Overview

This year, SZU-China has established a comprehensive and systematic prevention and control strategy of " prevention-detection-treatment" for rice sheath blight. In order to support our design, we conducted many experiments and finally got experimental results, which were sufficient to support our entire design. In the following, we will introduce our results in three modules: prevention, detection and treatment.

Prevention

Trichoderma atroviride transformation

To prevent Rice Sheath Blight, we choose Trichoderma atroviride as our chassis to overexpress Epl1, a T.atroviride derived eliciting plant response-like protein 1, which is also a hydrophobic protein and Prb 1, a type of serine protease playing great importance in mycoparasitism. Otherwise, we express Snakin 1, a potato derived cysteine-rich antimicrobial peptide inhibiting the growth of a variety of plant pathogens, especially R.solani. To achieve our goal, we first need to overproduce the three plasmids and then transfer them into T.atroviride for further expression and function verification.

1.Overproduction of three plasmids

Since we needed to transfer the plasmids into Trichoderma, which would require a large number of plasmids, we transferred pCAMBIA1302 recombinant plasmids with Epl 1, Prb 1, and Snakin 1 into E.coli DH5a, to amplify them in large quantities, thus obtaining a constant and large number of plasmids.

After transforming the recombinant pCAMBIA1302 plasmid into DH5a competent cells, the recombinants were screened by the kana resistance gene on the plasmid. Subsequently, we first performed colony PCR on the isolated colonies and selected the successfully transformed isolated colonies for simple amplification with the extracted plasmids. Then we verified them by PCR and double digestion. We designed three pairs of primers with theoretical PCR fragment sizes of Epl 1-565bp, Prb 1-1388bp, and Snakin 1-425bp, respectively. The PCR results of three plasmids are shown in Figure 1-1, and all the selected plasmids were in expected positions, consistent with the positions of the positive control.

Figure 1-1. 1% agarose gel stained with Epl 1, Prb 1, Snakin 1 intergration of pCAMCIA1302 in E.coli was checked by PCR . Agarose gel was used to validate the pcr results. This figure shows PCR results of Epl 1, Prb 1, and Snakin 1, respectively. E+, P+, S+ are positive controls for Epl 1, Prb 1, and Snakin 1(positive clone is the synthetic plasmid). The capitalized word with a number represents the sample we choose.

In the double digestion verification, we used EcoRI and Bgl II enzymes to cut the plasmid into two segments, the longer segment was 9729bp, and the shorter segments were (Epl 1) 1252bp, (Prb 1) 2065bp, and (Snakin 1) 1102bp, but after the PCR test when digested, we found that the plasmid band of Snakin 1 was always at a lower position. As shown in the electrophoresis diagram of Prb 1 plasmid in Figure 1-2, the lower plasmid is in the superhelical state, followed by a band in the target position, which is probably the linear band of the plasmid. Therefore, we decided to single-cleave our Snakin 1 plasmid at the same time. However, this time the obtained agorase gel results showed that the plasmid of Snakin 1 was in the position of the theoretical linear plasmid. The results are shown in Figure 1-2.

Figure 1-2. 1% agarose gel stained with epl 1, prb 1, snakin 1 intergration of pCAMCIA1302 in E.coli was checked by enzyme digestion. (A) The results of double digestion of Epl 1 plasmid. E5-0 and E6-0 are controls that were not treated with enzymes. E5-2 and E6-2 represent the results of double digestion of E5 and E6. (B) The results of double digestion of Prb 1 plasmid. P5-0 and P6-0 are controls that were not treated with enzymes. P5-2 and P6-2 represent the results of double digestion of P5 and P6. (C) The results of double digestion of Snakin 1 plasmid. S0 is control ,S1 is the result of single enzyme digestion and S2 is double digestion of Snakin 1.

These results show that the selected separated colonies are positive and we then amplified and cultured these bacteria, and then extracted the plasmids in bulk for subsequent transformation of Trichoderma.

2. OD550nm-spore count curve

When attempting transfermation of Trichoderma, we found that among other things, the quantification of Trichiderma material took a large amount of time. Because the number of spores required for transformation of Trichoderma is `10^7` spores/ml and we need to try a lot of times to succeed transforming Trichoderma, it is necessary to count the spores. Traditionally, spore counting is done by hematocrit, which adds a little more work to the already tedious task of transforming Trichodrma. At this point we thought of the instrument used in the laboratory to measure concentrations: a spectrophotometer. We then wondered if we could use a spectrophotometer to make measurements of the spore count of Trichoderma. After reviewing papers, we found that the idea was feasible and that the spores of Trichoderma atroviride were suitable at OD550 as the absorption nanometer value for counting, so we decided to make this spore counting curve. We measured the absorbance values at OD550 nm of the spores at each gradient concentration after counting them with a hemocytometer plate, and each concentration was repeated three times. Then, after processing, we obtained the following relevant equations:

In this equation, `y` represents the value of OD550, `X`represents the concentration of T.a spore(`10^7` per ml)

3.Genetic transformation of Trichoderma

To transfer recombinant plasmids into Trichoderma, we first tried nanomaterials-mediated transformation as well as using cell penetrating peptides to transfer the plasmids, but neither of them succeeded. After that, we tried a more traditional way protoplasted-mediated transformation. However, this CaCl2-PEG induction method didn't work. All of these methods and tries can be viewed in Protocol and Notebook. Finally, we decided to use Agrobacterium-mediated transformation (AMT).

We first transferred the three recombinant plasmids into agrobacterium GV3101 and these were screened by Kanamycin and colony PCR.

Figure 1-3. Agarose gel stained with epl 1, prb 1, snakin 1 integration of pCAMCIA1302 in GV3101 was checked by colony PCR. (A) E1 and E2 represent the isolated Epl 1 transformed GV3101 colony. Their were in expected positions. (B) P1 and P2 represent the isolated Prb 1 transformed GV3101 colony. Their were in expected positions. (C) S1 and S2 represent the isolated Snakin 1 transformed GV3101 colony. Their were in expected positions.

These gel results showed that the recombinant plasmids had already been transformed into agrobacterium GV3101 correctly.

Then we used positive agrobacterium GV3101 to transform T.atroviride. After several attempts and having got advice from our PI, we finally obtained the transformed T.atroviride. We selected the recombinant T.atroviride by 50ug/ml Hygromycin-B and PCR after extracting its genome. Each potential transformant was selected by 50ug/ml Hygromycin-B 4 times in case of unstable genetic inheritance caused by gene fragment inserting in cytoplasmic genome.

Figure 1-4. Agarose gel stained with epl 1, prb 1, snakin 1 integration of T.atroviride genome was checked by PCR. (A) 1% agarose gel stained with Prb 1 integration PCR results. P+ is positive control which is Prb 1 plasmid checked by PCR and P1 and P2 represent recombinants we chose. It is shown on the picture that P2 is consist with P+, which means P2 is positive.(B) 2% agarose gel stained with Snakin 1 integration PCR results. S+ is positive control which is Snakin 1 plasmid checked by PCR and S1 represents recombinant we chose.

According to our PCR results, we can initially confirm that we have transformed Prb 1 and Snakin 1 into T.atroviride successfully. Epl 1 transformant failed to grow up in the second time of selecting.

4.SDS-PAGE

After verifying that our plasmid was successfully transferred into T. atroviride, we need to further verify its expression activity in T. atroviride. We first used PRB 1 transformants as an example, Prb-1 transformants were expanded in Mini medium, and R. solani fungus powder obtained by grinding in liquid nitrogen was used as an inducer to induce Prb-1 expression in T.atroviride. After two days of culture, we extracted the whole protein of T.atroviride using a fungal protein extraction kit and verified it by SDS-PAGE. As shown in Figure 1-5, lane 1 is the protein extract of the Prb 1 transformant that was successfully transferred into the plasmid, lane 3 is the protein extract of the wild-type T.atroviride, and the total protein concentration was adjusted to the same for both groups of samples during loading. It can be seen that they have a relatively obvious band at the position of about 42.3kD, and from the perspective of depth, the band corresponding to the Prb-1 transformant is darker than that of the wild type, that is, the content is higher.

Figure 1-5. Result of SDS-PAGE（The picture on the right is the result of grayscale processing of the picture on the left to facilitate observation and comparison）

Due to the difficulty of selecting and purchasing antibodies derived from fungi, we did not use Western-blot for further verification. Instead, we determined the alkaline protease activity of the prb-1 transformants and compared it with that of the wild-type extract. The results of this experiment can be seen on the Proof of Concept page.

5. Suicide switch

5.1 Plasmid production and preliminary validation

We constructed the recombinant vector [Pcdh1-cdh1ls-MazEF-Tcdh1]-pCAMBIA1302. In order to produce the recombinant vector in larger scale for subsequent experiments, we transferred the recombinant vector into E.coli DH5`\alpha`. Transformants were clearly visible on the culture medium after 16 hours of incubation at 37^o C.

Figure 1-6. Transformation result.

Figure 1-7. 1% agarose gel stained with suicide switch (Pcdh1-cdh1ls-MazEF-Tcdh1) integration of pCAMCIA1302 in E.coli DH5`\alpha` was checked by PCR. The capitalized word with a number represents the sample we choose.

We further cultured the transformants and extracted the plasmids from them. Restriction enzyme XhoI was used for single digestion. There are two restriction sites of XhoI on the plasmid [Pcdh1-cdh1ls-MazEF-Tcdh1]-pCAMBIA1302, the theoretical sizes of bands after digestion are 1094bp and 10268bp. Electrophoresis was performed in a 1% agarose gel. The results showed successful single-enzyme digestion and correct plasmid extraction. However, a band of 1094bp size was not shown because a few plasmids were simultaneously cleaved twice by the enzyme Xhol.

Figure 1-8. 1% agarose gel stained with suicide switch (Pcdh1-cdh1ls-MazEF-Tcdh1) integration of pCAMCIA1302 in E.coli DH5`\alpha` was checked by single restriction enzyme digestion. M: 10000bp Marker. SS1: the plasmid [Pcdh1-cdh1ls-MazEF-Tcdh1]-pCAMBIA1302, which showed closed circular plasmid DNA (cc DNA) and open circular plasmid DNA (oc DNA). SS2: plasmid digested by restriction enzyme XhoI, which showed linear plasmid DNA (Linear DNA).

5.2 Genetic transformation of Trichoderma atroviride

To transfer recombinant plasmids into Trichoderma, we used Agrobacterium-mediated transformation (AMT) to integrate the circuit of Trichoderma suicide switch (on T-DNA fragment of plasmid pCAMBIA1302) into the genome of Trichoderma atroviride. Then we used positive Agrobacterium GV3101 to transform T.atroviride. We selected the recombinant T.atroviride by 50ug/ml Hygromycin B and PCR after extracting its genome. Unfortunately, due to lack of time, we did not carry out the subsequent characterization experiments.

6. Induction of Trichoderma atroviride conidiation

In order to explore the optimal conditions for inducing Trichoderma atroviride conidiation in commercial production, and to provide more spores for our subsequent experiments of engineered Trichoderma and TACE（Trichoderma’s acest carrier ever）, we conducted corresponding literature research and experimental exploration. Through literature, we found the following ways to induce Trichoderma conidiation:

1. During the growth of Trichoderma, white fluorescent lamps with light intensity of 600 LX were irradiated for a short time.
2. Cultivate Trichoderma in the dark for 1~2 days, which makes them grow more rapidly, and then expose them to white light to induce conidiation.
3. Mechanical cutting of Trichoderma on the culture medium causes mechanical injury to the mycelia, thus accelerating conidiation.

The above are general methods of Trichoderma SPP. For Trichoderma atroviride, it is necessary to try these methods to explore the best method for conidiation induction. Therefore, we set up the following groups to induce Trichoderma atroviride conidiation with different treatments.

Table 1-1. Experimental groups

The sporulation rate and sporulation quantity of each group were different by different treatments.

1. Light induction: Compared with groups A5/A6 and A1/A2/A3/A4, spores could be observed on the 4th day of consistent exposure to white light. Compared with A1/A2 and A3/A4, it had no effect on conidiation through short-term exposure of strong white light during culture in dark. Consistent exposure to white light is the best choice for conidiation.
2. Injury induction: Compared with groups A1/A3/A5 and A2/A4/A6, mechanical damage significantly promoted conidiation, and most spores were produced along cracks. Mechanical damage is more conducive to conidiation.
3. Conidiation in different inoculation methods: compared with group A and group B, conidiation is more significant in the groups using germinated spore solution to inoculate. In addition, it was easier to observe the growth of fungus in the groups which were inoculated by Trichoderma clumps.

Figure 1-9. Morphological changes of Trichoderma atroviride in groups A1, B1, A2 and B2 within 4 to 10 days.

Figure 1-10. Morphological changes of Trichoderma atroviride in groups A3, B3, A4 and B4 within 4 to 10 days.

Figure 1-11. Morphological changes of Trichoderma atroviride in groups A5, B5, A6 and B6 within 4 to 10 days.

Figure 1-12. Number of eluted spores in group A and group B on day 10.

Detection

LAMP-LFD detection system

1. LAMP Amplification

1.1 DNA extraction of Rhizoctonia solani (Evaluation of common laboratory extraction methods)

We compared different labortory methods including crude extraction, protocols of three easy methods, CTAB protocol, TPS protocol, which were used to extract DNA from R.solani in our experiments. Fungal universal primers ITS5, ITS4 and ITS6 and ITS4 were used to amplify ITS sequences of R.solani, respectively (Fig. 2-1A, 2-1B), and each amplification product was running in 3% agarose gel. Using each method, R.solani ITS sequences (650~750bp) were amplified, with bands in the expected position (Fig. 2-1). Protocols of three easy methods could be used to extract DNA rapidly. The corresponding bands of methods III and IV were bright. CTAB and TPS bands were brighter. However, crude extraction is not ideal compared with other methods. ITS sequences of R.solani are unable to be amplified in some samples (Fig. 2-1C).

Figure 2-1. ITS sequence of R.solani AG-3 DNA samples extracted by different laboratory extraction methods. (A). DNA templates amplified by fungal universal primers ITS5 & ITS4. (B). DNA templates amplified by fungal universal primers ITS6 & ITS4. (C). DNA templates amplified by fungal universal primers ITS6 & ITS4. EMII: DNA extracted by easy method II. EMIII: DNA extracted by easy method III. EMIV: DNA extracted by easy method IV. CTAB1, CTAB2: DNA extracted by CTAB protocol. TPS1, TPS2, TPS3, TPS4, TPS5: DNA extracted by TPS protocol. CE1~CE5: DNA extracted by crude extraction. +CE control: a positive control group of DNA extracted by crude extraction.

1.2 ITS sequence of R.solani and related PCR system

We use the following universal primers and related PCR system for fungi to amplify ITS sequences of R.solani though PCR reaction (Table 2-1). In our experiment, primers pair ITS1 & ITS4 was more suitable for R.solani AG-1, primers pairs ITS5 & ITS4 and ITS6 & ITS4 were more suitable for R.solani AG-3 (Figure 2-3,2-4).

Table 2-1. Primers for R.solani ITS sequences in PCR reaction

1.3 LAMP system

LAMP primers

We use the following primers to amplify ITS sequences of R.solani though LAMP reaction (Table 2-2).

Table 2-2. Primers for R.solani ITS sequences in LAMP reaction

Optimization of Mg(2+) concentration

The effect of LAMP amplification reaction was affected by Mg(2+) concentration. We set a Mg(2+) concentration gradient (5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM), selected R.solani AG-3 genome DNA as templates, and used primers (AG-3: RSAGF3, RSAGB3, RSAG-3FIP, RSAGBIP) for LAMP reaction. The amplification was carried out at 65^o C for 60 min. Each amplification product was running in 1.3% agarose gel (Fig. 2-2). The bands were brightest at a Mg(2+) concentration of 7 mM (Fig. 2-2B). DNA extracted by each method was successfully amplified to present a ladder-like band within this concentration of Mg(2+). Therefore, LAMP Reactions were performed with 7 mM Mg(2+) in subsequent experiments.

Figure 2-2. Optimization of Mg(2+) concentration. (A). DNA LAMP amplification products with Mg(2+) concentration of 5mM (left) and 6mM (left). (B). DNA LAMP amplification products with Mg(2+) concentration of 7mM and 8mM. (C). DNA LAMP amplification products with Mg(2+) concentration of 9mM and 10mM. Use Rhizoctonia solani AG-3 genome DNA as template. II: DNA extracted by easy method II. III: DNA extracted by easy method III. IV: DNA extracted by easy method IV. CTAB1, CTAB2: DNA extracted by CTAB protocol. TPS1, TPS2: DNA extracted by TPS protocol. +Control: An amplification product that is able to run out of the correct band. -Control: sterilized water was used instead of the DNA template.

1.4 Detection limit and sensitivity of LAMP protocol

We used R.solani AG-3 and AG-1 DNA extracted by different methods as templates, each template was serially diluted in sterile water to amplify its ITS sequence. For PCR reaction, we used primers (AG-3: ITS6 and ITS4; AG-1:ITS1 and ITS4) as well as corresponding reaction temperatures. For LAMP reaction, We used primers (AG-3: RSAGF3, RSAGB3, RSAG-3FIP, RSAGBIP; AG-1:RSAGF3, RSAGB3, RSAG-1FIP, RSAGBIP) with Mg(2+) concentration of 7mM and amplified for 60 mins at 65 ^o C.

Table 2-3. Dilution gradient of DNA samples extracted by different methods and the quality of DNA in the amplification reaction.

Figure 2-3. Detection limit and sensitivity of PCR protocol. A. DNA extracted by easy method III. B. DNA extracted by easy method IV. C. DNA extracted by CTAB protocol. D. DNA extracted by TPS protocol. Use Rhizoctonia solani AG-3 genome DNA as template. The number above the loading well indicates the order of magnitude of the corresponding DNA template content in nanograms.

Figure 2-4. Detection limit and sensitivity of PCR protocol. Use DNA extracted by TPS protocol. Use Rhizoctonia solani AG-1 genome DNA as template. The number above the loading well indicates the order of magnitude of the corresponding DNA template content in nanograms.

Figure 2-5. Detection limit and sensitivity of LAMP protocol. A. DNA extracted by easy method and easy method IV. B. DNA extracted by CTAB protocol and TPS protocol. C. DNA extracted using the four methods, corresponding to the lowest dilution. Use Rhizoctonia solani AG-3 genome DNA as template. The number above the loading well indicates the order of magnitude of the corresponding DNA template content in nanograms.

Figure 2-6. Detection limit and sensitivity of LAMP protocol. DNA extracted by CTAB protocol and TPS protocol. Use Rhizoctonia solani AG-1 genome DNA as template. The number above the loading well indicates the order of magnitude of the corresponding DNA template content in nanograms.

For more information about the LAMP system and implementation of LAMP in LFD for the detection of R.solani, please view more details on Proof of Concept.

Treatment

RNAi therapy

1. siRNA stage

After deciding to use RNAi technology to biocide Rhizoctonia solani, we need to consider which small RNA(sRNA) molecules can trigger the RNAi process. We first noticed that siRNA molecules can be double stranded RNA (dsRNA) cleaved by RNAse III (such as Dicer) in cells into 21~25bp double stranded RNA.

siRNA fragments are loaded into RISC, which consists of Argonaute-2, Dicer and TRBP proteins. Then the two siRNA chains are separated, one of which leaves the complex. The RISC complex carrying the antisense chain recognizes the target mRNA, and finally completes the mRNA cleavage and degradation. Due to its extensive clinical application and mature commercial system, we hope to realize our RNAi process through siRNA molecules.

So we found the first target gene, PG, which plays a key role in the process of starting infection of Rhizoctonia solani. According to the sequence of this target gene, we designed two different siRNA molecules (Table 3-1) and sent the sequence information to biological company for chemical synthesis. Based on the chemically synthesized molecules, we can preliminarily verify whether RNAi is feasible in the treatment of rice sheath blight.

Table 3-1. siRNA information targeting the PG gene of Rhizoctonia solani

2. shRNA stage

Because of the high cost of siRNA synthesis and the poor effect of silencing target genes, we hope to use fermentation technology to produce sRNA molecules in large quantities. We chose Escherichia coli, which is commonly used in fermentation engineering, to produce our sRNA molecules. In our expectation, sRNA molecules will be sprayed in farmland to play a role after production. The environmental factors of farmland are complex, and our sRNA needs to ensure its stable existence in the natural environment to ensure its continuous function. Therefore, we focus on shRNA, which is more stable than siRNA, in the selection of sRNA molecules, and expect it to play a more stable and effective silent role。

2.1 Selection of shRNA Molecular Target Genes

After screening, we finally selected several target genes as the silencing targets of our RNAi molecules. The information of these target genes is shown in Table 3-2. PG, RPMK1-1 and RPMK1-2 are key genes of Rhizoctonia solani in the process of infecting rice. Cohesin complex subunit Psm1 (Psm1), Importin beta-1 subunit(`\beta`-1), RNA pol III subunit C6 (C6) and Ubiquitin ligase E3 (E3) are essential genes for the survival of Rhizoctonia solani.

Table 3-2.Target genes of shRNA and their target sequence

After selecting the above target genes, we designed the corresponding shRNA molecule through the CDS sequence of the target gene. After BLAST and confirmation of its specificity in the total nucleic acid library, we assembled it through the sequence of XbaI restriction site - RNAi fragment - loop - reverse complementary fragment of RNAi fragment - XhoI restriction site, and connected it to the pET-28a (+) plasmid to obtain our shRNA expression vector.

2.2 IPTG induced production

We transformed the constructed shRNA expression vector into E. coli HT115 (DE3), and PCR after extracting the plasmid. Our specific primer can amplify a 260 bp band on the plasmid, and the results verify the successful transformation of the plasmid (Fig. 3-1).

Figure 3-1 Agarose gel electrophoresis after plasmid PCR
1-7: Plasmids extracted from Escherichia coli; 8: Plasmid control; M：DL500 marker
After IPTG induction, its RNA was extracted by Trizol method, and the results in Figure 2 were obtained after electrophoresis.

Figure 3-2 Electrophoresis of RNA extracted from HT115 (DE3)

It can be seen from the figure 3-2 that there is a brighter band between 50-100 bp in the induced sample lane than in the uninduced sample. The size of our shRNA is 59 bp, which proves that our shRNA extraction is successful and the shRNA produced meets the expected size.

2.3 shRNA can be absorbed by R.solani

After inoculated with R.solani, its hyphae will be on the leaves and invade the leaves. We can observe the extension of hyphae in leaves under different times of microscope (Fig. 3-3).

Figure 3-3 Infection of hyphae in leaves.
(a) The mycelium is in the leaf (40×). (b) The mycelium is in the leaf(100×). (c) The mycelium is in the leaf (400×).

The infected leaves were sprayed with Cy5 dye labeled gfp-shRNA and observed under laser confocal microscope. As shown in Figure 3-4, shRNA can effectively enter the mycelia during the process of infection of leaves by mycelia, which provides basic evidence for shRNA silencing in R.solani.

Figure 3-4 Adding GFP shRNA of Cy5 labeled bundled CNT to R.solani on leaves

3. Nanomaterials-shRNA stage

However, the instability of shRNA in the field environment cannot make our products play a better role. After understanding our expectations, our PI proposed that we could try to bind shRNA with nanomaterials for spraying, and provided CNT and LDH nanomaterials and their fabrication methods.

3.1 shRNA can enter R.solani after binding CNT

Scratch micro mycelia on the plate, add the gfp shRNA labeled with Cy5 dye after binding CNT to the mycelia, and observe the distribution of shRNA in the mycelia through laser confocal microscope after treatment. The results showed that shRNA could also effectively enter the cells of Rhizoctonia solani (Fig. 3-5).

Figure 3-5. Adding GFP shRNA of Cy5 labeled bundled CNT to R.solani

3.2 Research on LDH binding conditions

At the same time, in order to explore the optimal conditions for binding with LDH, we explored the effect of co incubation with LDH at different ratios, and confirmed the reasonable packing concentration by setting gradient in the range of 1:1-1:10. The binding mode of LDH and DNA/RNA is mainly electrostatic binding. The interaction between the two and the large molecular volume of LDH cause it to stay at the sample hole on the gel, resulting in the RNA bound with it to stay here but enter the edge gel. Therefore, a brighter fluorescence will appear around the sample hole. From the results in Figure 3-6, the groups wrapped in 1:1 and 1:3 ratios are not complete, and there are still obvious bands in the target region. Due to insufficient LDH concentration, some RNA cannot bind to LDH, so bright fluorescence appears at the target band. It can be inferred that the concentration of complete encapsulation is between 1:3 and 1:5.

Figure 3-6. Research on binding conditions with LDH

Due to the limited time, we only sprayed shRNA binding with CNT, but did not carry out the verification experiment of shRNA after binding LDH. In the future, we plan to complete this part and compare the silencing effect of shRNA after binding LDH and CNT respectively.

For more information about the verification of siRNA, shRNA, nanomaterials-shRNA in the RNAi process, please view more details on Proof of Concept.

4. R-body

4.1 Plasmid transformation and preliminary validation

We constructed the recombinant vectors [RebABDC]-pRSFDuet1 and [RebBAC]-pRSFDuet1, through which we compared the R-body production between the part of 2019 SZU-China and our improved part. We transferred the recombinant vector into E.coli Ht115(DE3). Transformants were clearly visible on the culture medium after 16 hours of incubation at 37^o C.

Figure 3-7. Colonial morphology of E.coli transformants (left: [RebABDC]-pRSFDuet1 transformants; right: [RebBAC]-pRSFDuet1 transformants).

We selected 8 single colonies on each culture medium and carried out colony PCR for plasmid amplification. The theoretical length of the amplified product was 1730bp (RebABDC) and 1340bp (RebBAC). Electrophoresis was performed in a 1% agarose gel. The results showed that all the colonies were positive transformants, which indicated that the recombinant vector was successfully transformed

Figure 3-8. 1% agarose gel stained with RebABDC and RebBAC integration of pRSFDuet1 in E.coli Ht115(DE3) was checked by PCR. The capitalized word with a number represents the sample we choose. M: 2000bp Marker. RA1~8: RebABDC transformants. RB1~8: RebBAC transformants.

After enlarged production of recombinant plasmids, we conducted double restriction enzyme digestion for further verification. Restriction enzyme NdeI & HindIII was used for digestion. For [RebABDC]-pRSFDuet1, the theoretical sizes of bands are 449bp and 4094bp; for [RebBAC]-pRSFDuet1, the theoretical sizes of bands are 98bp and 4196bp. Electrophoresis was performed in a 1% agarose gel. The results showed successful double-enzyme digestion and correct plasmid extraction.

Figure 3-9. 1% agarose gel stained with RebABDC and RebBAC integration of pRSFDuet1 in E.coli Ht115(DE3) was checked by restriction enzyme digestion. M: 10000bp Marker. pRA1, pRA2, pRB1, pRB2: the plasmids without enzyme digestion, which showed closed circular plasmid DNA (cc DNA) and open circular plasmid DNA (oc DNA). s-pRA1, s-pRA2, s-pRB1, s-pRB2: plasmid digested by restriction enzyme HindIII, which showed linear plasmid DNA (Linear DNA) with theoretical band size 4543bp and 4294bp for RA and RB, respectively. d-pRA1, d-pRA2, d-pRB1, d-pRB2: plasmid digested by restriction enzyme NdeI and HindIII, which showed theoretical band size 449bp and 4094bp for RA, 98bp and 4196bp for RB.

5. Rice cultivation

Since we needed to test the effectiveness of our product, we cultured rice（ZH11） in our laboratory culture room so that it can be used for infection by R.solani AG1-IA in subsequent experiments. We sowed the seeds under the soil after germination and selected rice leaves and stems from the 6th to 8th week as the material. Below are the approximate changes in rice plant height that we recorded during this time.

Figure 3-10. Height of ZH11 every 7 days. Every Monday is Seeding Day and we set every Thursday of the week to record the heights, including the first week which was the week we sowed the seeds.