CN104109682B - A kind of pectin lyase BnPL gene and promoter thereof and application - Google Patents

Abstract

The invention discloses a kind of pectin lyase BnPL gene and promoter thereof and application.The present invention uses homologous sequence method, on the basis of arabidopsis pectin lyase enzyme sequence, by cD NA RACE technology, clone obtains the c DNA of cabbage type rape pectin lyase BnPL gene, genomic DNA, code area, and utilizes genome walking method to obtain the promoter of BnPL gene on this basis.The present invention utilizes recombinant vector to carry out the prokaryotic expression of BnPL gene, shows that this gene has pectin lyase activity；By building the antisense expression vector of BnPL, illustrate that BnPL has the function promoting angle fruit cracking；Expressed by the promoters driven gus gene of BnPL gene, illustrate that the function of BnPL gene may be relevant to the separation of cell.These results are all expressed BnPL gene and are participated in cell separation process, utilize antisense technology operation BnPL gene can create the vegetable material at cracking resistance angle.

Description

A kind of pectin lyase BnPL gene and its promoter and application

technical field

The invention belongs to the fields of biotechnology and plant genetic engineering. It specifically relates to a pectin lyase BnPL and its coding gene and application.

Background technique

Rapeseed silique cracking is one of the main factors constituting the reduction of rapeseed yield, and it is also the most prominent problem in current mechanized harvesting. The loss caused by cleavage in production is generally 10%-20%, and the loss will be as high as 50% when the harvest period encounters rainy and high temperature weather. Rapeseed horn cracking is not conducive to mechanized harvesting. Improving the cracking horn resistance of rapeseed siliques is a major problem that needs to be solved urgently in the process of mechanized harvesting. Strengthening the research on the cracking horn resistance traits of rapeseed is of great significance to the sustainable development of rapeseed production.

Pectin is the main component of plant cell wall, which can play a role in the interconnection process between cells, and plays an important role in maintaining the structure of plant cell wall. In addition, pectin can also act on plant disease resistance traits and signal transduction. Pectin lyase gene is a general term for a class of enzymes that can degrade pectin. It exists widely in microorganisms and plant tissues. The elimination mechanism breaks the α-1,4 bond of galacturonic acid, which is a lyase gene that can degrade the cell wall. During the growth and development of plants, it is accompanied by the modification of the cell wall and the change and degradation of its components. The pectin lyase gene does this by degrading pectin in the cell wall. In plants, the pectin lyase gene was first isolated during tomato flower development and has flower developmental properties. The pectin lyase gene can degrade the pectin substance in the pollen cell wall, which leads to the relaxation of the pollen cell wall and the germination of the pollen tube. In Arabidopsis, the pectin lyase gene can not only affect tissue decay but also induce the disease resistance system of Arabidopsis plants.

In climacteric fruit, pectin lyase gene can affect fruit firmness. Two pectin lyase genes, Pel-1 and Pel-2, were isolated from the fruits of ripe bananas, both of which were expressed only in mature siliques, and were expressed in green siliques, leaves, and roots. Not expressed, indicating that the two pectin lyase genes play an important role in the ripening and softening of fruits. In strawberry, a vector of antisense expression pectin lyase gene driven by 35S promoter was constructed to transform strawberry. In the positive strawberry lines with antisense expression pectin lyase gene, the expression level It is 30% lower than the control, and the phenotype is manifested in that the yield is reduced, the content of pectin lyase in mature fruit is lower than that of the control, and the fruit is harder. The cytological structure of the two antisense gene-transferred transgenic lines was observed, and it was found that the gap between cells in the antisense pectin lyase gene-transferred fruit was smaller than that of the control, and the combination between two adjacent cells The ability is stronger, and the fruit is more resistant to storage. These experiments proved that the pectin lyase gene can control the connection between adjacent cells and play an important role in maintaining the binding ability between fruit cells.

Since brassica napus cleavage involves the degradation of detached cells, the pectin lyase gene may play a role in the development of rapeseed siliques. Therefore, the function of the pectin lyase gene was studied by reverse genetics. It is of great significance to create and cultivate rapeseed varieties with strong resistance to cracking horns.

Contents of the invention

The object of the present invention is to provide a pectin lyase BnPL gene, and the present invention provides the nucleotide sequence of this gene and the amino acid sequence of its protein. The gene is involved in the process of cell separation, and manipulation of the BnPL gene using antisense technology can create plant material resistant to cleavage horns.

Another object of the present invention is to provide a method for preparing the pectin lyase BnPL gene, which method finds three genes in the Brassica napus EST sequence database according to the sequence of the pectin lyase gene (AT5G15110) of Arabidopsis thaliana A well-matched sequence, primers were designed according to these three EST sequences, PCR was carried out with the Brassica napus cDNA as a template, and after the PCR product was sequenced, RACE primers were designed according to the amplified sequence fragments for RACE reaction, and the entire DNA was amplified. Long cDNA sequence, so as to obtain the full-length cDNA sequence. Specific primers were designed based on the full-length cDNA sequence, and the full-length sequence of the BnPL gene was amplified using rapeseed genomic DNA as a template.

Another object of the present invention is to provide a preparation method of the pectin lyase BnPL gene promoter, design primers according to the full-length sequence of the pectin lyase BnPL gene, and use the genome walking method in Brassica napus line R2 The promoter sequence of pectin lyase BnPL was amplified and assembled. Specific primers were designed according to the spliced sequence of the spliced pectin lyase BnPL promoter, and the full-length sequence of the gene promoter was amplified.

Another object of the present invention is to provide an application approach of pectin lyase BnPL gene in improving plant horn cleavage resistance.

Technical scheme of the present invention is realized by the following methods:

A pectin lyase BnPL gene, the nucleotide sequence of which is shown in SEQ ID NO.3, with a total length of 1416bp, encoding 471 amino acids in total.

A pectin lyase BnPL gene, the amino acid sequence of the expressed protein is shown in SEQ ID NO.4.

The application of the pectin lyase BnPL gene in improving the resistance of plants to cleavage.

The nucleotide sequence of the promoter of the pectin lyase BnPL gene is shown in SEQ ID NO.5.

The application of the pectin lyase BnPL gene promoter in the regulation of plant silique development.

1. A pectin lyase BnPL gene is obtained through the following steps:

Step 1, Cloning of the full-length cDNA sequence of the BnPL gene

a. According to the sequence of the Arabidopsis pectin lyase gene (AT5G15110), three well-matched sequences were found in the Brassica napus EST sequence database: GR462449.1, GR453547.1, GR463150.1, according to these three ESTs Sequence design primers were amplified to obtain a small fragment sequence, which was used as a template for primer design after splicing: PL-F-1 (5'-CGAGGAAGAGGACGACATTGAG-3') and PL-R-1 (5'-CGAATCCTCGGCATCCTCTG-3' );

b. PCR amplification was performed using the reversed cDNA of RNA in the developmental stage of Brassica napus strain R2 silique as a template, and the transformation was sent to Yingjun Biotechnology Service Co., Ltd. (Shanghai) for sequencing.

c. In order to obtain the full-length sequence of the gene, use RACE technology to amplify the full-length cDNA sequence, and use the amplified sequence fragment to design RACE primers: PL-RACE-5 (5'-TCCATCGGCCTGTCCTCTAAACC-3') and PL-RACE- 3 (5'-GTACAGGACCACAGGAGGCAAACG-3') for RACE reaction. The full-length cDNA sequence of the BnPL gene was obtained after sequencing and splicing of the amplified products: as shown in SEQ ID NO.2, the length is 1796bp, which includes a 5'UTR of 178bp, a 3'UTR of 172bp, and a polyA tail of 32bp .

Step 2, obtain the full-length DNA sequence of the BnPL gene

a. Design primers according to the full-length cDNA sequence of the gene. The primer combination is Mq-Pectate-5-3: (5'-CGGATCCATGGAGACAGCTAGGCTTTTC-3'), Mq-Pectate-3-1: (5'-CCGGAATTCTTAGCATGGTTTTCCGACC-3 ');

b. Using the genomic DNA of rapeseed line R2 as a template PCR to obtain the BnPL full-length gene fragment, the fragment is recovered and sequenced to obtain the sequence BnPL full-length DNA gene sequence, as shown in SEQ ID NO.3, its total length is 2468bp, composed of 3 It consists of 4 introns and 4 exons.

2. Acquisition of the promoter of pectin lyase BnPL gene

Step 1, according to the full-length sequence of pectin lyase BnPL gene, design 6 specific primers: SP1 (5'-ACTCATCGGTCTCCGCAACA-3'), SP2 (5'-GGAATCAAACTGGCAATACAAATCAC-3'), SP3 (5'-TTTCTGTTTGGTAGATGAACCGTATT -3'), SP11 (5'-TCGCAGACTAATGGGTTGGTAAA-3'), SP12 (5'-ACGACGCCGCTTGAGTG-3') and SP13 (5'-CCTTATAGACGCCAGCCACGA-3').

Step 2, using the rapeseed genomic DNA as a template, the nucleotide sequence of the genomic DNA, using the Genome-walking kit (Takara company) to amplify the rapeseed BnPL promoter sequence by genome walking:

For the first step, the AP2 primer provided by the kit was used as the upstream primer, and the SP1, SP2, and SP3 primers were respectively used as the downstream primers to perform 3 rounds of thermal asymmetric PCR reactions;

In the second step, the AP4 primer provided by the kit was used as the upstream primer, and the SP11, SP12, and SP13 primers were respectively used as the downstream primers to carry out three rounds of thermal asymmetric PCR reactions.

After sequencing the PCR fragments after the two steps, sequence assembly was performed to obtain the promoter sequence, the nucleotide sequence of which is shown in SEQ ID NO.5, and the total length of the sequence is 1955bp.

3. Carrier Construction

1) Construction of pectin lyase BnPL gene antisense vector

a. The primer combination used is Fanyi-3-1: (5'-CGGATCCTTAGCATGGTTTTCCGACC-3') and Fanyi-5-1: (5'-CCGGAATTCATGGAGACAGCTAGGCT-3');

b. Using rapeseed cDNA as a template to amplify, the fragments were recovered and inserted into the PMD18 simple T vector, and sequenced by M13 primers;

c. After confirming the integrity of the sequence, use BamHI/EcoRI to digest the recombinant vector, and also use BamHI/EcoRI to digest the original PBI121 vector to remove the GUS gene sequence;

d. Subcloning the two enzyme-cut fragments with the BnPL gene into the PBI121 expression vector, so that the foreign gene sequence replaces the position of the reporter gene GUS in the vector, where the sense vector is replaced in the forward direction, and the antisense vector is reverse direction instead.

2) Construction of pectin lyase BnPL gene promoter recombinant vector

a. the amplification product of primer MQ-promoter-5-101 and MQ-promoter-3-3 (sequence sees above) in the rape genomic DNA is reclaimed, inserted in the simple T carrier of PMD18, sequenced through M13 primer;

b. After confirming the integrity of the sequence, use ClaI/XbaI to digest the recombinant vector, and also use ClaI/XbaI to digest the original PBI121 vector to remove the 35S sequence;

c. Then clone the restriction fragment with the BnPL promoter into the PBI121 expression vector, so that the BnPL promoter replaces the 35S promoter sequence in the vector.

4. Plant transformation

The recombinant new plasmid PBI121 expression vector containing the target pectin lyase BnPL gene and its promoter was introduced into Agrobacterium (EHA105), and rape and Arabidopsis were transformed by Agrobacterium-mediated method, and Arabidopsis was transformed into flower buds by inflorescence dipping method , rape transformed hypocotyls by tissue culture method. The transgenic plants were screened using 1/2MS medium containing 50mg/L kanamycin, and verified by PCR. The primer combination used was Kan-5 and Kan-3, and the target gene amplified was kanamycin T3 transgenic plants were used for phenotypic analysis after two consecutive generations of selection.

The term "transgenic plant" used in the present invention refers to a plant containing an introduced target gene and capable of stably enhancing or inhibiting the expression of the introduced gene and producing specific biological traits.

The plants mentioned in the present invention include food crops such as rice, wheat, and corn that are greatly affected by high temperature and adversity, economic crops such as rapeseed, soybean, and cotton, and vegetable crops such as cucumbers and tomatoes that grow in summer.

Extracting plant leaf DNA is a commonly used molecular biology technique. There are also many mature techniques for extracting mRNA. The enzyme cutting, ligation, and Agrobacterium mediated enzymes used to construct the vector construction described in the present invention and transfect the vector into plants are commonly used. Methods such as transformation of rapeseed hypocotyls and infection of Arabidopsis inflorescences are also common techniques in this field.

The DNA Marker (100bp) used in the present invention, dNTPs (10mmol/L) are all purchased from Beijing Dingguo Biological Company; Total RNA extract Trizol and RNA reverse transcription kit are purchased from Invitrogen Company, and DNase is purchased from Promega Company, DNA recovery and purification kits were purchased from OMEGA Corporation. All other reagents were of domestic analytical grade. The cloning vector pMD18-T, Escherichia coli DH5α, and Ex Taq polymerase (5U/μL) were purchased from Treasure Bioengineering (Dalian) Co., Ltd. The PBI121 vector was preserved by the applicant, and the rapeseed line R2 was provided by the Rapeseed Heterosis Utilization Project of the Institute of Oil Crops, Chinese Academy of Agricultural Sciences.

The present invention clones the pectin lyase BnPL gene, constructs an antisense plant expression vector to transform rapeseed and Arabidopsis thaliana, and clarifies that the gene participates in the cell separation process in the development of plant siliques, and plays an important role in improving the performance of plant horn resistance. important role. Using the antisense technology, the applicant can use the gene to artificially create plant materials resistant to cleavage horns. In positive plants transformed with antisense vectors in Arabidopsis and rapeseed, the crack resistance of siliques was enhanced.

The present invention clones the pectin lyase BnPL gene and its promoter, constructs a plant expression vector containing the GUS gene driven by the promoter, and transforms Arabidopsis thaliana into anthers, petal stigmas and horns whose GUS activity matures only in the reproductive growth stage. It is detected in the fruit, especially at the top of the silique, the pseudoseptum and the junction of the silique and the stalk. It shows that the promoter of pectin lyase BnPL has tissue specificity, and its expression plays an important role in the regulation of plant silique development.

detailed description

For the molecular biology experimental methods not specifically described in the following examples, all refer to Sambrook et al.'s "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989), or Draper et al. (Blackwell Science Publishing House, 1988) the specific methods or conditions listed in the book, or in accordance with the conditions recommended by the manufacturer of the reagents used.

Embodiment 1 A kind of acquisition of pectin lyase BnPL gene

1.1 Extraction of total DNA and total RNA

The plant DNA was extracted by the CTAB method; the total RNA of the plant was extracted by the Triozol method, and the DNA contamination in the RNA was removed by DNase digestion. The quality of DNA and total RNA was detected by 1% agarose gel electrophoresis. Using Takara's M-MLV RTase cDNA kit, 2 μg of total RNA was reverse transcribed into cDNA according to its instructions.

1.2 Cloning of full-length cDNA sequence of BnPL gene

According to the sequence of Arabidopsis thaliana pectin lyase gene (AT5G15110), a homology search was carried out in the Brassica napus EST sequence database, and three well-matched sequences were found, and the EST numbers were GR462449.1 and GR453547.1 and GR463150.1, these three sequences were spliced with CAP3 software, and the obtained sequences were used as templates to design primers: PL-F-1 (5'-CGAGGAAGAGGACGACATTGAG-3') and PL-R-1 (5'-CGAATCCTCGGCATCCTCTG- 3'), PCR amplification was carried out using the reversed cDNA of RNA in the R2 silique of Brassica napus R2 as a template, and the reaction conditions were: 95°C, 2min; 94°C, 30s, 55°C, 30s, 72°C, 1min, total 34 cycles; finally, 72°C extension for 10 min; PCR products were detected and recovered by 1.2% agarose gel electrophoresis. The resulting fragment was cloned into the pMD18-T vector, transformed into Escherichia coli DH5α, the recombinants were screened by blue and white spots, and the correct positive clones identified by colony PCR were sent to Yingjun Biotechnology Service Co., Ltd. (Shanghai) for sequencing. The result was a 822bp clone sequence. In order to obtain the full-length sequence of the gene, the full-length cDNA sequence was amplified by RACE technology, and the amplified 822bp sequence was used to design RACE primers for RACE reaction. The primers for 5' and 3' RACE were PL-RACE-5(5 '-TCCATCGGCCTGTCCTTCTAAACC-3') and PL-RACE-3 (5'-GTACAGGACCACAGGAGGCAAACG-3'). The synthesis of the first strand of cDNA and the configuration and amplification of the PCR reaction system in the process of 3'RACE and 5'RACE were carried out according to the instructions of SMARTer RACE cDNA Amplification Kit of Clontech Company. The main components are: cDNA first strand 2.5 μL, RACE primer (10 μmol/L) 1.0 μL, UPM (10×) 5.0 μL, Master Mix 41.5 μL. The PCR reaction program is: 94°C, 30s, 72°C, 3min, 5 cycles; 94°C, 30s, 70°C, 30s, 72°C, 3min, 5 cycles; 94°C, 30s, 68°C, 30s, 72°C , 3min, 27 cycles; a total of 37 cycles. After the PCR reaction, 5.0 μL of 3'RACE and 5'RACE products were taken and detected on 1.2% agarose gel electrophoresis, and the RACE products were recovered by using the gel recovery kit of OMEGA Company, and cloned and sequenced after ligation with pMD18-T vector . The results showed that the length of the 3'RACE product was 1322bp, and the length of the 5'RACE product was 982bp. There is a 361bp sequence repeat in the 3'RACE and 5'RACE products, and the full-length sequence of the BnPL gene is obtained after splicing by CAP3 software, with a length of 1796bp, including 178bp 5'UTR, 172bp 3'UTR, and There is a 32bp polyA tail.

The full-length cDNA sequence of the BnPL gene, analyzed by ORF find software, shows that its open reading frame sequence is 179bp to 1594bp, the total length is 1416bp, and a total of 471 amino acids are encoded, and a rapeseed pectin lyase gene is obtained, and its base sequence is Nucleotide sequence shown in SEQ ID NO.1. Using the online domain analysis software http://smart.embl-heidelberg.de/smart analysis showed that the protein encoded by the gene had a pectin lyase gene domain (domain). This sequence was searched and compared with Blaxtp on the public database NCBI to find homologous proteins. The results showed that the BnPL of rapeseed had a high similarity with some pectin lyase gene proteins of Arabidopsis thaliana. In order to compare the homology relationship of pectin lyase gene sequences in different species, 24 pectin lyase gene proteins were extracted, including 6 Arabidopsis pectin lyase gene family proteins, involving 18 species. By constructing a phylogenetic tree, the results showed that BnPL was very consistent with the two proteins of Arabidopsis thaliana, and the corresponding gene numbers of Arabidopsis were At5g15110 and At3g01270. The sequences of Arabidopsis pectin lyase gene family proteins have diverged during the evolution process, which may lead to the functional differentiation of different pectin lyase genes.

1.3 PCR amplification of the full-length sequence of pectin lyase BnPL gene

In order to obtain the full-length DNA sequence of the Brassica napus BnPL gene, the primer combination was designed according to the cDNA sequence of the gene: Mq-Pectate-5-3:(5'-CGGATCCATGGAGACAGCTAGGCTTTTC-3'), Mq-Pectate-3-1:( 5'-CCGGAATTCTTAGCATGGTTTTCCGACC-3'). Using the genomic DNA of R2 as a template to amplify the full-length sequence of the BnPL gene, the PCR amplification system is: 20 μL reaction system, containing 8.75 μL of ddH2O, 2 μL of 10×LA PCR buffer, 4 μL of 2.5 mmol/L dNTP, 2 μL MgCl2 (25mM), 1μL PCR primer 10mmol/Lprimer, 1μL 100mmol/L template DNA, and finally add 0.25μL Takara LA Taq (5U/μL). The reaction conditions were: 95°C, 3min; 94°C, 45s; 50°C, 45s; 72°C, 3min; 34 cycles; finally, 72°C extension for 10min. The PCR product was detected by 1.2% agarose gel electrophoresis, and the obtained fragment was recovered for cloning and sequencing. As a result, a 2468bp genome sequence of BnPL was obtained. Use this sequence and cDNA sequence to analyze and compare on the website http://gsds.cbi.pku.edu.cn/ to determine the positions of exons and introns. The results show that the Brassica napus BnPL gene consists of 3 introns Son and 4 exons.

Amplification and vector construction of embodiment 2BnPL gene promoter

2.1 Amplification of BnPL gene promoter

Genome-walking kit (Takara Company) was used to amplify the rapeseed BnPL promoter sequence using rapeseed genomic DNA as a template. According to the full-length sequence of the BnPL gene, six specific primers SP1 (5'-ACTCATCGGTCTCCGCAACA-3'), SP2 (5'-GGAATCAAACTGGCAATACAAATCAC-3'), SP3 (5'-TTTCTGTTTGGTAGATGAACCGTATT-3'), SP11 (5'-TTTCTGTTTGGTAGATGAACCGTATT-3') were designed. '-TCGCAGACTAATGGGTTGGTAAA-3'), SP12 (5'-ACGACGCCGCTTGAGTG-3') and SP13 (5'-CCTTATAGACGCCAGCCACGA-3'). For the first step, the AP2 primer provided by the kit was used as the upstream primer, and the SP1, SP2, and SP3 primers were respectively used as the downstream primers. Genomic DNA of rapeseed line R2 was used to perform three rounds of thermal asymmetric PCR reactions. The last round of product was recovered and purified, and ligated with T-vector, and its length was found to be 1018bp by sequencing. Because the sequence is short, a second walk is performed on the basis of the sequenced sequence. In the second step, the AP4 primer provided by the kit was used as the upstream primer, and the SP11, SP12, and SP13 primers were respectively used as the downstream primers to carry out three rounds of thermal asymmetric PCR reactions. The sequence length of the third round of PCR primers was 1431bp. Due to the presence of repeated sequences between the two walks, a total of 2339bp leader sequences were obtained after splicing according to the repeated sequences. (The specific PCR process is carried out in strict accordance with the kit instructions. PCR product electrophoresis detection, recovery, cloning, and sequencing are the same as above.)

2.2 Vector construction of pectin lyase BnPL gene promoter

Using the genomic DNA of rapeseed line R2 as a template, according to the obtained 2339bp leader sequence design primer combination MQ-promoter-5-101 and MQ-promoter-3-3 for PCR, Mq-promoter-5-101: (5'-CATCGATGTGCTGATACCAGCGTAGTCA -3'); Mq-promoter-3-3: (5'-CTCTAGATTTTTTTTAATTTGATTACCCCTTTTTGCT-3'), to verify the authenticity of the promoter sequence obtained by Genome-walking. The PCR amplification system is: a total of 20 μL reaction system, containing 10.75 μL of ddH2O, 2 μL of 10×ExTaq buffer, 2 μL of 2.5 mmol/L dNTP, 2 μL of MgCl2 (25 mM), 1 μL of 10 mmol/L PCR primers, 1 μL of 100 mmol/L cDNA, 0.25 μL of Takara Ex Taq (5 U/μL). The reaction conditions are: 95°C, 2min; 94°C, 30s, 60°C, 30s, 72°C, 3min, a total of 34 cycles; finally, 72°C extension for 10min. The PCR products were detected by 1.2% agarose gel electrophoresis, and the resulting fragments were recovered for cloning and sequencing. The results show that the sequence of the walk is identical to the sequence obtained by conventional PCR amplification. Since the length of the sequence amplified by the primers MQ-promoter-5-101 and MQ-promoter-3-3 is 1955bp, the online promoter analysis software http: //www.fruitfly.org/seq_tools/promoter.html predicts that this fragment has the basic characteristics of a promoter and can represent the promoter sequence of pectin lyase BnPL.

Prokaryotic expression verification of embodiment 3BnPL gene function

According to the obtained cDNA sequence of BnPL, the primer combination was designed to amplify the coding region of the BnPL gene. -3'). The template is the cDNA of the silique development stage of the rape line R2, and the PCR amplification system is: a total of 20 μL reaction system, including 10.75 μL of ddH2O, 2 μL of 10×Ex Taq buffer, 2 μL of 2.5mmol/LdNTP, 2 μL of MgCl2 (25mM) , 1 μL of 10 mmol/L PCR primers, 1 μL of 100 mmol/L cDNA, 0.25 μL of TakaraEx Taq (5 U/μL). The reaction conditions are: 95°C, 2min; 94°C, 30s, 60°C, 30s, 72°C, 1min, a total of 34 cycles; finally, 72°C extension for 10min; PCR products were detected and recovered by 1.2% agarose gel electrophoresis, inserted Go to the PEASY-E prokaryotic expression vector (preserved in our laboratory), verify its insertion direction by PCR, and check positive colonies by colony PCR. The self-primers MQ-PL-5-1 and MQ-FUL-PL-3-1 were used for sequencing to confirm the correctness and completeness of the sequence. The positively inserted vector was transformed into Escherichia coli BL21 for prokaryotic expression. First use the LB plate containing ampicillin to screen the positive single clone, pick out the single clone and inoculate it into 2ml of LB liquid medium containing ampicillin, overnight at 37°C. Inoculate 0.5 ml of the overnight cultured bacterial solution into 50 ml of AMP-containing LB liquid medium and incubate at 37°C for 3 hours to make the OD600 reach 0.5. IPTG was added to make the total concentration reach 1 mM to induce expression, and culture was continued at 37°C. Take 1ml of the samples induced by IPTG for 2 hours, 4 hours, 6 hours, 8 hours and 10 hours respectively, centrifuge at 12000rpm for 0.5 minutes, discard the supernatant, add 100ul of 1 times SDS loading buffer, shake and mix well, Heat in boiling water for 5 minutes, centrifuge at 12,000 rpm for 0.5 minutes, and then perform electrophoresis, using a standard SDS-PAGE electrophoresis process for electrophoresis. After induction by IPTG, precipitation and electrophoresis of the induction solution showed that the recombinant protein produced a 62kD protein, and the recombinant protein induced by IPTG for 2 hours, 4 hours, and 6 hours could produce the target protein. And after ultrasonic treatment, the target protein can be detected in both the precipitate and the supernatant, indicating that the recombinant protein is a soluble protein.

Purification of recombinant protein using Ni column, the main process is: centrifuge the induced cultured Escherichia coli, resuspend in GuNTA-0 buffer, add 1mM PMSF to suspend the cells, add lysozyme, digest overnight at 4°C, use The wall was broken by ultrasonic wave, centrifuged at 10000rpm for 10 minutes, and the supernatant was slowly added to the Ni column for purification in sections, and the purification steps were carried out according to the instructions.

Enzyme activity detection adopts the standard galacturonic acid method, and the process is: get 0.5ml test solution and add 2.5ml of 0.07M Tris-HCl (PH=8) containing 0.3% polygalacturonic acid, and add 30ul. .1M CaCl2, react at 37 degrees for half an hour, then add 0.1ml of 1M NaOH to terminate the reaction; after heating in boiling water for 5 minutes, centrifuge at 10000rpm for 2 minutes, take the supernatant and measure the absorbance at 232nm. Set up a negative control, do not add the enzyme solution to be tested in the above process, and the other processes are completely the same as the above process. The soluble protein was purified by Ni column, and the enzyme activity was measured. The results showed that the recombinant protein had typical pectin lyase gene activity under the action of Ca ions.

Embodiment 4 plant transformation test

Mainly by constructing antisense expression vectors to carry out transgenic experiments of Arabidopsis and rapeseed, to verify the function of BnPL gene. The construction process was as follows: the primer combinations used were Fanyi-3-1: (5'-CGGATCCTTAGCATGGTTTTCCGACC-3') and Fanyi-5-1: (5'-CCGGAATTCATGGAGACAGCTAGGCT-3'). Using KOD Plus polymerase (TOYOBO), the PCR system is 20 μL, mainly including 11 μL of ddH2O, 2 μL of 10×PCR buffer for KOD Plus, 2 μL of 2mmol/L dNTP, 1 μL of 25 μmol/L MgSO4, 1 μL of PCR primers, 1 μL of 100mmol /L cDNA, and finally add 1 μL KOD Plus (1U/μL) enzyme. The reaction conditions are: 95°C, 2min; 94°C, 30s, 60°C, 30s, 72°C, 2min, a total of 34 cycles; finally, 72°C extension for 10min. The PCR product was detected and recovered by 1.2% agarose gel electrophoresis, inserted into the PMD18 simple T vector, sequenced by M13 primers, and the integrity of the sequence was confirmed, and the recombinant vector was digested with BamHI/EcoRI, and BamHI/EcoRI was also used. EcoRI digests the original PBI121 vector and removes the GUS gene sequence. Then subclone the enzyme-digested fragment carrying the BnPL gene into the PBI121 expression vector, so that the foreign gene sequence replaces the position of the reporter gene GUS in the vector. The recombined new PBI121 vector containing the antisense target BnP L gene was introduced into Agrobacterium (EHA105), wild-type Arabidopsis was transformed by inflorescence dipping method, and rapeseed hypocotyl was transformed by tissue culture method. The screening of transgenic plants was carried out using 1/2MS medium containing 50 mg/L kanamycin, and verified by PCR. The primer combination used was Kan-5 and Kan-3, and the target gene amplified was kanamycin Gene, the PCR amplification system used is: Dream PCR MIX 10 μL (purchased from Fermentas), DNA template 1 μL, forward and reverse primers 2 μL each (10 μM/L), ddH2O 5 μL. The reaction conditions were: 95°C, 2min; 94°C, 30s, 60°C, 30s, 72°C, 1min, a total of 34 cycles; the last extension was 10min at 72°C; PCR products were detected by 1.2% agarose gel electrophoresis. Transgenic positive plants were selected for two consecutive generations, and T3 transgenic plants were used for phenotypic analysis.

RNA was extracted from flower buds of transgenic plants, cDNA was obtained by reverse transcription, and the expression level of pectate lyase gene in transgenic plants containing antisense BnPL gene expression vector was detected by primer combination pectate-75-f and pectate-244-r. Phenotype identification was performed on the plants expressing the antisense gene, and the wild type was used as a control. Compared with the wild-type Arabidopsis, the siliques of the progeny of the antisense-expressed transgenic positive Arabidopsis plants were mutated, and the siliques of most plants became shorter, and the siliques did not crack immediately after yellow ripening, even after prolonged storage. , The whole plant is placed for a long time without silique cracking. However, wild-type Arabidopsis siliques will all naturally crack once they mature and turn yellow. The siliques of the transgenic rapeseed plants containing the antisense BnPL gene expression vector changed slightly, and the length of the siliques of the antisense BnPL gene plants decreased slightly compared with the non-transgenic control. Using the random collision method, it was found that the siliques of the transgenic plants had significantly enhanced resistance to silique cracking. After 10 minutes of collision, only a few siliques of the transgenic plants were broken, while almost all siliques of the non-transgenic control plants were broken.

Example 5 Validation of promoter tissue-specific expression

The promoter of the BnPL gene is used to construct the expression vector. The construction process is as follows: first use the primers MQ-promoter-5-101 and MQ-promoter-3-3 to carry out PCR with the DNA of the rape line R2 as the template, and use 1.2% agarose for the PCR product It was detected and recovered by gel electrophoresis, and inserted into the simple T vector of PMD18. Sequencing with M13 primers and confirming the integrity of the sequence, the recombinant vector was digested with ClaI/XbaI, and the original PBI121 vector was also digested with ClaI/XbaI to remove the 35S sequence. Then subclone the restriction fragment with BnPL promoter into the PBI121 expression vector, so that the BnPL promoter can replace the 35S promoter sequence in the PBI121 vector.

Transgenic positive plants were analyzed by GUS staining. Preparation of GUS mother liquid mixture: the total volume is 1L, mainly containing 15.601g of sodium dihydrogen phosphate; Use NaOH to adjust the pH value to 7.0, and store the brown reagent bottle in a refrigerator at 4°C in the dark. Preparation of X-Glux: Weigh 0.06g of dry powder and dissolve it in 1.5ml of N,N-dimethylformamide, wrap it in tin foil and store it in a -20°C refrigerator for later use.

Put different tissues of positive plants into a 10ml centrifuge tube, add 8ml of GUS mother liquor mixture, then add 100μl of X-Glux solution, and then add 800μl of methanol, completely submerge the plant tissue, and leave it overnight at 37°C. Pour off the dye solution overnight, first decolorize the material with 75% ethanol, then decolorize with absolute ethanol, change the ethanol every 1 hour, until the tissue is transparent, generally need 2 to 3 times, and then place the tissue in 50 % ethanol for 5 minutes, finally put the transparent tissue into pure water, and observe the staining of the tissue under a microscope.

Observation results showed that GUS activity was not detected in roots, stems, and leaves, but GUS activity was only detected in flowers and siliques, and GUS activity could not be detected in the young flower bud stage, only after the flower buds opened, in the open petals and The activity of GUS can be detected in the mature anthers, and the GUS activity can be detected in the silique peel and DZ zone in the silique stage. It shows that the GUS activity is only detected in the tissues of the reproductive growth stage, but not in the tissues of the vegetative growth stage, indicating that the promoter of BnPL has tissue specificity, and mainly regulates the expression of genes in anthers, stigmas and siliques, Especially in the expression in siliques, the expression level is higher in the silique skin, the detachment area and the top of the immature siliques, and the joint between the siliques and the receptacle. These are the parts closely related to cell separation. It is speculated that the function of the BnPL gene Associated with the segregation of cells, it plays an important role in the development of siliques in plants.

Claims (3)

1. a pectin lyase BnPL gene, it is characterised in that: its nucleotides sequence is classified as shown in SEQ ID NO.3.

A kind of pectin lyase BnPL gene the most according to claim 1, it is characterised in that: described pectin lyase The expressing protein amino acid sequence of BnPL gene is as shown in SEQ ID NO.4.

3. the application in improving plant cracking resistance angle property of a kind of pectin lyase BnPL gene described in claim 1.