CN102604948B - Separation and application of salicylic acid-induced citrus sinensis osbeck promoter GSTU19P - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering. The invention specifically relates to separation and identification of a promoter. A salicylic acid-induced promoter GSTU19P can be separated from GSTU19 genes which are separated from citrus sinensis osbeck, the nucleotide sequence of the promoter is as shown in SEQ ID NO: 1 (sequence identity number: 1), the length of the nucleotide sequence is 249bp, and the promoter has obvious salicylic acid induced activity. The induced promoter GSTU19P can be used for plant stress resistance or secondary metabolism and other physiological processes.

Description

Isolation and application of a salicylic acid-induced navel orange promoter GSTU19P

technical field

The invention belongs to the technical field of plant genetic engineering. It specifically relates to the cloning and spatial expression of a salicylic acid (SA)-inducible promoter and a GSTU19 gene promoter in navel orange. The isolation of the promoter provides a controllable expression regulatory element for agricultural production and biological research. The inducible promoter plays an important role in physiological processes such as plant resistance to stress and secondary metabolism.

Background technique

Agriculture is a country's basic industry, and the supply of food has become particularly important as the world's population continues to grow. Traditional agricultural production mainly depends on fertilization and pesticide weeding and pest control, which not only increases the cost of agricultural products, but also brings a great burden to the environment. The improvement of crop quality is now more and more dependent on the continuous development of molecular biology techniques. Many genetic resources have become an important weapon for the improvement of crop varieties by transgenic technology, but these genes are often expressed by constitutive promoters in transgenic plants. This expression method violates the natural physiological activity rules of plants and not only fails to achieve the desired results. If the intended purpose of the transgene is reached, it may even cause great harm to the physiological activities of the transgenic plant itself. For example, the overexpression of the disease resistance gene Pto in tomato (Li, et al.2002) and the overexpression of the disease resistance-related gene NPR1 in Arabidopsis (Blanco, et al.2005) both have adverse effects on the growth of transgenic plants. There are side effects, such as false lesions and short plants. Based on the impact of transgenes on plant growth and safety considerations, people hope to have sufficient safety guarantees while transgenic, so it is the best choice to use inducible promoters and promoters that are not expressed in edible parts to drive the expression of foreign genes.

The successful transcription of genes requires the participation of a series of factors such as promoters, RNA polymerases, and transcription factors. The TATA and CAAT boxes in eukaryotes determine the initiation site and efficiency of transcription, and are responsible for binding to RNA polymerase to initiate the basic transcription process. Other components that drive gene expression in specific time and space are called cis-acting elements. The structures of cis-elements driving specific expression vary due to different functions, and a unified structure cannot be given. The structures of these elements may have their own characteristics, and they play a decisive role in the specific expression of genes . The existence of these specific elements brings certain difficulties to the isolation of specific promoters, but it is these functional regions that determine the specificity of the promoter.

Glutathione transferases (GSTs) are a class of soluble proteins that exist widely in organisms, and they are encoded by a large gene family. According to their gene sequence characteristics, GSTs can be divided into seven categories: Phi, Tau, Theta, Zeta, Lambda, DHAR (dehydroascorbic acid) and TCHQD (tetrachlorohydroquinone dehalogenase). Among them, Tau-like GSTs (GSTU) are unique to plants and play important roles in physiological processes such as stress resistance and secondary metabolism. Previous studies have found that GST is induced by many factors, and salicylic acid is one of them. Salicylic acid (SA) is an endogenous phenolic small molecule compound with low content in plants (Raskin 1992), and the content in monocotyledonous plants is higher than that in dicotyledonous plants (Raskin, et al.1990) . SA has important physiological functions in plants, including affecting the opening and closing of stomata, seed germination, fruit production, ion absorption, heat production, flowering, sex differentiation and inhibition of ethylene biosynthesis, etc. Therefore, SA and its salts are considered to be a Plant hormones with important application value. SA can induce a variety of plants to develop resistance to viruses, fungi and bacterial diseases (Fan Zhijin, et al.2004). SA is also one of the signaling molecules that induce systemic acquired resistance (SAR) (Yalpani and Raskin 1993), and is involved in and involved in plant hypersensitive response (hypersensitive reaction, HR) and SAR response, and in plant SAR signal transduction plays a key role in. As a plant hormone and signaling molecule, SA directly or indirectly regulates the expression of related genes during plant development.

From the above research results, it can be seen that GSTs genes play an important role in physiological processes such as resistance to stress and secondary metabolism. SA can induce the early activation and expression of GSTs genes. Several cis-acting elements indirectly regulated by SA have been isolated. At the same time, salicylic acid is a class of small molecular substances in plants, and is considered to be a new plant hormone. From the perspective of biosafety, the prospect of such promoters being used in field is optimistic. In the invention, the navel orange is used as the material, the leaves are treated with salicylic acid, the up-regulated GSTU19 gene is isolated and cloned, and its promoter is isolated by a chromosome walking method. Through the scanning of the promoter element analysis software, the regulatory elements related to the induction of expression in the promoter sequence were determined, and then the expression vector of the three deletion sequences of the promoter was constructed, and the function was performed by vacuum infiltration method mediated by Agrobacterium and transient expression in protoplasts After verification, it was concluded that the promoter of GSTU19 gene contained salicylic acid-inducible related elements in the -700bp to -300bp sequence, and its promoter was an inducible promoter induced by SA.

Contents of the invention

The purpose of the present invention is to isolate an inducible promoter regulated by salicylic acid (SA) from red navel orange. In this way, useful gene resources are further provided for crop variety improvement and plant stress resistance. After salicylic acid was used to treat navel orange leaves, the expression of the GSTU19 gene was significantly up-regulated, and then the promoter of the gene (GSTU19P) was isolated and transformed into Arabidopsis by constructing a vector. Arabidopsis protoplasts could induce its expression, so the promoter There is potential for important applications in field crops.

The present invention is realized through the following technical solutions:

The applicant isolated a promoter of the GSTU19 gene whose expression was upregulated after being induced by salicylic acid from navel orange by gene cloning technology. Its nucleotide sequence is shown in the sequence table SEQ NO: 1, and the sequence length is 2072bp. Applicants named this promoter GSTU19P.

The promoter GSTU19P of the present invention is induced to express by salicylic acid. The expression of GSTU19 gene in red navel orange is induced by salicylic acid, so the promoter GSTU19P of GSTU19 gene was cloned by chromosome walking method. Through bioinformatics analysis, it was found that there were a variety of genes related to salicylic acid induction in the GSTU19P promoter. Elements, such as W-box bound by WRKY transcription factors, ACGT elements recognized by bZIP-like proteins, and ARR1 binding elements. The transient expression of this promoter in Arabidopsis protoplasts and the results of salicylic acid-induced treatment experiments also showed that whether the GSTU19P promoter drives the expression of GFP or GUS is affected by salicylic acid induction. It shows that GSTU19P of the present invention is a salicylic acid-inducible promoter.

The main steps for obtaining the promoter shown in the present invention are as follows:

(1) Set the gradient of salicylic acid (SA) treatment concentration, and select the appropriate SA concentration to treat the leaves of navel orange.

(2) Screen up-regulated genes by fluorescent quantitative PCR.

(3) The nucleotide sequence of GSTU19P promoter was cloned by chromosome walking technique.

(2) Analyze the structure of the promoter using bioinformatics methods.

(4) Construct GSTU19P expression vector and transform Arabidopsis.

(5) Resistance screening and transplanting of transgenic Arabidopsis thaliana.

(6) GUS activity detection was carried out on the positive plants.

(7) Transient expression transformation of Arabidopsis protoplasts.

The GSTU19P promoter cloned in the invention can further provide useful gene resources for genetic improvement of plant strains and improvement of stress resistance of plants. The application of the promoter of the present invention also includes regulating the resistance of navel orange to stress or secondary metabolism through the response to salicylic acid.

More detailed technical details of the invention are provided by the following examples, but are not intended to limit the protection scope of the invention:

Description of drawings

Sequence listing SEQ ID NO: 1 is the nucleotide sequence of the GSTU19P promoter isolated and cloned in the present invention.

Figure 1: The technical roadmap of the present invention.

Figure 2: Morphology of navel orange leaves treated with different concentrations of salicylic acid (7 days after treatment). In the figure: CK is the control, namely pure water treatment; 1 is 10mmol/L SA treatment; 2 is 5mmol/L SA treatment.

Figure 3: Real-time quantitative PCR detection of GSTU19 gene expression after SA treatment. In the figure: blue indicates water treatment group samples; purple indicates 5mmol/LSA treatment group samples; numbers indicate time points of re-sampling after treatment.

Figure 4: Analysis of GSTU19P structure using PLACE software (open access software).

Figure 5: Schematic diagram of constructing missing fragments to verify GSTU19P sequence missing fragments.

Figure 6: Schematic diagram of the construction of the expression vectors pCAMBIA1391+GSTU19P and pCAMBIA1301+GSTU19P vectors of the present invention.

Figure 7: Schematic diagram of the construction of the expression vector pCAMBIA1302+GSTU19P vector of the present invention.

Figure 8: Protoplasts of Arabidopsis thaliana transformed with promoter-deleted vectors and GFP to detect promoter response to SA activity.

Figure 9: Protoplasts of Arabidopsis thaliana transformed with promoter-deleted vector and GUS to detect promoter response to SA activity.

Detailed ways

Example 1 Selection and isolation of salicylic acid-induced candidate genes

This embodiment uses 0mmol/L, 1mmol/L, 5mmol/L, 4 concentrations of 10mmol/L salicylic acid to process adult red navel orange (kind is " early red ", from Huazhong Agricultural University citrus planting resource nursery, This kind has been awarded the Chinese plant variety right, the variety right number is CN20060194.6, the date of authorization: on May 1, 2008). Every two plants constituted a replicate. Processing time is 8:00 am. Observe every day, observe the leaf situation after one week, research has determined that 5mmol/L SA is the concentration (seeing Fig. 2) that handles red navel orange tree body.

DNA凝胶回收试剂盒(购自Omega公司，美国)回收特异带，提取步骤参照使用说明。回收纯化的DNA溶液与pMD18-T载体(购自宝生物工程大连有限公司即TaKaRa公司)进行连接反应，按说明书操作，连接反应体系中插入GSTU19基因与pMD18-T载体的摩尔比为3∶1连接反应总体积是10μl，其中包括5μl的2×缓冲液(购自宝生物工程大连有限公司)，4.5μl纯化的PCR产物，0.5μl T载体。16℃过夜连接。取10μl连接产物，采用热击法(Sambrook，et al.2002)转化大肠杆菌DH5α，在含有50mg/L氨苄霉素的LB固体平板中筛选阳性克隆，挑取5个克隆测序(由上海联合基因公司完成)，测序结果表明，本发明克隆GSTU19基因全长为1060bp(见SEQ ID NO：2所示)。为分析GSTU19基因是否对水杨酸的处理响应，采用实时定量PCR分析该基因在水杨酸不同处理下的表达。结果表明，GSTU19表达量却在4小时内快速上升，且在8小时时达到最高点，是初始状态的9倍多(图3)，表明它是一个水杨酸应答候选基因。Search the citrus EST database (HarvEST: Citrus ver.0.51) with GSTU as the keyword, and get 5 similar sequences, which are composed of DNAstar (public software) to form a unigene, use the sequence to design primers with Primer Premier 5.0, and use RT-PCR Method extends full-length. 5mmol/L SA treated navel orange leaves to extract RNA and reverse transcribe, and the obtained first-strand cDNA was used to amplify the full length of GSTU19 gene. RNA was extracted using a Trizol kit (purchased from Invitrogen, operated according to the operating instructions provided by the kit), and the extracted 3 μg total RNA sample was treated with 1 U of DNaseI (Amplification Grade, purchased from Invitrogen) for 15 minutes at room temperature. , add 1 μl EDTA (25 mM), and incubate at 65° C. for 10 minutes. The first-strand cDNA was synthesized using an MBI reverse transcription kit (product number: K1621, purchased from Fermentas, operated according to the instructions of the kit). Amplified gene GSTU19 primer pair: Forward, 5'-TTCTGTCACAATGGCGGACG-3'; Reverse primer: 5'-GGAATAAGCAGGCAGCACGA-3'. The 25 μl reaction system included 100 ng cDNA, 1× buffer, 2.5 mM MgCl 2 , 0.25 mM dNTP, 0.5 U Taq polymerase (the aforementioned buffer and Taq polymerase were purchased from Fermentas, Lithuania) plus 0.5 μM of the above primers. The PCR reaction was completed on the ABI 9700 (Applied Biosystem) amplification instrument according to the following procedures: 94°C, 5 minutes, denaturation at 94°C for 30 seconds, annealing at 60°C for 50 seconds, extension at 72°C for 90 seconds, 35 cycles; °C extension for 15 minutes. Produce a single PCR band product, after 1% agarose gel electrophoresis, use

DNA gel recovery kit (purchased from Omega, USA) was used to recover specific bands, and the extraction steps refer to the instructions for use. The recovered and purified DNA solution was ligated with the pMD18-T vector (purchased from Treasure Bioengineering Dalian Co., Ltd., namely TaKaRa Company), and operated according to the instructions. The molar ratio of the GSTU19 gene inserted into the ligated reaction system and the pMD18-T vector was 3:1 The total volume of the ligation reaction was 10 μl, which included 5 μl of 2× buffer (purchased from Treasure Bioengineering Dalian Co., Ltd.), 4.5 μl of purified PCR product, and 0.5 μl of T vector. Ligate overnight at 16°C. Take 10 μl of the ligation product, transform Escherichia coli DH5α by heat shock method (Sambrook, et al. 2002), screen positive clones on LB solid plates containing 50 mg/L ampicillin, pick 5 clones for sequencing (provided by Shanghai United Gene completed by the company), the sequencing results showed that the full length of the cloned GSTU19 gene of the present invention is 1060bp (see SEQ ID NO: 2). In order to analyze whether the GSTU19 gene responds to salicylic acid treatment, real-time quantitative PCR was used to analyze the expression of the gene under different salicylic acid treatments. The results showed that the expression level of GSTU19 increased rapidly within 4 hours, and reached the highest point at 8 hours, which was more than 9 times that of the initial state (Figure 3), indicating that it is a salicylic acid response candidate gene.

Cloning and bioinformatics analysis of embodiment 2GSTU19 promoter

The promoter of GSTU19 gene (GenomeWalkerTM Universal Kit, Clontech, USA) was obtained by chromosome walking method, and two antisense primers were designed according to the GSTU19 cDNA sequence of citrus in the HarvEST-citrus database: GSP1 and GSP2, and two sense primers Primers AP1 and AP2 (see Table 2), with GSP1 and AP1 as a pair of primers, according to the reaction conditions in Table 1, the first round of PCR reaction was performed. The PCR system is: 2.5μl 10×LA Taq Buffer, 0.5μl 10mM dNTP, 0.5μl 10μM GSP1 and AP1 primers, 1U LA Taq enzyme, 0.5μl DNA, add deionized water to make up to 25μl. In order to increase the specificity of the reaction, the PCR product of the first round was diluted 50 times as the template, and GSP2 and AP2 were used as the primer pair, and the second round of amplification was carried out by nested PCR. The PCR reaction system is: 2.5μl 10×LA Taq Buffer, 0.5μl 10mM dNTP, 0.5μl 10μM GSP2 and AP2 primers, 1U LA Taq enzyme, 0.5μl DNA, add deionized water to make up to 25μl.

Table 1 PCR reaction conditions

DNA凝胶回收试剂盒(购自Omega公司，美国)回收特异带，提取步骤参照使用说明。回收纯化的DNA溶液与pMD18-T载体(购自宝生物工程大连有限公司即TaKaRa公司)进行连接反应，按说明书操作，连接反应体系中回收片段与pMD18-T载体的摩尔比为3∶1连接反应总体积是10μl，其中包括5μl的2×缓冲液(购自宝生物工程大连有限公司)，4.5μl纯化的PCR产物，0.5μl T载体。16℃过夜连接。取10μl连接产物，采用热击法(Sambrook，et al.2002)转化大肠杆菌DH5α，在含有50mg/L氨苄霉素的LB固体平板中筛选阳性克隆，挑取5个克隆测序(由上海联合基因公司完成)。本发明克隆GSTU19P全长序列为2072bp(见SEQ ID NO：1所示)。使用PLACE(http://www.dna.affrc.go.jp/PLACE/signalscan.html)在线软件(Higo，et al.1998)分析GSTU19P的2072bp的序列，发现靠近3’端具有许多TATA-box和CAAT-box元件，且在-200bp内有三个GATA-box(见图4)。在-300bp内存在一类诱导性元件——TGAC。TGAC是W-box的核心元件(见图4)，W-box是WRKY转录因子所特异识别的结合位点，存在于许多胁迫诱导基因和病程相关蛋白(PR)的启动子中(Xie，et al.2005)。还有ocs元件(TGACG)，是存在于许多GST启动子且与水杨酸诱导相关的诱导元件(Chen andSingh 1999)。在-1000bp以外的启动子远端还存在着许多诱导性的启动子元件(见图4)，诸如ARR1-bindingelement、GTAC(GTAC是CuRE(铜响应元件)的核心序列)(Quinn，et al.2002)、TTATCC(一种糖效应元件(SRE))(Tatematsu，et al.2005)、还有许多MYB类蛋白的识别位点(见图4)。After PCR finishes, utilize 1% agarose gel electrophoresis to detect PCR product, utilize

DNA gel recovery kit (purchased from Omega, USA) was used to recover specific bands, and the extraction steps refer to the instructions for use. The recovered and purified DNA solution was ligated with the pMD18-T vector (purchased from Treasure Bioengineering Dalian Co., Ltd., namely TaKaRa Company), and operated according to the instructions. In the ligation reaction system, the molar ratio of the recovered fragments to the pMD18-T vector was 3:1 The total reaction volume was 10 μl, including 5 μl of 2× buffer solution (purchased from Treasure Bioengineering Dalian Co., Ltd.), 4.5 μl of purified PCR product, and 0.5 μl of T vector. Ligate overnight at 16°C. Take 10 μl of the ligation product, transform Escherichia coli DH5α by heat shock method (Sambrook, et al. 2002), screen positive clones on LB solid plates containing 50 mg/L ampicillin, pick 5 clones for sequencing (provided by Shanghai United Gene company completed). The full-length sequence of the clone GSTU19P of the present invention is 2072bp (see SEQ ID NO: 1). Using PLACE (http://www.dna.affrc.go.jp/PLACE/signalscan.html) online software (Higo, et al.1998) to analyze the 2072bp sequence of GSTU19P, it was found that there are many TATA-boxes near the 3' end and CAAT-box elements, and there are three GATA-boxes within -200bp (see Figure 4). In -300bp, there is a kind of inducible element——TGAC. TGAC is the core element of W-box (see Figure 4). W-box is the binding site specifically recognized by WRKY transcription factors, and exists in the promoters of many stress-induced genes and disease process-related proteins (PR) (Xie, et al. al. 2005). There is also the ocs element (TGACG), an inducible element present in many GST promoters and associated with salicylic acid induction (Chen and Singh 1999). There are also many inducible promoter elements (see Figure 4) at the distal end of the promoter beyond -1000bp, such as ARR1-bindingelement, GTAC (GTAC is the core sequence of CuRE (copper response element)) (Quinn, et al. 2002), TTATCC (a sugar response element (SRE)) (Tatematsu, et al. 2005), and many recognition sites of MYB-like proteins (see Figure 4).

Embodiment 3 utilizes GSTU19P promoter to carry out genetic transformation to Arabidopsis

1) Vector construction: In order to verify the spatial expression of GSTU19P, according to the multiple cloning site and GSTU19P sequence of the pCAMBIA1391 vector (purchased from CAMBIA Laboratory in Australia), the amplification design was designed with Primer Premier 5.0 software according to the general principle of primer design 3 Vectors with different fragment sizes (Figure 5 and Figure 6) are 329bp (numbered as Del 1), 702bp (numbered as Del 2), and 1856bp (numbered as Full-length, respectively). The primers for the construction of the Del1 vector are F1 and R1, the primers for the construction of the Del2 vector are F2 and R1, and the primers for the construction of the Full-length vector are F3 and R1 (see Table 2). Carry out PCR amplification with the DNA of red meat navel orange (kind is " early red ") as template, and the extraction method of DNA is according to the method (Cheng, et al.2003) reported by Cheng Yunjiang. The annealing temperature for PCR amplification was 60°C. The PCR reaction conditions are: 95°C, 5min; 95°C, 30s; 56°C, 30s; 72°C, 90s; 72°C, 10min. 35 cycles. Double enzyme digestion system: the total reaction volume is 20 μl, which contains 10 μl of purified PCR product, 2 μl of 10×G buffer (purchased from MBI Company), 1 μl of EcoR I and Sal1, and 6 μl of double distilled water. Purify and recover after enzyme digestion at 37°C overnight. Double enzyme digestion system of pCAMBIA1391 vector: the total reaction volume is 20 μl, which contains 8 μl of pCAMBIA1391 vector DNA obtained through plasmid extraction, 2 μl of 10×G buffer (purchased from MBI Company), 1 μl of EcoR I and Sal1, and double distilled water 8 μl. Purify and recover after enzyme digestion at 37°C overnight. In the ligation reaction system, the molar ratio of the different fragments inserted into GSTU19P to the vector pCAMBIA1391 was 3:1, and the total reaction volume was 10 μl, which contained 1 μl of 10×Buffer, 1 μl of T4 DNA ligase, 4 μl of the recovered product of double enzyme digestion of GSTU19P, and 4 μl of the pCAMBIA1391 vector 2 μl of recovered product by double enzyme digestion and 2 μl of double distilled water. React at 16°C for 14-16 hours. The ligation product was transformed into Escherichia coli strain DH5α, positive clones were screened on LB solid plate containing 50 mg/L kanamycin, the plasmid was extracted for enzyme digestion and PCR identification, sequencing confirmed that there was no mutation, and a recombinant clone containing the inserted target fragment was obtained. They were named pCAMBIA1391-Full-length, pCAMBIA1391-Del1, pCAMBIA1391-Del2 recombinant vectors, and the recombinant vectors pCAMBIA1391 series were introduced into Agrobacterium EHA105 by freeze-thaw method (Sambrook, et al. 2002).

2) Transformation of Arabidopsis thaliana: Inflorescence infection method (Zhang, et al. 2006) was used to transform Arabidopsis thaliana: when the Arabidopsis plants grew to 8-15 cm and more inflorescences appeared, the infection could be carried out. Inoculate Agrobacterium (EH105) with the target fragment (pCAMBIA1391-Full-length, pCAMBIA1391-Del1, pCAMBIA1391-Del2) into 3 mL liquid LB medium, shake the bacteria for 24 hours at 28°C and 250 r/min. Inoculate 1 mL of overnight culture solution into 250 mL of LB medium containing 100 mL of liquid, shake the bacteria at 28°C, 250 r/min, until OD600 in the logarithmic growth phase = 0.8-1.6. The cultured bacteria liquid was divided into 50mL centrifuge tubes, and the bacteria were collected by centrifugation at 4000r/min for 15min. Add 5% sucrose to resuspend the cells, gently pipette repeatedly to disperse the cells into single cells, and add 0.05% Sillwet L-77 before transformation. Dip the inflorescences of Arabidopsis thaliana into the bacterial solution containing Agrobacterium for 30sec~2min, and cultivate the infected Arabidopsis thaliana in the dark for 24 hours, then turn to normal growth. Arabidopsis thaliana seeds can be harvested in batches after they mature, and the harvested seeds are placed in a 2 mL centrifuge tube and stored at 4°C. First, heat and dissolve the solid 1/2MS medium. When the temperature of the medium drops below 60°C, add 50mg/ml carbenicillin and 50mg/mL hygromycin B to make the final concentration 75mg/L carbenicillin and 75mg/L Hygromycin B. Spread it on the plate after mixing, and blow it on the ultra-clean workbench for more than half an hour. Soak the Arabidopsis seeds in 70% alcohol for 1 min, then sterilize the seeds with 2% sodium hypochlorite solution for 4-5 min, wash with sterile water for 3-4 times, suspend the Arabidopsis seeds with sterilized 0.1% agar, and use a pipette The liquid container was repeatedly sucked and beaten evenly on the resistant flat plate. Seal with a Parafilm film, at a temperature of 25°C, a relative humidity of 75% to 80%, and 16 hours of light per day. When the true leaves grow to 4-5 pieces, wash off the residual culture medium on the root, transplant it into the bud culture medium (Pei Lei Company in Zhejiang), cover the film for 2 days to keep it moist, and then carry out follow-up experiments according to the needs of the experiment. Harvest the seeds by dividing the plants when they are mature. Del1 and Del2 respectively obtained 10 and 15 transgenic positive seedlings of T1 generation identified by PCR. The same screening method was used to screen resistant seedlings of T2 generation, and the positive seedlings of T2 generation were used to analyze the expression pattern of the promoter.

3) Result analysis: through GUS tissue staining of positive seedlings. The results showed that GUS gene was strongly expressed in the first pair of leaves and the bottom of the stem driven by different fragment lengths of the GSTU19P promoter, and each fragment could respond to the induction of SA. Among them, the mature leaf tips of Del 2 positive seedlings also expressed strongly. The deletion promoter Del 1 was highly expressed before and after treatment, indicating that the -300bp promoter fragment already had a strong function of initiating transcription. For Del 2, although there is relatively strong expression, it can be seen that there is no GUS gene expression in the stem of Del 2. Compared with Del 1 and Full-length, this phenomenon may be due to the existence of elements controlling tissue-specific expression

Example 4 Transient expression of Arabidopsis mesophyll protoplast transformation using GSTU19P promoter

Construction of the promoter vector: Design 3 fragments with different sizes, respectively 1856bp (numbered as Full-length), 702bp (numbered as Del 2), 329bp (numbered as Del 1), and load the above fragments into the vectors pCAMBIA1301 and On pCAMBIA1302 (purchased from the CAMBIA laboratory of Australia); it is named pCAMBIA1301-Full-length, pCAMBIA1301-Del1, pCAMBIA1301-Del2, pCAMBIA1302-Full-length, pCAMBIA1302-Del1, pCAMBIA1302-Del2 (see Fig. 6 and Fig. 7 ), the used primers of the pCAMBIA1301 series vectors (Full-length, Del1 and Del2) and pCAMBIA1302 series vectors (Full-length, Del1 and Del2) in the present embodiment, the technical procedures for amplification conditions, vector construction and the transformation of Agrobacterium Method is described with embodiment 3. Arabidopsis mesophyll protoplasts were transformed using the PEG-calcium-mediated method (Yoo, et al. 2007) (Fig. 8). The specific method is: select a robust Arabidopsis plant that has grown to 3-4 weeks old, take the 5th to 7th true leaves, cut off a 0.5-1mm wide tissue in the middle of the blade with a blade, and quickly remove the cut tissue. Put it into the enzymatic hydrolysis solution and submerge completely. Use a vacuum desiccator (Shanghai Jinghong Experimental Equipment Co., Ltd. DZF-6050, operate according to the instructions) to vacuum infiltrate for 30 minutes. Enzymolysis reaction at room temperature for more than 3 hours. The quality of the extracted protoplasts was examined under a microscope. Then dilute the enzymatic solution with an equal volume of W5 Solution, and filter the non-enzymolyzed leaf tissue with a 75 μm blood nylon membrane filter. Centrifuge at 100 g for 2 min at room temperature, and remove the supernatant. Count the number of protoplasts with a hemocytometer. Resuspend with W5 Solution to make the cell concentration 2×10 ⁵ cells/ml, and place on ice for 30 min. Remove the supernatant and resuspend with MMG Solution to make the cell concentration 2×10 ⁵ cells/ml to obtain the mesophyll protoplasts of Arabidopsis thaliana and place them at room temperature. Add 10 μL of plasmid DNA (10-20ug) to a 2ml centrifuge tube, then add 100 μL of Arabidopsis mesophyll protoplasts (about 2×10 ⁴ ), and mix gently. Add 110 μl of 30% polyvinyl alcohol (PEG) solution and mix gently. Place at room temperature for 5-15 minutes. Dilute with 440μl W5 Solution, mix upside down to terminate the reaction. Centrifuge at 100 g for 2 min at room temperature. Remove the supernatant, add Arabidopsis thaliana mesophyll protoplasts into a 6-well culture plate containing 1mL WI Solution, incubate at room temperature for 3-8h, add 0.1mmol/L salicylic acid after transformation, incubate for 16h, and collect the transformation by centrifugation at 100g for 2min Later protoplasts. All the drug configuration and operation methods in this example are in accordance with the method reported by Yoo et al. (Yoo, et al. 2007).

The Arabidopsis thaliana mesophyll protoplasts transformed with pCAMBIA1302 series were used for GFP fluorescence observation, and the results are shown in FIG. 8 . It can be seen from Figure 8 that the protoplasts transformed with pCAMBIA1302 empty vector had the strongest fluorescence intensity, indicating that although the GSTU19 promoter has been able to activate the expression of the GFP gene, the intensity is not as strong as that of the CaMV35S promoter. Comparing the two groups of pictures treated with salicylic acid and untreated, it can be found that the fluorescence intensity of the control in Del 1 is greater than that of the treatment group; while in Del 2 group, the expression of GFP after salicylic acid treatment is stronger, indicating the promoter fragment of -300bp It already has the function of initiating transcription; and there may be a salicylic acid inducible element between -700~-300bp (Fig. 8).

At the same time, in this example, the Arabidopsis thaliana mesophyll protoplasts transformed with pCAMBIA1301 series were used for quantitative analysis of GUS activity in parallel, and the transformation method was the same as that of pCAMBIA1302. The fluorescent quantitative determination of GUS activity refers to Jefferson's method (Jefferson, et al.1987), and its result is as shown in Figure 9, as can be seen from Figure 9, in the control group that does not have salicylic acid treatment, the GUS expression intensity of pCAMBIA1301 empty vector is stronger Compared with the other three recombinant vectors, it shows that the GSTU19P promoter can promote gene expression without any treatment, but the activity is not as strong as that of the CaMV35S promoter (pCAMBIA1301 empty vector is used to promote GUS expression by the CaMV35S promoter).

The nucleotide sequences of the related primers designed by the present invention in table 2

Comparing the control group and the treatment group, under the premise that the CaMV35S promoter is a constitutively expressed promoter, no matter whether salicylic acid is administered or not, the promoter activity will not be affected (see group 1301 in Figure 9). In the Del1 deletion group, the expression of GUS was slightly increased after salicylic acid treatment; in the Del 2 deletion group, the expression of GUS was stronger after salicylic acid treatment, which was 5 times that of the group without salicylic acid treatment; while in the Full- In the length group, the expression of GUS before and after treatment was the same, indicating that the -300bp promoter fragment of the GSTU19P promoter of the present invention has the function of initiating transcription; and there may be salicylic acid between -700～-300bp Acid-inducible elements (Figure 9).

main reference

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Claims (3)

1. A promoter of the red navel orange GSTU19 gene induced by salicylic acid, the nucleotide sequence of which is shown in the sequence table SEQ ID NO:1. 2. The application of the promoter described in claim 1 in the genetic improvement of Arabidopsis thaliana. 3. The application according to claim 2, which comprises the application in regulating Arabidopsis resistance to stress or secondary metabolism through the response to salicylic acid.

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