bio-Rad) with BSA as a standard). Incubation of wells was administered with 1: 200 dilution of rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) in PBS-T (1 hr. at 37 °C). PBS-T was used to wash the wells three times then the wells were incubated (1 hr. at 37°C) with a 1:1000 dilution in PBS-T of anti-rabbit IgG alkaline phosphatase conjugate (Sigma A3687). For each well, 100 μl p-nitrophenyl phosphate liquid substrate (Sigma N7653) was used to develop the plates. For halting the reactions, 1N HCL was used. ELISA reader (BioTekElx 800) identified the absorbance at 405 nm. A sample was considered positive (transgenic) if absorbance at 405 nm was greater than or equal to three times the average of negative (non-transgenic) samples.0.5, 1.5, 2.5, 5, 10 and 20 ng μl-1 of purified insulin was added to 6 wells to measure protein expression and their absorbance was used for drawing calibration curve. 7

The average absorption of the holes is compared based on a standard curve which has been designed in advance. By doing so, the protein concentration in each hole is determined. And, based on the total solution protein concentration of each sample, which has been obtained by Bradford reagent, the ration percentage of recombinant protein to the TSP is calculated.

Electrochemiluminescence (ECL) Assay

For doing the Electrochemiluminescence (sandwich) test, the specific insulin kit of ECL was used.

Purifying the Recombinant Human Pro-Insulin Protein from Strawberry Plant Tissues

In this research, protein A infused to the human pro-insulin gene. Protein A with a molecular weight of 56 kDa found in the cell wall of various stains of the bacteria Staphylococcus aureus. With the existence of FC area (Fab Heavy Chain) in the protein structure makes it possible for protein A to bind with immunoglobulin molecules, especially IgG. This important feature is used for purifying immunoglobulin in mammals [7]. In this research, for purifying human pro-insulin fusion A, which is only a proportion of protein A, was used. Fusion A has a weight of about 8 kDa. The reason that the total sequence of protein A was not used was the prediction of purification and avoiding bacterial infection during the bioassay experiment. It had been cloned in the target structure for more stability. The purification was conducted by the use of the kit IgG Sepharose 6 Fast Flow from GE healthcare company and according to the protocol of this company.

Animal Studies to Determine Hormone Activity

For conducting this experiment, adult male Wistar rats with the weight range of 250-300 gr were used. These rats were purchased from the Diabetes Center of Iran and were put in standard conditions in terms of light (12 hrs. of light and 12 hrs. of darkness), free access to water and food, and temperature (20- 22 °C). According to the National Specific Ethical Guidelines for Biomedical Research issued by Ministry of Health and Medicinal Education (MOHME) of Iran in 2005, all animal studies were confirmed by the ethical committee of Diabetes Center of Iran. For making animals diabetic, STZ was injected with a dose of 55 s.c. mg/ kg [6]. STZ by stimulating the production of H2O2 in beta cells, can cause DNA fragmentation and, 8 consequently, destruction of pancreas beta cells . Before STZ injection, by the use of tail prick technique, a blood drop was taken from the animal’s tail. By the use of Pars Azmoon kit, fasting blood sugar, cholesterol, triglyceride, HDL, and LDL were measured. Three days after STZ injection, the animals in which the amount of blood sugar was over 300 mg/ dl and which showed symptoms of weight loss (50- 80 gr), polydipsia, and polyuria were considered diabetic.

Analysis of Strawberry Plants of the Second Generation (T1) Containing Human Pro- Insulin Gene

The obtained seeds from transgenic plants and those of non-transgenic ones (as control) were cultured in this process, the best treatment (i.e. 36 N sulfuric acid from Razi petro chemistry

-1 -1 -1 company), 2 mg l GA3, and ½ MS medium including 15 gr l of Sucrose, 5 gr l of agar with PH=5.7 with the antibiotic hygromycin with concentrations of 20, 40, and 60 mg l-1 were used. The seeds were cultured on a medium that contained hygromycin and by observing the plants which were able to germinate; we could distinguish transgenic plants from non-transgenic ones.

Analysis of the Generation T1 Transgenic Plants at the DNA Level For PCR with specific primers of the human pro-insulin gene, several genomic DNA samples were used which were taken from the obtained plants of the selective media. Both Quality and quantity of these samples were suitable.

Analysis of the Generation T1 Transgenic Plants at the Protein Level

Those plants in which the presence of pro-insulin gene was confirmed by PCR were used to analyze the expression at the level of protein. First, protein was extracted through the method of Guy (1992) in four times of 8:00 AM, 12: PM, 6:00 PM, and 12:00 AM . Then, the presence of pro-insulin recombinant protein was evaluated in the plants of generation T1 by the use of Dot blot, ELISA, and ECL.

Statistical Analysis

Normality tests for data were assessed using Minitab 16 and SPSS 18, and Minitab 16 and was used for conducting statistical analyses of the experiment. The P value of 5 % was used as the criterion for comparing variances and means of treatments. The diagram related to the data was drawn using Excel 2010. As a result, the following experiments were conducted: 9

Gene Transformation via A. tumefaciens

For doing so, independent experiments with a factorial design and a completely random base with five replications were established on each of the cultivars. Then, in order to find the best cultivar in terms of gene transformation percentage, the effect size for each cultivar was determined. The studied factors were the strain of A. tumefaciens in seven surfaces and three types of explants (leaf, petiole, and terminal bud). The studied feature was the gene transformation percentage measured on the basis of the regeneration of the explant on the selection medium.

Gene Transformation via A. rhizogenes

For gene transformation the petiole explant was used. This experiment was independently established for each cultivar based on a completely random design with five replications. Then, in order to find the best cultivar for the production of hairy roots, slicing interactions were established on the basic design of the cultivars. The studied factor was the strain of A. rhizogenes in four surfaces and the evaluated feature was the percentage of hairy roots production. After that the transgenic of the hairy roots were confirmed, regeneration from the transgenic hairy roots was established in seven independent experiments (one for each cultivar). The studied factors included type of tissue culture medium (MS, ½ MS (Merk company)) and growth hormones (BAP and TDZ cytokines (0.5, 1, 1.5, 2 mg l-1 ), auxins (IAA, IBA, and NAA(0.1, 0.2, 0.5, 0.7, 1 mg l-1) (Duchefa company), likewise, with and without the presence of vitamin B5 in the tissue culture medium. The evaluated variable was regeneration of the complete plant of strawberry from the transgenic hairy roots.

Bioassay

For doing this test, factorial experiments with a completely random design with 6 treatments were conducted. The studied factors were as follows:

1. The type of plant tissue from which pro-insulin has been purified: a. Transgenic plants’ leaves which were the result of A. tumefaciens inoculation. b. Transgenic fruit. 10 c. Transgenic hairy roots which were the result of A. rhizogenes inoculation. d. Transgenic plants’ leaves which were the result of regeneration of transgenic hairy roots.

2. The injected concentration of purified human pro-insulin recombinant protein in three levels (20, 40, and 60 ng).

3. Time of blood sampling after injection (1, 3, 8, 12, and 24 hrs.).

Also, considerations for control treatments were as follows: a. Injection of the recombinant human insulin, neutral protamine Hagedorn (NPH) (commercial), with a concentration of 38.46 ng. b. Injection of ddH2O. c. Injection of protein A for investigating its lack of influence on decreasing blood sugar (three concentrations were considered for injection: 20, 40, and 60 ng) d. Injection of the produced pro-insulin to healthy rat in three concentrations: 20, 40, and 60 ng.

And the studied features were glucose, cholesterol, triglyceride, high-concentration lipoprotein (HDL-C), and low-concentration lipoprotein (LDL-C).

Supplementary References

1. Guy C, Haskell D, Neven L, Klein P, Smelser C (1992) Hydration-state-responsive proteins link cold and drought stress in spinach. Planta 188:265-270

2. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685

3. Banttari E, Goodwin P (1985) Detection of potato viruses S, X and Y by enzyme-linked immunosorbent assay on nitrocellulose membranes (Dot-ELISA). Plant Disease 69:202-205 11

4. Clark MF, Adams A (1977) Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of general Virology 34:475-483

5. Coskun O, Ocakci A, Bayraktaroglu T, Kanter M (2004) Exercise training prevents and protects streptozotocin-induced oxidative stress and β-cell damage in rat pancreas. The Tohoku journal of experimental medicine 203:145-154

6. Hajduch E, Darakhshan F, Hundal H (1998) Fructose uptake in rat adipocytes: GLUT5 expression and the effects of streptozotocin-induced diabetes. Diabetologia 41:821-828

7. Strambio-de-Castillia C, Tetenbaum-Novatt J, Imai BS, Chait BT, Rout MP (2005) A method for the rapid and efficient elution of native affinity-purified protein A tagged complexes. Journal of Proteome Research 4:2250-2256

Supplementary Figure Captions

Supplementary Fig. S1. Human Pro-INS gene transformation to strawberry via A. tumefaciens. a The strawberry inoculation leaves via A. tumefaciens: three weeks after the inoculation, the leaf cells which have died and the tissue which have become brown and the transgenic direct shoots of strawberry which have been lost on the leaves and the surrounding tissue as a result of antibiotic. b The possibly transgenic rooted plantlet which is resistant to hygromycin antibiotics. c The terminal bud which has been inoculated via A. tumefaciens. d The possible transgenic plant of strawberry transformed to the vase. e Hybrid transgenic plant of strawberry. f-g The possible transgenic plant of strawberry in the flowering stages. h-i The possible transgenic plant of strawberry in fruiting stages.

Supplementary Fig. S2. a The hairy roots which were the result of A. rhizogenes inoculation. b Reproduction the hairy roots which were the result of A. rhizogenes inoculation, after 5 days. c 15 days. d 25 days. e The diagram of the regeneration percentage of 7 cultivars of strawberry in different culture mediums of auxins and cytokinins in the MS or ½ MS medium and presence or 12

absence of B5 vitamins in the medium (there are 145 medium compounds and only those medium compounds to which cultivars responded within the compound limitation of have been drawn here). f Regeneration of the complete plant of strawberry from transgenic hairy roots. g Comparison of the transgenic plants obtained by Agrobacterium-mediated transformation. Transgenic hybrid plants obtained from A. tumefaciens inoculation. h Transgenic plant obtained from the regeneration of transgenic hairy roots. i Transgenic plant obtained from A. tumefaciens inoculation (Gaviota cultivar).

Supplementary Fig. S3. a The blood sugar level in diabetic rat after injection of protein A. b The diagram of comparison of the blood sugar level after the injection of purified pro-insulin (20, 40, and 60 ng concentration), NPH, and ddH2O (as controls). c The blood sugar level in healthy rats after injecting a compound of pro-insulin purified from transgenic hairy root, leaf, fruit, and regenerated leaf.

Supplementary Fig. S4. Change in the amount of cholesterol, triglyceride, HDL, and LDL after injection of protein A to diabetic rats

Supplementary Fig. S5. Change in the amount of cholesterol, triglyceride, HDL, and LDL after injection of purified pro-insulin (Pro-Insulin which has been purified from transgenic hairy root, leaf, fruit, and regenerated leaf) to healthy rats

Supplementary Fig. S6. The diagram of the comparison of the amount of cholesterol; triglyceride; HDL; and LDL after the injection of purified pro-insulin (20, 40, and 60 ng concentrations), NPH, and ddH2 to diabetic rats 13

Supplementary Fig. S7. a PCR analysis of transgenic plants (T1) and the quality of the extracted DNA. M: 1 kb marker, Lane 1: ddH2O, Lane 2: positive control. Lane 3-7: transgenic plants. Lane8: wild type plant. Lane 9-12: samples of extracted DNA. b Dot blot of transgenic plants of the second generation. WT )wild-type plant(, C+ )positive control(, T1 (transgenic line). c ELISA test for confirming the expression of human pro-insulin gene of T1. d Genes inside the T-DNA surrounded by left and right borders were inserted into the plant genome and contain: CaMV35S, CaMV35S promoter; Pro A+ INS, Protein A-Pro-insulin fusion; HYG(R), hygromycin phosphotransferase; NOS, nopalin synthase terminator; MCS, Multiple cloning site

Supplementary Tables:

Supplementary Table S1. Determination of the lethal dose of hygromycin for selection

Concentration of antibiotics To reaction of the explant to the concentration of Cultivar Explant Hygromycin (mg/1-1) antibiotics 15 The halt of the regeneration process Leaf and petiole Camarosa 35 The loss of the explant Terminal bud 70 The loss of the explant 20 The halt of the regeneration process Leaf and petiole Gaviota 35 The loss of the explant Terminal bud 75 The loss of the explant 15 The halt of the regeneration process Leaf and petiole Sleva 40 The loss of the explant Terminal bud 80 The loss of the explant 20 The halt of the regeneration process Leaf and petiole Paros 40 The loss of the explant Terminal bud 80 The loss of the explant

20 The halt of the regeneration process Leaf and petiole Pajaro 45 The loss of the explant Terminal bud 90 The loss of the explant

20 The halt of the regeneration process Leaf and petiole Apline 50 The loss of the explant Terminal bud 100 The loss of the explant

12 The halt of the regeneration process Leaf and petiole Sarian 30 The loss of the explant Terminal bud 65 The loss of the explant

Supplementary Table S2. Correlation between traits of blood after human pro-insulin injection (purification from four types of plant tissues) 14

Blood sugar Cholesterol Triglyceride HDL-C LDL-C

Cholesterol 0.801 ** Triglyceride 0.064 0.039 HDL-C -0.865 ** -0.859 ** -0.094 LDL-C 0.854 ** 0.747 ** 0.097 -0.854 ** weight 0.069 0.065 0.016 -0.067 0.064