CN103695439B - Gold mandarin orange FcWRKY70 gene and the application in raising drought tolerance in plants thereof - Google Patents

Abstract

The present invention provides kumquat FcWRKY70 gene and its application in improving plant drought tolerance. The applicant isolated and cloned a multiple stress-induced WRKY transcription factor FcWRKY70 from kumquat (Fortunella. crassifolia), and its sequence is SEQ ID NO. 1, the encoded protein is shown in SEQ ID NO.2. The applicant introduced the gene into tobacco, lemon and kumquat respectively, and the obtained overexpression transgenic plants had significantly improved tolerance to drought stress, and the tolerance of the interference transgenic plants to drought stress had been significantly weakened. The present invention provides new genetic resources for plant anti-abiotic stress molecular design and breeding, and provides new genetic resources for the implementation of green agriculture and water-saving agriculture. The development and utilization of the genetic resources is conducive to reducing agricultural production costs and promoting the efficient development of agricultural industries .

Description

Kumquat FcWRKY70 Gene and Its Application in Improving Plant Drought Tolerance

technical field

This patent belongs to the field of plant genetic engineering. It specifically involves the isolation and cloning of a multiple stress-induced WRKY transcription factor FcWRKY70 from kumquat (Fortunella. The tolerance to drought stress was significantly improved, and the tolerance of the intervention transgenic plants to drought stress was significantly weakened.

Background technique

During the growth process, plants are often affected by various adverse environmental stresses, such as drought, salt, cold damage, high temperature, nutrient deficiency, and pests and diseases, which seriously affect their growth and development (Gong and Liu 2013). It is worth noting that global crop yields are reduced by approximately 20% each year due to drought (Wang et al 2003).

To adapt to various environmental stresses, plants undergo a series of changes in physiological and biochemical processes (Krasensky and Jonak, 2012). For example, changing the metabolic process to accumulate a series of metabolites maintains cellular stress and resists stress (Nishizawa et al., 2008; Verbruggen and Hermans 2008; Livingston et al., 2009; Shi et al., 2010; Fu et al. , 2011a,b). In addition, a large number of genes have altered transcription levels, either being stress-induced or transcriptionally repressed (Seki et al., 2003). Some stress response genes encode functional proteins that promote the accumulation of beneficial metabolites in plant cells and protect cells from damage (Chen and Murata 2011; Wang et al., 2011). Another part of genes perform the function of transcriptional regulation, modifying the expression of target genes, such as transcription factors, protein kinases, phosphorylases (Su et al., 2010; Mao et al., 2011; Seo et al., 2012; Cui et al. al.,2012)

Transcription factors play an integral role in the transcriptional recombination of stress-responsive genes (Singh et al., 2002). Plant genome contains a large number of transcription factors, among which WRKY protein is considered to be a large gene family composed of a huge number of members unique to plants (Eulgem et al., 2000). Bioinformatics analysis shows that there are very abundant WRKY proteins in some species whose genome sequences have been published, including 74 in Arabidopsis, 109 in rice, 66 in papaya, 104 in poplar, and 68 in sorghum (Eulgem and Somssich 2007; Ross et al., 2007; Pandey and Somssich 2009). Since the first WRKY protein was discovered in sweet potato, researchers have identified more and more WRKY proteins in plants.

So far, a large number of studies have shown that WRKY proteins play key roles in different physiological and biochemical processes, such as signal transduction, metabolism, lignification, etc. (Ren et al., 2008; Guillaumie et al., 2010; Mao et al. , 2011; Yu et al., 2013). In addition, increasing evidence indicates that WRKY transcription factors are involved in plant responses to biotic and abiotic stresses (Liu et al., 2013). For example, the three members of Arabidopsis family II a subfamily, AtWRYK18, AtWRKY40 and AtWRKY60, form redundant, antagonistic and different complex modes of action in terms of structure and function, and respond to different fungal and bacterial diseases ( Xu et al., 2006; Shen et al., 2007; Pandey et al., 2010). AtWRKY6 and AtWRKY42 can regulate the expression of PHOSPHATE1 (PHO1) and improve the tolerance of transgenic Arabidopsis to low phosphorus stress (Chen et al., 2009). Two family III WRKY transcription factors, WRKY70 and WRKY54, cooperatively negatively regulate stomatal opening and closing in Arabidopsis to regulate osmotic stress (Li et al., 2013). In rice, there are also research reports on a pair of alleles of WRKY transcription factors that have opposite effects in bacterial blight, bacterial streak disease, drought, and salt stress (Tao et al., 2009; Tao et al., 2011 ). In view of the important role of WRKY transcription factors in the process of plant adversity stress, this type of gene can be used as an effective gene resource to create transgenic plants with enhanced resistance through transgenic models. So far, a large number of studies have shown that overexpression of similar genes can significantly enhance the stress resistance of plants (Fu at al., 2011b; Hao et al., 2011; Su et al., 2010).

In previous studies, we discovered a multiple stress-responsive EST, amplified the full length by RACE technology, and found that the gene has a WRKY domain and is a potential WRKY transcription factor. Bioinformatics analysis showed that this gene belongs to family III WRKY transcription factors and is most similar to Arabidopsis WRKY70, so we named it FcWRKY70. It is an unknown new gene, encoding a small molecule polypeptide product without any known conserved domain. Studies have found that the expression of FcWRKY70 can be significantly induced by exogenous addition of SA and ABA in canker sores, and it can also be induced by drought and salt treatment, which is a very good gene resource for resistance breeding.

Contents of the invention

The object of the present invention is to isolate and clone a WRKY transcription factor induced by multiple adversities, FcWRKY70, whose nucleotide sequence is shown in SEQ ID NO: 1, and its encoded amino acid sequence is shown in SEQ ID NO: ID NO: 2.

Another object of the present invention is to provide an application of the Kumquat FcWRKY70 gene in improving plant resistance to drought stress. It is introduced into tobacco, lemon and Kumquat through Agrobacterium-mediated genetic transformation, and its performance under dehydration and drought stress is identified. It provides new genetic resources for the design and breeding of plant stress-resistant molecules, and provides new genetic resources for the implementation of green agriculture and water-saving agriculture. The development and utilization of this genetic resource is conducive to reducing agricultural production costs and achieving environmental friendliness.

In order to achieve the above object, the present invention adopts the following technical measures:

Kumquat wild-type cDNA was used as a template and amplified with the following primers:

Forward primer: 5'-CATGCCATGGATCATAATGAAAATGGAGGCCGGTC-3';

Reverse primer: 5'-GGGTCACCAGTTGCAAAGGACTAGCGGTAGCAG-3'.

Obtain a new kumquat gene FcWRKY70, its nucleotide sequence is as shown in SEQ ID NO: 1, the full length of FcWRKY70 is 1196bp, wherein 83-1069bp is the coding region of the gene; its encoded amino acid sequence is as shown in SEQ ID NO: 2 According to the results, it contains an open reading frame of 987bp, encodes 328 amino acids, has an isoelectric point of 5.89, and a predicted molecular weight of 36.78kD.

The genetic transformation method mediated by Agrobacterium is used to transform tobacco and lemon, and the obtained transgenic plants are verified by biological function, which shows that the cloned FcWRKY70 gene of the present invention has the function of improving resistance to dehydration and drought stress. In the Example section of the present invention, we describe the isolation, functional verification and application of Kumquat FcWRKY70.

Compared with the prior art, the present invention has the following advantages:

Drought, low temperature, salinization and other adversities will be accompanied by severe plant dehydration process, forming physiological drought, affecting the normal growth and development of plants and the geographical distribution of crops. Traditional agriculture relies heavily on water resources, especially in high temperature seasons, which restricts the development of the agricultural industry. Relying on the invention, the drought tolerance ability of the plants can be effectively improved, so that they can adapt to the drought environment. Once effective genetically modified materials are obtained, the consumption of irrigation water can be effectively reduced in production, resources can be saved, and production costs can be reduced. On the other hand, the present invention can be used to improve rootstock materials or others according to actual production requirements, and has wide application and is easy to be accepted and approved.

Introduce the gene into plants through Agrobacterium-mediated genetic transformation, identify its function under different stress conditions, provide new genetic resources for plant stress-resistant molecule design and breeding, and provide new opportunities for implementing green agriculture and water-saving agriculture genetic resources.

Description of drawings

Fig. 1 is a technical flow chart of the present invention.

Fig. 2 is a schematic diagram of the expression of the FcWRKY70 gene of the present invention under different stress treatments.

Fig. 3 is a schematic diagram showing the subcellular localization fluorescence of the FcWRKY70 gene of the present invention.

Among them, A-B in Figure 3 is the imaging of GFP gene (control) in bright field and dark field; C-E in Figure 3 is the imaging of FcWRKY70 gene in bright field (C), dark field DAPI staining (D), and dark field GFP (E) image below.

Fig. 4 is a schematic diagram of identification of transcriptional activation of FcWRKY70 gene of the present invention.

Fig. 5 is a schematic diagram of the construction of the FcWRKY70 gene vector of the present invention.

A in Fig. 5 is a schematic diagram of constructing an overexpression vector; B in Fig. 5 is a schematic diagram of constructing an interference vector.

Fig. 6 is a schematic diagram of transformation of lemon with FcWRKY70 gene of the present invention and regeneration process.

A in Figure 6 is the co-cultured stem section, B in Figure 6 is the stem section of the screening culture; C in Figure 6 is the elongation and propagation of resistant buds on the elongation medium, and D in Figure 6 is the resistant buds Carry out rooting culture. The transformation process of tobacco and kumquat is similar.

Fig. 7 is a schematic diagram of PCR identification of the FcWRKY70 gene transgenic material of the present invention.

A in Fig. 7 is the PCR identification map of the regenerated plant obtained after FcWRKY70 transformed tobacco (the top is the PCR amplification map of the FcWRKY70 specific primer, and the bottom is the PCR amplification map of the HPTII gene specific primer);

B in Fig. 7 is the PCR identification diagram of the regenerated plant obtained after FcWRKY70 transforms lemon (upper is the PCR amplification diagram of FcWRKY70-specific primers, and the lower is the PCR amplification diagram of HPTII gene-specific primers);

C in Fig. 7 is the PCR identification diagram of the regenerated plant obtained after FcWRKY70 transformation of kumquat (upper is the PCR amplification diagram of 35S forward primer and FcWRKY70 specific primer, and the lower is the PCR amplification diagram of NPTII gene specific primer);

D in Fig. 7 is the expression level analysis of the target gene in the tobacco transgenic line, Fig. 7E is the expression level analysis of the target gene in the lemon transgenic line, and 7F is the expression level analysis of the target gene in the kumquat transgenic line;

Fig. 8 is a schematic diagram showing the drought resistance comparison between the FcWRKY70 transgenic tobacco seedlings (1#, 4#) of the present invention and the wild type.

A in Fig. 8 is a comparison of the phenotype of the transgenic tobacco isolated seedlings before and after natural dehydration for 60 minutes and the difference of trypan blue staining;

Among Fig. 8, B is the comparison of the relative water loss rate in the process of natural dehydration of each line of in vitro seedlings for 60 minutes;

C in Fig. 8 is the phenotype comparison before and after water control of the potting material;

Figure 8D is a comparison of the relative soil water content of various materials after water control; Figure 8E is the MDA value measured by sampling materials of each series after water control.

Fig. 9 is a schematic diagram showing the dehydration ability comparison between the FcWRKY70 transgenic lemon seedlings (28#, 43#) of the present invention and the wild type.

A in Figure 9 is a comparison of the relative water loss rate between the FcWRKY70 transgenic material and the wild type during natural dehydration for 6 hours;

Figure 9B is a comparison of the differences in NBT dyeing of various materials after dehydration; Figure 9C is a comparison of the differences in DAB dyeing of various materials after dehydration.

Fig. 10 is a schematic diagram showing the comparison of the dehydration ability of FcWRKY70 transgenic kumquat seedlings (20#, 22#) and the wild type.

Detailed ways

The present invention will be described in detail below in combination with specific implementations. From the following descriptions and these implementations, those skilled in the art can ascertain the essential characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention, so that it can be applied to various Use and Condition.

Example 1:

Isolation, Cloning and Expression Analysis of FcWRKY70 Gene

In the previous study, we compared the citrus EST database HarvEST (http://harvest.ucr.edu) with the chip probe Cit.7937.1.S1_at sequence, and obtained a target EST sequence. In order to obtain its full-length sequence information, we adopted RACE technology. According to the known fragment information, design 3'Race and 5'Race primers respectively, as follows:

3'Race outer primer-GS:5'AGTGCCCACATCACCGAAAA 3'

3'Race inner primer-GS:5'TGGGGATGTGATTTCTGGAGT 3'

5'Race primer-GS:5'AAATCTTTTTCGGTGATGTGGGCA 3'

First, the total RNA in kumquat leaves was extracted using the RNAiso Plus kit (the kit was purchased from TAKARA Company, and the operation method was in accordance with the instructions). Then take 1 μg of total RNA sample, add 3'Race reaction adapter, and use 3'Full RACE CoreSet Ver.2.0Kit kit to synthesize the first-strand cDNA (the kit was purchased from TAKARA company, see the kit instruction manual for the synthesis of the first-strand cDNA ). The obtained first-strand cDNA was used for nested PCR reaction to amplify the 3' end sequence of the target gene. The components in the Outer PCR 50μl reaction system are as follows:

PCR reaction in Bio-Rad Alpha ^TM Unit Block Assembly For DNA The following procedures were completed on the Systems thermal cycler: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 120 seconds (20 cycles in total); extension at 72°C for 10 minutes after the cycle was completed. After the Outer PCR amplification, the product was stored at -20°C for Inner PCR amplification.

The components in the Inner PCR 50μl reaction system are as follows:

PCR reaction in Bio-Rad Alpha ^TM Unit Block Assembly For DNA The following procedures were completed on the Systems thermal cycler: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 120 seconds (30 cycles in total); and extension at 72°C for 10 minutes after the cycle was completed. Obtain a PCR band product after two PCR reactions, after 1.2% agarose gel electrophoresis, use Axygen DNA Gel Extraction Kit gel recovery kit (purchased from Axygen Company) to recover the target band (specific steps refer to the instructions for use) ). Recover the purified DNA solution and pMD18-T carrier (purchased from TAKARA company) for ligation reaction (operated according to the instructions), the total volume of the ligation reaction is 10 μl, including 5 μl of Solution Ⅰ (all in the vector kit), and 4.5 μl of purified PCR product And 0.5μl T vector, 16 ℃ overnight. Then transform Escherichia coli DH5α competent by heat shock method with all the ligation products, screen positive clones on LB solid plate containing 100 mg/L ampicillin, pick 4-6 clones and sequence them.

The kit used for the 5'Race reaction was SMARTer ^TM RACE cDNA Amplification Kit (purchased from Clontech). 3 μg of total RNA sample was used to synthesize the first-strand cDNA. For details, please refer to the kit instructions. The resulting first-strand cDNA was used in a Touchdown PCR reaction to amplify the 5' cDNA end of the target gene. The PCR reaction was amplified using Advantage 2 Polymerase Mix (purchased from Clontech Company), and the addition of each component was referred to the instructions. PCR reaction in Bio-Rad Alpha ^TM Unit Block Assembly For DNA The following procedures were completed on the Systems thermal cycler: denaturation at 94°C for 30 seconds, extension at 72°C for 3 minutes (5 cycles in total); denaturation at 94°C for 30 seconds, annealing at 70°C for 30 seconds, and extension at 72°C for 3 minutes (5 cycles in total). ); denaturation at 94°C for 30 seconds, annealing at 68°C for 30 seconds, and extension at 72°C for 3 minutes (25 cycles in total); after the cycle was completed, extend at 72°C for 10 minutes. Subsequent experiments were the same as 3'Race, that is, the obtained PCR products were recovered by electrophoresis and connected to T vectors, and transformed into competent Escherichia coli DH5α, and finally 4-6 positive clones were picked and sequenced.

In order to analyze whether the FcWRKY70 gene can respond to different environmental stimuli, real-time quantitative PCR was used to analyze the expression of the gene under different stress treatments. The results showed that Xcc, ABA, SA, dehydration, and salt of X. citrus could all induce the expression of this gene, while low temperature had no obvious induction on its expression (see Figure 2), indicating that it is a multiple stress response gene that can simultaneously respond to biological Adversity and abiotic stress.

Example 2:

Subcellular localization of FcWRKY70 gene

Online localization prediction indicated that FcWRKY70 is localized in the nucleus. In this study, tobacco epidermis was used to study the subcellular localization of FcWRKY70 gene. Here we choose pCAMBIA1302 vector to construct the positioning vector of FcWRKY70 gene. The entire ORF (reading frame) of the FcWRKY70 gene was amplified by RT-PCR, and Nco I single enzyme cutting sites were added to both ends of the amplification primers. At the same time, Nco I was used to single-enzyme digest the product recovered from PCR and the pCAMBIA1302 vector, and then the digested product was recovered separately, and the gene fragment and the vector fragment were recombined and connected to obtain the pCAMBIA1302-FcWRKY70-GFP recombinant plasmid. The recombinant material and control plasmid (pCAMBIA1302) were then transformed into Agrobacterium GV3101 respectively.

Agrobacterium infection of tobacco epidermis is carried out as follows:

1. Bacterial cell activation: dip a small amount of Agrobacterium GV3101 bacterial liquid containing the recombinant plasmid and the control plasmid, streak on the Km/LB plate, and incubate at 28°C for 1-2 days to activate the bacterial cells.

2. Pick a single clone from the streaked plate, inoculate it in 50ml Km/LB liquid medium, culture at 28°C, 250rpm, until OD600=0.4-0.6, at this time, the growth of the bacteria is in the logarithmic phase, and the activity high.

3. Transfer the bacterial liquid into a 50ml centrifuge tube, centrifuge at 4000rpm for 10min, and collect the bacterial cells to the bottom of the tube.

4. Remove the supernatant, suspend the cells with 10 mM MES (pH 5.6, 10 mM MgCl ₂ , 150 μM AS (acetosyringone)) buffer to make OD600 = 1.0, and set aside.

5. In the ultra-clean bench, take 40-60d sterile tobacco seedlings (wild type, Nicotiana nudicaulis) as the material, clamp the moderately sized leaves into a sterile petri dish, and resuspend them with a disposable syringe that removes the needle. The bacterial liquid is injected into the tobacco leaves (both sides are acceptable, and the back of the leaf has more stomata, so it is easier to inject the bacterial liquid).

6. Use sterile filter paper to suck off the excess bacterial solution on the leaves, spread the leaves on the MS medium pre-matched with filter paper with the back side down, and cultivate in the dark at 28°C.

After 48 hours, observe the localization of fluorescence with a NIKON ECLIPSE 90i microscope. The results showed that there was fluorescence in the whole cell when the control vector was transformed (Figure 3, upper right), while the fluorescence in cells transformed with the recombinant vector could only be detected in the nucleus (Figure 3, lower right), indicating that FcWRKY70 is a nuclear localized protein.

Analysis of transcriptional activation of FcWRKY70 gene

Transcription activation activity is a basic feature of transcription factors. Here we use the pGBKT7 vector for recombinant construction to verify whether FcWRKY70 has transcription activation activity. Primers with BamH I and Pst I restriction sites were designed to amplify the full-length ORF of the FcWRKY70 gene, and the product was double-digested by BamH I and Pst I, and then subcloned into pGBKT7 that was also digested by BamH I and Pst I On the vector, the fusion expression vector pGBKT7-FcWRKY70 was obtained. After the sequence was confirmed to be correct, the fusion expression vector and the empty vector (pGBKT7) were respectively transformed into yeast strain AH109, and then cultured on different deletion media. The results showed that the yeast cells transformed with the empty vector could only grow on the deletion medium SD/-Trp, while the positive yeast cells transformed with the fusion vector could grow on SD/-Trp/-ade and SD/-Trp/-ade/- Growth in the deletion medium such as His, (Fig. 4), shows that FcWRKY70 has transcription activation activity and is a typical transcription factor.

Embodiment 3:

Plant Transformation Overexpression Vector Construction

According to the analysis of the multiple cloning site of pCAMBIA1301 vector and the restriction site on the coding region sequence of FcWRKY70 gene, Nco I and BstE II were selected as endonucleases. Firstly, the full-length of FcWRKY70 was obtained by amplifying the wild-type cDNA of Kumquat as a template, and the PCR product and the pCAMBIA1301 vector were digested with Nco I and BstE II respectively and reconnected into the target vector.

The total volume of the PCR product double enzyme digestion system was 20 μl, which contained 16 μl of recovered PCR products, 2 μl of 10×K buffer (purchased from Takara), and 1 μl each of Nco I and BstE II. The total volume of the pCAMBIA1301 vector double enzyme digestion system was 40 μl, which contained 8 μl of pCAMBIA1301 plasmid, 4 μl of 10×K buffer (purchased from Takara), 1 μl each of Nco I and BstE II, and 24 μl of double distilled water. After enzyme digestion at 37°C overnight, they were purified and recovered separately. The ligation reaction system contained 1 μl of 10×T4 ligation buffer, 1 μl of T4 DNA ligase, 3 μl and 1 μl of FcWRKY70 gene and vector pCAMBIA1301, respectively, and made up to 10 μl with double distilled water, and ligated overnight at 16°C. Then transform Escherichia coli DH5α competent cells with all the ligation products, and screen on LB solid plates containing 100mg/L kanamycin, and send the positive monoclonals detected by PCR for sequencing, and extract the recombinant plasmids from monoclonals with correct sequences. The recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101 by freeze-thaw method (refer to Sam Brook, translated by Huang Peitang, "Molecular Cloning Experiment Guide" third edition, Science Press, 2002) and preserved.

Plant transformation intervention vector construction

In order to obtain the interfering transgenic plants, the pHellsgate2 interfering vector was used for recombination construction. First, a fragment of about 300 bp was selected at the 5' end of FcWRKY70 to design primers, and a general primer at the attB site was added before the specific primer as a primer for amplification of the target fragment. The target fragment was amplified by using the wild-type cDNA of kumquat as a template, and the PCR product was recovered and then recombined and connected to the pHellsgate2 vector by BP reaction to construct the target vector.

The general primer sequences are as follows:

attB1: 5'-GGGGACAAGTTTGTACAAAAAAAGCAGGCT-3'

attB2: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3'

The kit used for BP reaction is BP Clonase ^™ II Enzyme Mix (purchased from Invitrogen). The reaction system was 6 μl, in which 100 ng of purified PCR product and 100 ng of pHellsgate2 plasmid with attP site were added, 1 μl of B/P Clonase was added, TE buffer (pH 8.0) was added to 6 μl, and the reaction was carried out overnight at 25°C. All the reactants were transformed into competent Escherichia coli DH5α by heat shock method, and applied to LB solid plates containing spectinomycin (Spec) 100 mg/L to screen positive clones. Use the 35S promoter sequence primer and the reverse specific primer as amplification primers to perform PCR detection and analysis on the positive clones. The positive clones detected by PCR are further verified by single enzyme digestion with XbaI and XhoI respectively. If the two groups of single enzyme digestion fragments are of the same size And it is about 200bp larger than the target fragment, then the target fragment in the positive clone is correctly connected. Correctly connected positive monoclonals were sent for sequencing, and single clones with correct sequences were extracted from recombinant plasmids, and the recombinant plasmids were extracted by freezing and thawing (referring to Sam Brook, translated by Huang Peitang, "Molecular Cloning Experiment Guide" third edition, Science Press, 2002). The plasmid was transformed into Agrobacterium tumefaciens GV3101 and preserved.

Embodiment 4:

tobacco genetic transformation

The genetic transformation steps of tobacco mediated by Agrobacterium tumefaciens are as follows:

1. Cultivation of Agrobacterium tumefaciens: Take the Agrobacterium tumefaciens (including overexpression vector) bacterial liquid preserved in the ultra-low temperature refrigerator, streak and activate it on the LB plate added with 100mg/L kanamycin, scrape the streaked bacteria Spots were added to liquid MS basic medium, cultured with shaking at 180 rpm at 28°C, and dipped when the concentration of the bacterial solution reached OD ₆₀₀ =0.4-0.6.

2. Dip dyeing: Take sterile wild-type tobacco material, cut its leaves into 0.5cm×0.5cm size, put them into the cultured Agrobacterium tumefaciens bacteria liquid, infect for 8-10 minutes, and shake continuously during this period.

3. Co-cultivation: Take the tobacco leaves after dipping, blot the excess bacterial liquid on the leaves with sterile filter paper, spread the back of the leaves downward on the MS basic medium containing 0.3g/L NAA and 2.25g/L 6-BA Incubate in the dark at 25°C for 3 days.

4. Screening culture: After 3 days of co-culture, the tobacco leaves were washed once with an aqueous solution containing 400mg/L cephalosporin, then rinsed with sterile water for 3-5 times, and then transferred to a medium containing 2.5mg/L hygromycin+400mg /L cephalosporin+2.25mg/L6-BA+0.3mg/L NAA MS basic medium for culture.

5. Rooting culture: when the adventitious buds grow to about 2-3cm, transfer them to MS medium supplemented with 2.5mg/L hygromycin (or not) and 400mg/L cephalosporin+0.3mg/L NAA to induce roots generate.

6. Tobacco seedlings are transferred to soil culture: the transformed seedlings after rooting are full of culture bottles, taken out from the rooting medium, the medium on the transformed seedlings is washed with tap water, and planted in sterilized nutrient soil.

See Table 1 for the medium used for tobacco transformed seedlings.

Table 1 The formula of medium used for tobacco transformed seedlings

7. Preliminary identification of positive transgenic tobacco

The FcWRKY70-transformed tobacco-resistant shoots were obtained according to the above-mentioned method, and DNA was extracted to identify positive transgenic materials by PCR method. The specific method is as follows: design primer HPTII and gene-specific primer PCR amplification to identify positive seedlings (Table 1). Harvest the seeds (T1 generation seeds) after transplanting the plants identified as transgenic by PCR, vernalize them at 4°C for 3 days, take a few seeds in a 1.5ml centrifuge tube, soak the seeds in 70% alcohol for 30 seconds, and then use sterilized double steamed Wash once with water, then add 1ml 2.5% NaClO to disinfect the surface for 7 minutes, shake fully, discard NaClO, wash 3 times with sterilized double distilled water, and finally suspend the sterilized seeds with 1ml 0.1% agarose, and lay them out on the surface added with 2.5mg/ On the L hygromycin (Hyg) MS basic medium, the results show that the resistant seedlings obtained by the present invention can grow normally on the resistant medium and are green, while the non-resistant seedlings turn yellow and die. Positive transgenic materials were used for resistance function identification.

Example 5:

Genetic transformation of lemons and kumquats

1. Take lemon and kumquat seeds, soak them in 1M NaOH for 20 minutes, wash them with clean water, then sterilize them by soaking them in 2% NaClO for 15-20 minutes on an ultra-clean workbench, wash them three times with sterile water, and then sterilize them on the surface. The seeds of the bacteria were peeled off the seed coat under sterile conditions, sowed on MT solid medium, cultured in the dark for 3-4 weeks and then cultured in the light for 1 week for transformation.

2. Take the Agrobacterium containing the recombinant vector (the overexpression vector is transformed into lemon, the interference vector is transformed into kumquat) and the bacteria solution is streaked on the antibiotic containing 100 mg/L (the overexpression vector is kanamycin, and the interference vector is spectinomycin). On the solid LB medium, cultivate in the dark at 28°C for two days; pick a single colony and streak it on a new plate again, and culture in the dark at 28°C for 2-3 days; scrape off the grown bacteria with a scalpel, and inoculate on different In liquid MT medium containing antibiotics, shake culture at 28°C at 200 rpm to OD ₆₀₀ =0.6-0.8, add AS (acetosyringone) to a final concentration of 100 μM (20 mg/L), and set aside.

3. Take the epicotyls of sterile seedlings, and cut them obliquely into 1-1.5cm long stems in an ultra-clean workbench, and temporarily put the cut stems in a sterilized empty triangular flask (add a small amount of water to moisturize), and wait until After the Agrobacterium culture was completed, it was used for transformation.

4. Soak the cut explants in the prepared Agrobacterium bacterium solution (Agrobacterium transforming lemon containing overexpression vector, transforming kumquat containing Agrobacterium interfering vector) and infect for 20 minutes, during which it is constantly shaken several times. After the infection, the excess bacterial solution was blotted with sterile absorbent paper, the explants were spread on the co-culture medium, and cultured in the dark at 25°C for 3 days.

5. After 3 days of co-cultivation, wash the explants 3-5 times with sterile water, then blot the surface moisture with sterile absorbent paper, and transfer to the screening with 2.5mg/L hygromycin and 400mg/L cephalosporin On the culture medium, cultured in the dark at 25°C for 4 weeks and then transferred to the light condition. When the resistant buds are >0.5cm, cut off the resistant buds and transfer them to the budding medium to promote their differentiation. When the resistant bud > 1.5cm long, it is transferred to the rooting medium to induce rooting. The transformation process of lemon and kumquat is shown in Figure 6.

Common media formulations:

LB solid medium: peptone 10g/L+yeast extract 5g/L+NaCl 10g/L+agar 15g/L

Lemon and kumquat co-culture medium: MT+1.0mg/L BA+20mg/L AS

Lemon screening medium: MT+1.0mg/L BA+400mg/L Cef+4mg/L Hyg

Lemon sprouting medium: MT+1.0mg/L BA

Kumquat selection medium: MT+2.0mg/L BA, 1.0mg/L NAA+1.0mg/L KT+400mg/L Cef+25mg/L Km)

Lemon and kumquat rooting medium: 1/2MT+0.5mg/L NAA+0.1mg/L IBA+0.5g/L activated carbon

During the transformation process, 7.5 g/L agar + 35 g/L sucrose were added to each medium, and the pH was adjusted to 5.8.

Embodiment 6:

Molecular and physiological identification of transgenic plants

1 DNA extraction from leaves of tobacco, lemon and kumquat

Put an appropriate amount of leaves into a 1.5ml centrifuge tube, add liquid nitrogen, and grind thoroughly; then add 1ml cetyltriethylammonium bromide (abbreviated as CTAB) DNA extraction buffer (recipe: 100mM Tris -HCl, pH 8.0, 1.5M NaCl, 2mM EDTA, pH 8.0, 1% PVP (polyvinylpyrrolidone), 2% CTAB, 2% mercaptoethanol), incubate at 65°C for 60-90 minutes (take out the centrifuge tube every 15 minutes) Gently upside down and mix); 12000rpm centrifuge for 10 minutes; take 600μl supernatant, add an equal volume of chloroform, mix upside down for 10 minutes; /10 volume of 3M NaAc (sodium acetate), mix well, place at -20°C for 30 minutes, centrifuge at 12000rpm for 20 minutes; discard the supernatant, wash the precipitate 3 times with 1 ml of 75% ethanol, and add an appropriate amount of 1×TE solution to dissolve.

2 PCR detection of positive transgenic plants

PCR amplification was performed using primers Hyg and NPT II and gene-specific primers. The primer sequences, reaction procedures and systems are shown in Table 1, Table 2 and Table 3, respectively. Firstly, Hyg primers were used to identify the regenerated tobacco plants by PCR. 17 transgenic lines could amplify the Hyg gene fragments. Using gene-specific primers for PCR, 14 transgenic lines could amplify fragments of the expected size (Fig. 7A ), indicating that they are positive transgenic lines. Figure 7B shows the PCR identification of some lemon-resistant seedlings. It was found that 10 resistant seedling materials could amplify the Hyg gene fragments, and they were amplified by PCR with gene-specific primers, among which 7 lines could simultaneously amplify fragments of the expected size (Figure 7B), indicating that they were all transgenic plants. Figure 7C shows the PCR identification of some kumquat resistant seedlings. A total of 15 resistant seedling materials can amplify fragments of the NPT II gene. When using 35S forward primers and gene-specific reverse primers for amplification, 4 of them Lines can amplify fragments of the expected size, indicating that they are positive transgenic lines.

Table 1. Primer sequence information

Table 2, PCR reaction program

Table 3. PCR reaction system

Embodiment 7:

Semi-quantitative RT-PCR detection of FcWRKY70 gene expression

In this study, semi-quantitative RT-PCR was used to analyze the expression of the exogenous gene FcWRKY70 in transgenic tobacco, lemon and kumquat plants, and the RNA extraction of transgenic lines was performed using Trizol kits. The steps are as follows:

1. Pre-cool the mortar at -80°C, take an appropriate amount of leaf material in the mortar, and grind it into powder with liquid nitrogen. 2. Take a 1.5ml centrifuge tube and add 1ml Trizol buffer (to lyse cells and protect RNA), add an appropriate amount of ground powder into the buffer, shake vigorously for 2 minutes, let stand for 10 minutes, centrifuge at 12000g at 4°C for 15 minutes. 3. Take the supernatant, add 200 μL chloroform, shake vigorously for 2 minutes, let stand at room temperature for 10 minutes, centrifuge at 12000 g at 4°C for 15 minutes. 4. Repeat step 3. 5. Take the supernatant, add 500 μL isopropanol, mix slowly, let stand for 10 minutes, centrifuge at 12,000 g at 4°C for 10 minutes. 6. Discard the supernatant, add 1ml of 75% ethanol (made with sterilized DEPC water) to wash the precipitate 3 times. Air-dry the precipitate, add 30-50 μl DEPC water to dissolve, and store at -80°C. 7. Take 2 μl to detect the concentration and quality of RNA.

Take 2 μl of RNA to synthesize the first-strand cDNA. The kit used is First Strand cDNA Synthesis Kit (purchased from Toyobo). For specific operations, refer to the kit manual. The primers used for semi-quantitative analysis were FcWRKY70 gene-specific primers (F: 5'-CCAAGCAAGTAAGCAGGTTCA-3'; R: 5'-TGACTCCAGAAATCACATCCC-3'), and the reaction program was listed in Table 2 (annealing temperature 56°C). The tobacco ubiquitin gene and citrus actin gene were used as internal reference genes respectively, and the primers were as follows:

Ubiqutin-F: 5'-GGTGTTTCCAGTGGCGGACG-3'

Ubiqutin-R: 5'-TCCTCCCCTCAGCTACGGGGTAT-3'

Actin-F: 5'-CCAAGCAGCATGAAGATCAA-3'

Actin-R: 5'-ATCTGCTGGAAGGTGCTGAG-3'

The expression levels of 4 transgenic lines of tobacco, 3 transgenic lines of lemon, and 5 transgenic lines of kumquat were selected for analysis. The results showed that the expression levels of FcWRKY70 in all 7 selected lines in overexpressed transgenic tobacco and lemon were significantly enhanced. (Fig. 7D,E). In the interfering transgenic kumquats, the expression of FcWRKY70 in the five transgenic lines all showed different degrees of weakening ( FIG. 7F ). Among these different transgenic materials, two transgenic lines were selected for subsequent transgenic resistance evaluation experiments.

Embodiment 8:

Drought Resistance Evaluation of FcWRKY70 Transgenic Plants

In order to evaluate whether FcWRKY70 can change the drought resistance of transgenic plants, transgenic tobacco, lemon and kumquat materials were subjected to different degrees of drought stress, so as to observe the phenotypic differences of each material and evaluate its drought resistance.

Leaves of tobacco transgenic lines (1# and 4#) and wild-type detached seedlings were naturally dehydrated at room temperature. After 60 minutes, the leaves of wild-type materials showed obvious wilting symptoms, and similar phenotypes did not appear on the transgenic leaves. At the same time, the leaves of each line were stained with trypan blue after dehydration. The wild-type leaves showed dark blue staining, while the transgenic leaves were slightly light blue (Figure 8A), indicating that after natural dehydration, the cell death program of the transgenic lines was much lower than that of the wild type. On the other hand, during the 60-minute dehydration process, each line showed different degrees of water loss. After 60 minutes, the relative water loss rate of the wild type reached 39.3%, and the transgenic lines were 30.5% and 24.6%, which were significantly lower than the wild type. , indicating that the water holding capacity of the transgenic line is better than that of the wild type (Fig. 8B). Accordingly, it was shown that FcWRKY70 could significantly improve the anti-dehydration ability of transgenic materials.

In order to further investigate the phenotype of the transgenic material under long-term drought stress, the 50-day-old tobacco material entered the water control period after 1 week of normal watering. As shown in Figure 8C, FcWRKY70 does not affect the normal phenotype of the transgenic material (left in the figure), but the phenotype difference between the wild type and the transgenic material after water control is very obvious (right in the figure), the wild type material is wilted and yellow, while the transgenic material Although the growth of the material was also inhibited to a certain extent, it did not show any wilting symptoms and still maintained good leaf tension. Random samples were taken to test the soil moisture content, and the test results negated the possibility that the difference in underground water supply caused the difference in phenotype (Fig. 8D). The difference in malondialdehyde (MDA) content between the wild type and the transgenic material further indicated that after water control, the degree of cell damage in the transgenic material was significantly lower (Fig. 8E). The above results indicated that FcWRKY70 effectively changed the phenotype of the transgenic tobacco material under dehydration and drought stress, and improved its resistance.

In order to provide more abundant and powerful experimental evidence for the role of FcWRKY70 in drought resistance, different citrus materials were also used to enhance or weaken the expression of transgenes, providing effective genetic resources for citrus molecular breeding. The leaves of the overexpressed lemon materials (28#, 43#) and the wild-type materials were naturally dehydrated at room temperature. The materials of each line showed different degrees of water loss. After 6 hours, the water loss rate of the wild-type materials reached 30.5%. The series materials showed strong water holding capacity, and the water loss rates were 26.7% and 22.3%, respectively (Fig. 9A). After dehydration, the materials of each line were taken for NBT and DAB staining analysis. Half of the leaves of the wild-type material were stained dark blue, while there were only sporadic blue spots on the leaves of the transgenic material ( FIG. 9B ). The same results were presented in DAB staining. As shown in Figure 9C, the transgenic lemon leaves evenly distributed sporadic light brown staining spots, while the wild-type leaves had a large area of dark brown staining. The staining results showed that after 6 hours of natural dehydration, more active oxygen accumulated in the wild-type material, causing serious cell damage, while the transgenic material effectively removed excess active oxygen and maintained the normal function of cells. On the other hand, the design weakens the expression of FcWRKY70 in kumquats, as shown in Figure 10, taking the effective weakening line (20#, 22#) and the wild-type material at room temperature for natural dehydration. After 6 hours, the relative water loss rate of the wild-type The relative water loss rate of 20# and 22# was 63.5% and 60.0%, respectively, which was significantly higher than that of the wild type, indicating that the water holding capacity of 20# and 22# materials was significantly weakened compared with the wild type. Studies in citrus have shown that overexpressing FcWRKY70 can significantly improve the dehydration resistance of lemon materials; conversely, weakening the expression of FcWRKY70 can significantly weaken the dehydration resistance of kumquat materials, indicating that FcWRKY70 can effectively change the tolerance of transgenic citrus materials to drought stress sex.

SEQUENCE LISTING

the

<110> Huazhong Agricultural University

the

<120> Kumquat FcWRKY70 Gene and Its Application in Improving Plant Drought Tolerance

the

<130> Kumquat FcWRKY70 Gene and Its Application in Improving Plant Drought Tolerance

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<160> 4

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<170> PatentIn version 3.3

the

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Claims (4)

1. An isolated gene, whose sequence is shown in SEQ ID NO.1. 2. The protein encoded by the gene of claim 1, whose sequence is shown in SEQ ID NO.2. 3. The application of the gene according to claim 1 or the protein according to claim 2 in improving the drought tolerance of kumquat or lemon. 4. The application of the gene according to claim 1 or the protein according to claim 2 in improving the drought tolerance of tobacco.