CN114381466A - A gene GbC4H encoding cinnamic acid-4-hydroxylase derived from cotton and its application - Google Patents

Abstract

The invention provides a coding gene GbC4H of cinnamic acid-4-hydroxylase from cotton and application thereof, belonging to the technical field of genetic engineering; the nucleotide sequence of the coding gene GbC4H is shown in SEQ ID NO. 2. The coding gene GbC4H of the cinnamic acid-4-hydroxylase can regulate and control the blight resistance of cotton and can also regulate and control the accumulation of flavonoids in the cotton. The invention uses gene silencing technology to promote the expression silencing of the coding gene GbC4H, which can cause the content of flavonoids to be reduced, so that the silenced cotton is more likely to be infected by fusarium oxysporum and is easy to catch diseases.

Description

A gene GbC4H encoding cinnamic acid-4-hydroxylase derived from cotton and its application

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a cotton-derived cinnamic acid-4-hydroxylase encoding gene GbC4H and applications.

Background technique

Monooxygenase refers to an enzyme that can directly activate molecular oxygen, so that an oxygen atom is added to the substrate molecule. In plants, monooxygenases affect the biosynthesis of various metabolites including fatty acids, phenylpropanoids, alkaloids, and terpenoids. Cinnamate-4-hydroxylase (C4H) is the first phenylpropane pathway monooxygenase identified from Arabidopsis thaliana. At present, the sequence and expression pattern of C4H have been reported in various plants such as poplar, tea, rapeseed, garlic, skullcap, cloverleaf, bird's tongue, sweet potato and angelica. For example, patent CN112410354A discloses a cinnamic acid-4-hydroxylase gene ThC4H derived from cloverleaf and its application. As one of the key enzymes in the metabolic pathway of phenylpropane, this gene can be used to produce resveratrol. In addition, CN102220353B discloses a novel coding sequence for cinnamic acid-4-hydroxylase (C4H) protein expressed in sweet potato, the gene is expressed in sweet potato, and the obtained transgenic sweet potato has The content of anthocyanins and their precursor compounds was increased. These reports provide important information for the further study of C4H in the synthetic pathway of phenylpropane.

Currently, there is no report that the gene encoding cinnamic acid-4-hydroxylase is associated with the accumulation of flavonoids.

SUMMARY OF THE INVENTION

In view of this, the object of the present invention is to provide a cinnamic acid-4-hydroxylase encoding gene GbC4H derived from cotton and its application, the cinnamic acid-4-hydroxylase encoded by the encoding gene GbC4H of the present invention can be regulated The ability of cotton to resist fusarium wilt can also regulate the accumulation of flavonoids in cotton.

The present invention provides a cotton-derived cinnamic acid-4-hydroxylase encoding gene GbC4H, whose nucleotide sequence is shown in SEQ ID NO.2.

The present invention also provides a recombinant vector into which the encoding gene GbC4H described in the above scheme is inserted.

The present invention also provides a recombinant bacteria, comprising the recombinant vector described in the above scheme.

The present invention also provides the application of the encoding gene GbC4H described in the above scheme or the recombinant vector or the recombinant bacteria; the application includes the application of any one or several of the following:

1) Regulate the resistance of cotton to fusarium wilt;

2) Regulate the flavonoid pathway of cotton;

3) Regulate the accumulation of flavonoids in cotton;

4) Fusarium wilt resistant cotton breeding;

5) Construction of transgenic cotton plants with reduced resistance to fusarium wilt.

Preferably, the flavonoids include flavonoids antibacterial substances.

The present invention also provides a method for improving the resistance to fusarium wilt of cotton and/or promoting the accumulation of flavonoids in cotton, comprising the following steps: overexpressing the encoding gene GbC4H in the above scheme in cotton.

The present invention also provides the application of a reagent for silencing the cinnamic acid-4-hydroxylase or the encoding gene GbC4H in the above scheme in constructing a transgenic cotton plant with reduced resistance to fusarium wilt and/or reduced accumulation of flavonoids .

Preferably, the reagent comprises a recombinant Agrobacterium; the recombinant Agrobacterium comprises a VIGS silencing vector encoding a specific fragment of the gene GbC4H; the nucleotide sequence of the specific fragment encoding the gene GbC4H is as shown in SEQ ID NO.35 Show.

Preferably, the original vector of the VIGS silencing vector is pTRV2 vector.

The present invention provides a cotton-derived cinnamic acid-4-hydroxylase encoding gene GbC4H, whose nucleotide sequence is shown in SEQ ID NO.2. The encoding gene GbC4H of the cinnamic acid-4-hydroxylase of the present invention can regulate the accumulation of flavonoids in cotton, thereby regulating the ability of cotton to resist fusarium wilt. In the embodiment of the present invention, the gene silencing technology is used to silence the expression of the encoding gene GbC4H, which will reduce the content of flavonoids, and make the silenced cotton more susceptible to Fusarium wilt infection and disease.

Description of drawings

Fig. 1 is the clone of GbC4H gene;

Figure 2 is the expression pattern of GbC4H gene in 06-146 and Xinhai 14 under hormone (MeJA and SA) treatment;

Figure 3 shows the results of GbC4H gene silencing efficiency detection and identification of resistance to Fusarium wilt, among which, A: positive control silencing phenotype, silencing CLA1 (cloroplastos alterados 1) as a positive control, positive control (pTRV2::CLA1) cotton plants Showing an albino phenotype; B: GbC4H gene silencing efficiency test, when the expression level was not infected with Fusarium wilt at 0 h, the expression level decreased by 86.6%, indicating that GbC4H gene silencing was successful; C: Empty control plants and plants with silenced GbC4H gene were resistant to Fusarium wilt Phenotypes of GbC4H gene-silenced plants (pTRV2::GbC4H) and empty control plants (pTRV2::00) after infection with F.

Fig. 4 is the detection of flavonoid metabolic pathway downstream genes and cotton fusarium wilt-related genes in GbC4H gene silenced plants;

Figure 5 shows the establishment of the standard curve of rutin in cotton and the content and antibacterial status of flavonoids in GbC4H gene-silenced plants; wherein, A: establishment of the standard curve of rutin in cotton, linear regression equation y=0.1322x+0.0968, R ² = 0.9925, that is, the content of flavonoids = (A508-0.0968)/0.1322*100; B: the content of flavonoids before and after infection of Fusarium wilt plants with empty control plants and plants with silent GbC4H gene, pTRV2::00a: empty control Flavonoid content in plants; pTRV2::00b: flavonoid content in plants infected with Fusarium wilt of empty control plants; pTRV2::GbC4Ha: flavonoid content in plants with silenced GbC4H gene; pTRV2::GbC4Hb: silenced GbC4H gene The flavonoid content in the plant after the plant was infected with Fusarium wilt; C: the antibacterial status of flavonoids, a: The sterile water and the same amount of Fusarium wilt were mixed at a ratio of 1:1 to set as the control group; b: The extracted flavonoids and the same amount of Fusarium wilt were mixed at a ratio of 1:1 to set up the experimental group, and placed at room temperature for 24 hours.

Detailed ways

The present invention provides a cotton-derived cinnamic acid-4-hydroxylase encoding gene GbC4H, the nucleotide sequence is shown in SEQ ID NO.2, specifically:

atggatctcctcttcttggagaaggccctcctgggccttttcgtggcggtggtactagccatcaccatctctaagcttcgtggcaagcggttcaagctccctcctggaccattacccgtgccggtgttcggcaactggctccaagtgggtgatgacttgaaccaccgcaacttgacagatttggccaagaaatacggtgacatatttttacttcgaatgggacagcgtaatctagtggtggtgtcttcacctgagctagccaaagaggtgctccactcgcagggagtggagttcggctcaagaactaggaacgtagtgtttgatatattcacgggtaagggtcaagacatggttttcacggtgtacggagagcattggaggaaaatgaggcggatcatgacggtaccattttttaccaacaaggttgtgcaacagtacaggtttggatgggaggacgaggctgctcgtgtagtggaggacgtgaggaaaaatcccgaggcagccaccaacggaatcgttttgaggaggagattgcagctgatgatgtacaacaacatgtacagaatcatgttcgacacaagattcgagagtgaggatgatcctttgtttgttaggctcaaggctttgaacggggagaggagccggttgactcagagttttgaatacaattacggggattttattccaatcttaaggcccttcctcagaggatacttgaagatctgtaaggaggttaaggacaggaggttgcagctcttcaaggaccatttcgtcgaagagaggaagaaacttggaagcacaaaaagcatgaacaacgatggattgaaatgtgccatagatcatattttcgatgctcaacagaagggggaaatcaatgaggacaacgttctctatattgtcgagaatatcaatgttgccgcaattgagacgacactatggtcgatcgagtggggcattgcggagctggtgaaccaccctgaaatccagaagaagctgcggc atgaacttgacactgttctaggacctggtaaccagatcactgaacctgacacccacaaacttccctaccttcaggctgtgatcaaggagactttgaggttacgaatggcaattcctctactcgtgccccacatgaacctgcatgatgcgaaattgggtggctatgatatccctgctgagagcaaaatcttggtaaatgcatggtggcttgccaacaaccctgctaactggaaaaatcccgaagaatttaggcctgaaaggttcttcgaagaggaagccaaggttgaggccaacggcaatgatttccgctacctcccctttggcgtggggagaagaagttgcccaggaattattcttgcattgcccatccttggtattactttgggtcgtttggtacagaattttgagctcttgcctccccctgggcaatctcaaattgataccacggagaaaggtggacagttcagtcttcatattttgaagcattccaccattgttgctaagccaaggcaattttaa。 In the present invention, the length of the reading frame of the encoding gene GbC4H is 1518 bp.

The amino acid sequence of the cinnamic acid-4-hydroxylase encoded by the encoding gene GbC4H of the present invention is shown in SEQ ID NO.1, specifically:

In the present invention, the cinnamic acid-4-hydroxylase contains a complete P450 (PF00067) domain.

The present invention also provides a recombinant vector into which the encoding gene GbC4H described in the above scheme is inserted. In the present invention, the recombinant vector includes an overexpression vector, a knockout vector or a silencing vector encoding the gene GbC4H.

In the present invention, the overexpression vector is transferred into crops by means of genetic engineering, so that a plant that produces a large amount of flavonoids can be obtained, and the new variety of the transgenic plant can also resist Fusarium wilt.

In the present invention, the silencing vector is preferably a VIGS silencing vector; the original vector of the VIGS silencing vector is pTRV2 vector.

The present invention also provides a recombinant bacteria, comprising the recombinant vector described in the above scheme. In the present invention, the original bacteria of the recombinant bacteria is preferably Agrobacterium, more preferably Agrobacterium GV3101.

The present invention also provides the application of the encoding gene GbC4H described in the above scheme or the recombinant vector or the recombinant bacteria; the application includes the application of any one or several of the following:

1) Regulate the resistance of cotton to fusarium wilt;

2) Regulate the flavonoid pathway of cotton;

3) Regulate the accumulation of flavonoids in cotton;

4) Fusarium wilt resistant cotton breeding;

5) Construction of transgenic cotton plants with reduced resistance to fusarium wilt.

In the present invention, the flavonoids include flavonoid antibacterial substances; the flavonoid antibacterial substances have a C6-C3-C6 structure.

The present invention also provides a method for improving the resistance to fusarium wilt of cotton and/or promoting the accumulation of flavonoids in cotton, comprising the following steps: overexpressing the cinnamic acid-4-hydroxylase described in the above scheme or the Coding gene GbC4H.

The present invention also provides the application of a reagent for silencing the cinnamic acid-4-hydroxylase or the encoding gene in the above scheme in constructing a transgenic cotton plant with reduced resistance to fusarium wilt and/or reduced accumulation of flavonoids.

In the present invention, the reagent includes recombinant Agrobacterium; the recombinant Agrobacterium comprises a VIGS silencing vector encoding a specific fragment of the gene GbC4H; the nucleotide sequence of the specific fragment encoding the gene GbC4H is as shown in SEQ ID NO.35 shown, specifically:

atggatctcctcttcttggagaaggccctcctgggccttttcgtggcggtggtactagccatcaccatctctaagcttcgtggcaagcggttcaagctccctcctggaccattacccgtgccggtgttcggcaactggctccaagtgggtgatgacttgaaccaccgcaacttgacagatttggccaagaaatacggtgacatatttttacttcgaatgggacagcgtaatctagtggtggtgtcttcacctgagctagccaaagaggtgctccactcgcagggagtggagttcggctcaagaactaggaacgtagtgtttgatatattcacgggtaagggtcaaga。

In the present invention, the original vector of the VIGS silencing vector is pTRV2 vector. The present invention has no particular limitation on the construction method of the silencing vector, and conventional methods in the art can be used.

In one embodiment of the present invention, the construction method of described VIGS silencing vector, the steps are as follows:

1) using the cDNA of cotton as a template, and using GbC4H-F and GbC4H-R as primers, carry out the first PCR amplification to obtain the GbC4H gene;

The nucleotide sequence of the GbC4H-F is shown in SEQ ID NO.3; the nucleotide sequence of the GbC4H-R is shown in SEQ ID NO.4;

2) inserting the GbC4H gene into the pLB-Simple Vector vector to obtain the first recombinant vector;

3) with the first recombinant vector as a template, with pTRV2-GbC4H-F and pTRV2-GbC4H-R as primers, carry out the second PCR amplification to obtain the specific fragment of the GbC4H gene; the pTRV2-GbC4H-F The nucleotide sequence is shown in SEQ ID NO.5; the nucleotide sequence of the pTRV2-GbC4H-R is shown in SEQ ID NO.6;

4) inserting the specific fragment of the GbC4H gene into the pTRV2 vector to obtain the second recombinant vector;

5) The second recombinant vector is transformed into Agrobacterium.

In the present invention, the cotton is preferably Sea Island cotton.

In the present invention, the cotton cDNA is used as a template, and GbC4H-F and GbC4H-R are used as primers to carry out the first PCR amplification to obtain the GbC4H gene. In the present invention, the procedure of the first PCR amplification is 98°C, 5min; 98°C, 30s, 57°C, 30s, 72°C, 1min 40s, 35 cycles; 72°C, 10min; the first PCR The amplified systems were: PrimeSTAR MaxPremix (2x): 25 μl, cotton cDNA: 2 μl, GbC4H-F: 1 μl, GbC4H-R: 1 μl and _H2O : 21 μl.

After the GbC4H gene is obtained, the present invention inserts the GbC4H gene into the pLB-Simple Vector vector to obtain the first recombinant vector. In the present invention, the Lethal Based Simple Fast Cloning Kit is preferably used to insert the GbC4H gene into the pLB-Simple Vector vector.

After obtaining the first recombinant vector, the present invention uses the first recombinant vector as a template, and uses pTRV2-GbC4H-F and pTRV2-GbC4H-R as primers to perform second PCR amplification to obtain a specific fragment of the GbC4H gene; The nucleotide sequence of the pTRV2-GbC4H-F is shown in SEQ ID NO.5; the nucleotide sequence of the pTRV2-GbC4H-R is shown in SEQ ID NO.6. In the present invention, the system for the second PCR amplification is preferably: PrimeSTAR Max Premix (2x): 25 μl, the first recombinant vector template: 2 μl, pTRV2-GbC4H-F: 1 μl, pTRV2-GbC4H-R: 1 μl and H ₂ O: 21 μl.

After obtaining the specific fragment of the GbC4H gene, the present invention inserts the specific fragment of the GbC4H gene into the pTRV2 vector to obtain a second recombinant vector; the insertion site is preferably EcoRI.

After the second recombinant vector is obtained, in the present invention, the second recombinant vector is transferred into Agrobacterium; the transfer method is preferably a freeze-thaw method.

The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

The materials used in the experiment were sea island cotton (Gossypium barbadense L.) resource material 06-146 (resistance to fusarium wilt) and Xinhai No. 14 (susceptibility to fusarium wilt). 06-146 An excellent line cultivated by the Economics Institute of Xinjiang Academy of Agricultural Sciences using compound hybridization technology. Its variety characteristics are: zero branching, high lint, and high resistance to type II fusarium wilt. For details, please refer to ([1] Chai Yanjun . Yield, Fusarium Wilt Resistance Genetic Analysis and QTL Mapping of Sea Island Cotton F_(2:5) Population [D]. Xinjiang Agricultural University, 2013.). Xinhai 14 is the preferred line of Xinhai No. 10 × Junhai No. 1 used by the Agricultural Science Institute of the First Division of Agriculture.

The seeds of 06-146 and Xinhai No. 14 were provided by the Xinjiang Key Laboratory of Crop Genetic Improvement and Germplasm Innovation, and were planted in cotton with a photoperiod of 16h (light)/8h (dark), a temperature of 25°C, and a relative humidity of 60% to 70%. in the cultivation room. The Fusarium wilt strain used in the test was Fusarium oxysporum, physiological race No. 7. The seeds and fusarium wilt strains of these materials were provided by the Key Laboratory of Cotton, Xinjiang Agricultural University. The cotton tobacco mosaic virus-induced gene silencing vectors pTRV1, pTRV2, silencing control vectors pTRV2::CLA1 and GV3101 Agrobacterium strains used in the experiment were all provided by the Cotton Key Laboratory of Xinjiang Agricultural University.

Max DNAPolymerase)购自宝生物工程有限公司，限制性内切酶购自赛默飞世尔科技公司。反转录试剂盒、荧光定量PCR Mix、同源重组酶和普通PCRMix购自镇江爱必梦生物科技有限公司。引物合成及基因测序均由上海生工生物工程有限公司完成。Plant polysaccharide and polyphenol RNA extraction kit, pLB zero background rapid cloning kit, Escherichia coli strain DH5a, agarose gel DNA recovery kit, plasmid mini kit and DNAMark were purchased from Beijing Tiangen Biochemical Technology Co., Ltd. High-fidelity DNA polymerase (

Max DNA Polymerase) was purchased from Bao Bioengineering Co., Ltd., and restriction enzymes were purchased from Thermo Fisher Scientific. Reverse transcription kit, fluorescence quantitative PCR Mix, homologous recombinase and common PCR Mix were purchased from Zhenjiang Aibimeng Biotechnology Co., Ltd. Primer synthesis and gene sequencing were performed by Shanghai Sangon Bioengineering Co., Ltd.

Example 1 Cloning of cotton GbC4H gene

The total RNA of cotton was extracted with a plant polysaccharide polyphenol RNA extraction kit, and cotton cDNA was synthesized with a reverse transcription kit. According to the gene sequence of GbC4H (GB_D13G2721) (from Cotton Functional GenomicsDatabase (https://cottonfgd.org) website), Primer Premier 5 software was used to design specific amplification primers GbC4H-F (5′-cctcgaagccaaatggatc-3′ , SEQ ID NO. 3) and GbC4H-R (5'-agagaacaagttaaaattgccttg-3', SEQ ID NO. 4). The amplification program was 98°C, 5 min; 98°C, 30s, 57°C, 30s, 72°C, 1min, 40s, 35 cycles; 72°C, 10min. The sea island cotton GbC4H fragment was amplified from the cDNA of the resource material 06-146 cotton, and constructed on the pLB-Simple Vector according to the instructions of the pLB zero background rapid cloning kit. Positive clones were sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing.

The total RNA of Fusarium wilt material 06-146 was extracted and reverse transcribed into cDNA. The primers GbC4H-F and GbC4H-R were used for amplification, and the amplified product was 1518bp (Fig. 1). After the gel was recovered and connected to the cloning vector, the sequenced gene fragment was 1518 bp long, which was 99.87% similar to the sequence of the reference genome Hai7124 numbered GB_D13G2721. The amino acid sequence analysis of GbC4H found that the sea island cotton GbC4H protein contained a complete P450 (PF00067 ) domain.

Example 2 Construction of VIGS silencing vector of GbC4H gene

Primers pTRV2-GbC4H-F (5'-tgagtaaggttacc gaattc atggatctcctcttcttggaga-3', SEQ ID NO. 5) and pTRV2-GbC4H-R (5'-ggaggccttctaga gaattc tcttgacccttacccgtgaa- 3', SEQ ID NO. 6), the underline is the EcoRI enzyme cleavage site. A specific fragment of 347bp of GbC4H gene was amplified by PCR and constructed into pTRV2 vector. The constructed pTRV2::GbC4H vector was transformed into Agrobacterium GV3101 by freeze-thaw method.

Example 3 Cotton RNA extraction and expression pattern analysis of GbC4H gene

计算法进行基因相对表达量的分析。Cotton seedlings of 06-146 and Xinhai 14 cultured for 3 weeks were treated with cotton fusarium wilt infection, MeJA (100 μmol/L) and SA (100 μmol/L) induction treatment, MeJA and SA solutions were sprayed with a sprayer, control group Spray with distilled water as a control and cover with a clear cover to moisturize. Under the three treatment conditions, samples were taken at 0, 4, 8, 12, 24, and 48 h, and the samples were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for later use. The total RNA of cotton was extracted with a plant polysaccharide polyphenol RNA extraction kit, and cotton cDNA was synthesized with a reverse transcription kit. Real-time PCR was performed with the SYBR Green dye method using the cDNAs under the three treatments as templates. The reaction conditions were 95 °C for 3 min; 95 °C for 15 s, 60 °C for 1 min, 40 cycles. The PCR primer sequences were RTGbC4H-F: 5'-aacctgacacccacaaacttcc-3' (SEQ ID NO. 7) and RTGbC4H-R: 5'-caaccttggcttcctcttcg-3' (SEQ ID NO. 8). Using cotton UBQ7 as an internal reference, the primer sequences are: F: 5'-gacctacaccaagcccaagaag-3' (SEQ ID NO. 9) and R: 5'-tgagcccacacttaccacaatagt-3' (SEQ ID NO. 10). The biology was repeated 3 times, using

Computational method was used to analyze the relative expression of genes.

Under the conditions of spraying MeJA and SA, the expression of GbC4H gene increased in both 06-146 and Xinhai 14. Under the condition of spraying MeJA, the expression of GbC4H gene increased to 06-146 after 8 h of treatment. 3.3 times. In Xinhai 14, the expression of GbC4H gene was up to 1.5 times after treatment for 48h (Fig. 2). Under SA treatment, the expression levels of 06-146 and Xinhai 14 increased to the highest at 4h and 24h, which were about 2.8 times and 1.7 times, respectively.

Example 4 Fusarium wilt infection treatment of GbC4H gene silenced plants

Select cotton seedlings (06-146) planted in a cotton culture room with a photoperiod of 16h (light)/8h (dark), a temperature of 25°C, and a relative humidity of 60% to 70% for 8 days. Cotton seedlings were injected, and then the injected cotton seedlings were cultured in the dark for 24 h in a cotton culture room. After dark cultivation is completed, continue to cultivate under suitable conditions. After the cotton seedlings were whitened, samples were taken from the experimental group and the control group to detect the silencing efficiency. At the three-leaf stage of cotton seedlings, the experimental group and the control group were treated with fusarium wilt infection respectively. Repeat 3 times, each injection and count at least 30 strains. The disease was counted according to the 5-level standard, and the disease index was calculated.

The GbC4H gene of sea island cotton 06-146 was silenced by VIGS technology, and CLA1 (cloroplastosalterados 1) was silenced as a positive control. Positive control (pTRV::CLA1) cotton plants exhibited an albino phenotype 15 days after injection. Silencing efficiency test found that the expression of GbC4H gene was significantly reduced, and the expression level decreased by 86.6%, indicating that GbC4H gene silencing was successful. Plants with silenced GbC4H gene (pTRV2::GbC4H) and empty control plants (pTRV2::00) were observed after 28 days after infection with Fusarium wilt, and the plants with silenced GbC4H gene and empty control plants had yellow leaves, Wilting and defoliation, the plants with silenced GbC4H gene had more severe yellowing and wilting than the empty control plants (Figure 3). The statistical analysis of disease index showed that the disease index (33.9) of the experimental group with silenced GbC4H gene was significantly higher than that of the empty control. group's disease index (28.6). Taken together, it was shown that the silencing of the GbC4H gene in sea island cotton 06-146 significantly reduced the resistance of cotton seedlings to Fusarium wilt.

Example 5 Detection of downstream genes of metabolic pathways and cotton fusarium wilt-related genes

At the three-leaf stage of GbC4H gene-silenced cotton seedlings, the experimental group and the control group were treated with fusarium wilt, respectively, and samples were taken at 0, 4, 8, 12, 24, and 48 h, respectively. GbCHI01, GbCHI05, GbCHI06, GbCHI09, GbDFR, GbF3'H, GbANR, GbFLS and GbANS) and cotton wilt-related genes (GbERF-like and Gbar_D03G002290) were used for expression detection. The assays were performed and analyzed according to the method in Example 3 above, and the quantitative primers are shown in Table 1.

Table 1 Quantitative primers

In order to study the effect of GbC4H gene silencing on the downstream genes of the flavonoid pathway, the expression levels of the downstream synthetic genes in the metabolic pathway were detected. 'H, GbANR, GbFLS and GbANS) in cotton seedlings with silenced GbC4H gene were infected by cotton wilt, and their expression levels all decreased to varying degrees (Fig. Accumulation is initiated from the GbC4H gene. The reduction of GbC4H gene expression will affect the downstream gene synthesis of flavonoid antibacterial substances to inhibit cotton fusarium wilt. In order to study the effect of GbC4H gene silencing on cotton fusarium wilt-related genes, the expression levels of GbERF-like and Gbar_D03G002290 previously reported were detected and analyzed. Among them, GbERF-like gene is a gene involved in the resistance to Fusarium wilt of sea island cotton mediated by SA. Under the condition that cotton seedlings with silenced GbC4H gene were infected by cotton fusarium wilt, the expression of GbERF-like gene was up-regulated to varying degrees ( Figure 4), the study showed that the silencing of the GbC4H gene may reduce the antibacterial substances, thereby activating the SA-mediated disease resistance pathway of the GbERF-like gene. Gbar_D03G002290 (Gh_D03G0209) is a key gene for resistance to Fusarium wilt of Upland cotton. The homologous gene Gbar_D03G002290 in Sea Island cotton was down-regulated under the condition that Sea Island cotton 06-146 was infected by Fusarium wilt (Figure 4). Under the condition that the cotton seedlings with silenced GbC4H gene were infected by Fusarium wilt, the expression level of Gbar_D03G002290 first decreased in the absence of Fusarium wilt infection and increased after infection with Fusarium wilt. The results showed that the silencing of the GbC4H gene may affect the expression of the Gbar_D03G002290 gene, but after stress, the increase of the expression of the Gbar_D03G002290 gene may be related to other pathways of resistance to Fusarium wilt.

Example 6 Extraction and antibacterial situation of flavonoids

At the three-leaf stage of the GbC4H gene-silenced cotton seedlings, the test group and the control group were treated with fusarium wilt, respectively, and the stems and leaves above the cotyledon base of the GbC4H gene-silenced cotton seedlings were sampled at 0 h and 96 h, respectively, for the experiment. The materials were dried in a constant temperature electric heating box at 60°C for 2 hours, and then pulverized by a mortar and passed through a 40-mesh sample sieve. The powder was collected in a sampling ziplock bag, marked and placed in a dryer for use. A total of 3 biological replicates were collected for each treatment. According to the extraction characteristic that the flavonoids are easily soluble in ethanol and hot water, the extraction solution was prepared by heating in a 60% ethanol water bath. Accurately weigh 0.05g of the prepared cotton sample into a 10mL centrifuge tube, add 60% ethanol, mark the centrifuge tube and heat it in a constant temperature water bath at 70°C for 4h to fully extract the flavonoids in the sample. 10mL volumetric flask, dilute to volume with 60% ethanol. 2mL of the prepared sample extraction solution was placed in a 10mL volumetric flask, 0.5mL of 5% sodium nitrite solution was added, mixed for 6 minutes, 0.5mL of 10% aluminum nitrate solution was added, and 6 minutes was added after mixing, and finally 4% sodium hydroxide solution was added. 5mL, and dilute to volume with 60% ethanol solution, and after mixing, let stand for 15min to obtain the sample reaction mixture. The extracted flavonoids and the same amount of Fusarium wilt were mixed in a ratio of 1:1 to set up the test group, and sterile water and the same amount of Fusarium wilt were mixed in a ratio of 1:1 to set up the control group. After the seed treatment was placed at room temperature for 24 hours, 20 μl of the mixed solution was drawn and dropped in the center of the PDA solid medium, and then incubated in an inverted 28°C incubator for observation after 3 days. The results of the antibacterial test are shown in Figure 5. The results show that flavonoids can inhibit the growth of Fusarium oxysporum.

Example 7 Establishment of rutin standard curve and determination of total flavonoids

Weigh an appropriate amount of rutin standard product, dissolve it with 60% ethanol to constant volume, and prepare a rutin standard solution (0.2 mg/mL). Pipette 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mL of rutin standard solution (0.2 mg/mL) into a 10 mL volumetric flask, and prepare the rutin standard reaction mixture according to method 2.8, using UV-Vis spectroscopy The photometer performs spectral scanning and selects the peak position (the absorption peak position in this experiment is at 508 nm). Taking the rutin concentration (mg/mL) as the abscissa and the absorbance at 508 nm as the ordinate, draw a standard curve and obtain the linear regression equation y=ax+b. The absorbance value at 508 nm of the reaction mixture of each prepared sample was measured, and the total flavonoid content of the unit sample was calculated according to the linear regression equation.

According to the standard curve of rutin, a linear regression equation was established (Fig. 5) y=0.1322x ⁺ 0.0968, R2=0.9925, that is, the content of flavonoids=(A508-0.0968)/0.1322*100. Statistics of flavonoid content data show that the content of flavonoids in uninoculated control plants is higher than that in uninoculated plants with silent GbC4H gene, and it is found that the content of flavonoids in inoculated control plants is lower than The content of flavonoids in inoculated GbC4H gene-silenced plants (Fig. 5). The experimental results show that the GbC4H gene is involved in the gene synthesis of flavonoids, but it is not the only gene that controls the synthesis of flavonoids. The synthesis of flavonoids is regulated by multiple genes. Flavonoids are not only antibacterial, but also related to a variety of other biological processes. Previous studies on the main synthases in the flavonoid metabolic pathway are relatively clear, but the regulatory network of the flavonoid metabolic pathway is complex. It plays an important role in various biological processes, and the study of its regulatory network is of great significance for elucidating the role of metabolites in biological processes.

The present invention explores the function of the GbC4H gene by cloning the GbC4H gene, silencing the GbC4H gene in the disease-resistant material 06-146 by tobacco mosaic virus and detecting the content of flavonoids, and further explores the regulation of flavonoids by the GbC4H gene in the flavonoid pathway of sea island cotton The reaction mechanism between the accumulation of similar substances and resistance to fusarium wilt provides theoretical basis and genetic resources for the resistance to fusarium wilt of sea island cotton. This study found that silencing of GbC4H in sea island cotton 06-146 significantly reduced the resistance of cotton seedlings to Fusarium wilt. At the same time, it was found that the expression levels of the downstream genes of the metabolic pathway decreased to varying degrees under the condition that the cotton seedlings with silenced GbC4H gene were infected by cotton wilt. The expression of GbERF-like gene in SA signaling pathway was up-regulated, while the expression of the homologous gene Gbar_D03G002290 in sea island cotton was down-regulated. The experiment showed that the accumulation of flavonoids synthesized by the flavonoid pathway may start from the GbC4H gene, and under the condition of simultaneous action of downstream genes, flavonoids were synthesized to inhibit cotton wilt. In the exploration of the regulatory network, it was found that the SA signaling pathway was involved in the mechanism of cotton wilt resistance, and the regulatory network of upland cotton and sea island cotton may be different.

It can be seen that in the future, the cinnamic acid-4-hydroxylase GbC4H gene, which regulates the content of flavonoids, is transferred into crops through genetic engineering, and plants that produce a large amount of flavonoids can be obtained, and this new variety of transgenic plants can also Resist Fusarium wilt.

Although the above embodiment has made a detailed description of the present invention, it is only a part of the embodiments of the present invention rather than all of the embodiments, and people can also obtain other embodiments according to the present embodiment without creativity, and these embodiments are all It belongs to the protection scope of the present invention.

sequence listing

<110> Xinjiang University

<120> A gene GbC4H encoding cinnamic acid-4-hydroxylase derived from cotton and its application

<141> 2022-01-13

<160> 35

<170> SIPOSequenceListing 1.0

<210> 1

<211> 505

<212> PRT

<213> Artificial Sequence

<400> 1

Met Asp Leu Leu Phe Leu Glu Lys Ala Leu Leu Gly Leu Phe Val Ala

1 5 10 15

Val Val Leu Ala Ile Thr Ile Ser Lys Leu Arg Gly Lys Arg Phe Lys

20 25 30

Leu Pro Pro Gly Pro Leu Pro Val Pro Val Phe Gly Asn Trp Leu Gln

35 40 45

Val Gly Asp Asp Leu Asn His Arg Asn Leu Thr Asp Leu Ala Lys Lys

50 55 60

Tyr Gly Asp Ile Phe Leu Leu Arg Met Gly Gln Arg Asn Leu Val Val

65 70 75 80

Val Ser Ser Pro Glu Leu Ala Lys Glu Val Leu His Ser Gln Gly Val

85 90 95

Glu Phe Gly Ser Arg Thr Arg Asn Val Val Phe Asp Ile Phe Thr Gly

100 105 110

Lys Gly Gln Asp Met Val Phe Thr Val Tyr Gly Glu His Trp Arg Lys

115 120 125

Met Arg Arg Ile Met Thr Val Pro Phe Phe Thr Asn Lys Val Val Gln

130 135 140

Gln Tyr Arg Phe Gly Trp Glu Asp Glu Ala Ala Arg Val Val Glu Asp

145 150 155 160

Val Arg Lys Asn Pro Glu Ala Ala Thr Asn Gly Ile Val Leu Arg Arg

165 170 175

Arg Leu Gln Leu Met Met Tyr Asn Asn Met Tyr Arg Ile Met Phe Asp

180 185 190

Thr Arg Phe Glu Ser Glu Asp Asp Pro Leu Phe Val Arg Leu Lys Ala

195 200 205

Leu Asn Gly Glu Arg Ser Arg Leu Thr Gln Ser Phe Glu Tyr Asn Tyr

210 215 220

Gly Asp Phe Ile Pro Ile Leu Arg Pro Phe Leu Arg Gly Tyr Leu Lys

225 230 235 240

Ile Cys Lys Glu Val Lys Asp Arg Arg Leu Gln Leu Phe Lys Asp His

245 250 255

Phe Val Glu Glu Arg Lys Lys Leu Gly Ser Thr Lys Ser Met Asn Asn

260 265 270

Asp Gly Leu Lys Cys Ala Ile Asp His Ile Phe Asp Ala Gln Gln Lys

275 280 285

Gly Glu Ile Asn Glu Asp Asn Val Leu Tyr Ile Val Glu Asn Ile Asn

290 295 300

Val Ala Ala Ile Glu Thr Thr Leu Trp Ser Ile Glu Trp Gly Ile Ala

305 310 315 320

Glu Leu Val Asn His Pro Glu Ile Gln Lys Lys Leu Arg His Glu Leu

325 330 335

Asp Thr Val Leu Gly Pro Gly Asn Gln Ile Thr Glu Pro Asp Thr His

340 345 350

Lys Leu Pro Tyr Leu Gln Ala Val Ile Lys Glu Thr Leu Arg Leu Arg

355 360 365

Met Ala Ile Pro Leu Leu Val Pro His Met Asn Leu His Asp Ala Lys

370 375 380

Leu Gly Gly Tyr Asp Ile Pro Ala Glu Ser Lys Ile Leu Val Asn Ala

385 390 395 400

Trp Trp Leu Ala Asn Asn Pro Ala Asn Trp Lys Asn Pro Glu Glu Phe

405 410 415

Arg Pro Glu Arg Phe Phe Glu Glu Glu Ala Lys Val Glu Ala Asn Gly

420 425 430

Asn Asp Phe Arg Tyr Leu Pro Phe Gly Val Gly Arg Arg Ser Cys Pro

435 440 445

Gly Ile Ile Leu Ala Leu Pro Ile Leu Gly Ile Thr Leu Gly Arg Leu

450 455 460

Val Gln Asn Phe Glu Leu Leu Pro Pro Pro Gly Gln Ser Gln Ile Asp

465 470 475 480

Thr Thr Glu Lys Gly Gly Gln Phe Ser Leu His Ile Leu Lys His Ser

485 490 495

Thr Ile Val Ala Lys Pro Arg Gln Phe

500 505

<210> 2

<211> 1518

<212> DNA

<213> Artificial Sequence

<400> 2

atggatctcc tcttcttgga gaaggccctc ctgggccttt tcgtggcggt ggtactagcc 60

atcaccatct ctaagcttcg tggcaagcgg ttcaagctcc ctcctggacc attacccgtg 120

ccggtgttcg gcaactggct ccaagtgggt gatgacttga accaccgcaa cttgacagat 180

ttggccaaga aatacggtga catattttta cttcgaatgg gacagcgtaa tctagtggtg 240

gtgtcttcac ctgagctagc caaagaggtg ctccactcgc agggagtgga gttcggctca 300

agaactagga acgtagtgtt tgatatattc acgggtaagg gtcaagacat ggttttcacg 360

gtgtacggag agcattggag gaaaatgagg cggatcatga cggtaccatt ttttaccaac 420

aaggttgtgc aacagtacag gtttggatgg gaggacgagg ctgctcgtgt agtggaggac 480

gtgaggaaaa atcccgaggc agccaccaac ggaatcgttt tgaggaggag attgcagctg 540

atgatgtaca acaacatgta cagaatcatg ttcgacacaa gattcgagag tgaggatgat 600

cctttgtttg ttaggctcaa ggctttgaac ggggagagga gccggttgac tcagagtttt 660

gaatacaatt acggggattt tattccaatc ttaaggccct tcctcagagg atacttgaag 720

atctgtaagg aggttaagga caggaggttg cagctcttca aggaccattt cgtcgaagag 780

aggaagaaac ttggaagcac aaaaagcatg aacaacgatg gattgaaatg tgccatagat 840

catattttcg atgctcaaca gaagggggaa atcaatgagg acaacgttct ctatattgtc 900

gagaatatca atgttgccgc aattgagacg acactatggt cgatcgagtg gggcattgcg 960

gagctggtga accaccctga aatccagaag aagctgcggc atgaacttga cactgttcta 1020

ggacctggta accagatcac tgaacctgac acccacaaac ttccctacct tcaggctgtg 1080

atcaaggaga ctttgaggtt acgaatggca attcctctac tcgtgcccca catgaacctg 1140

catgatgcga aattgggtgg ctatgatatc cctgctgaga gcaaaatctt ggtaaatgca 1200

tggtggcttg ccaacaaccc tgctaactgg aaaaatcccg aagaatttag gcctgaaagg 1260

ttcttcgaag aggaagccaa ggttgaggcc aacggcaatg atttccgcta cctccccttt 1320

ggcgtgggga gaagaagttg cccaggaatt attcttgcat tgcccatcct tggtattact 1380

ttgggtcgtt tggtacagaa ttttgagctc ttgcctcccc ctgggcaatc tcaaattgat 1440

accacggaga aaggtggaca gttcagtctt catattttga agcattccac cattgttgct 1500

aagccaaggc aattttaa 1518

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 3

cctcgaagcc aaatggatc 19

<210> 4

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 4

agagaacaag ttaaaattgc cttg 24

<210> 5

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 5

tgagtaaggt taccgaattc atggatctcc tcttcttgga ga 42

<210> 6

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 6

ggaggccttc tagagaattc tcttgaccct tacccgtgaa 40

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 7

aacctgacac ccacaaactt cc 22

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 8

caaccttggc ttcctcttcg 20

<210> 9

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 9

gacctacacc aagcccaaga ag 22

<210> 10

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 10

tgagcccaca cttaccacaa tagt 24

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 11

tctcttttgg gtcatggaat tac 23

<210> 12

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 12

cttcgtattt gtcgtcagct gcc 23

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 13

ggttacaggt gattttgaga 20

<210> 14

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 14

ccttggcctg aaatagtga 19

<210> 15

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 15

tgaacctgaa gttgtagggc attta 25

<210> 16

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 16

ctgccaatcg gtccctca 18

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 17

cgttacaggt gattttgaga 20

<210> 18

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 18

ccttggcctg aaatagtga 19

<210> 19

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 19

tgggtatggc gtattatttg tct 23

<210> 20

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 20

ttgccttctt ctgttgacct tt 22

<210> 21

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 21

cagcaatgac gcaacgaatc 20

<210> 22

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 22

agtgtttctt cgtcggagtc g 21

<210> 23

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 23

ctccactgtt ccctcctcgt a 21

<210> 24

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 24

aaataaaaca ggtaaagggg agc 23

<210> 25

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 25

gaggaaggtg gtggataaac tgt 23

<210> 26

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 26

tcgccaatgt gaataataag gg 22

<210> 27

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 27

agcaaaggct aaacaggaaa catc 24

<210> 28

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 28

cttttctttc gtcgggcttt c 21

<210> 29

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 29

accgtcacaa gttatgaaac cgct 24

<210> 30

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 30

ctatcagatt cctcgtaaac agtcgtc 27

<210> 31

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 31

cggctctggg ttcattggt 19

<210> 32

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 32

ctcagggtcc tcggactcg 19

<210> 33

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 33

tcggcgagta ttcgtgacc 19

<210> 34

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 34

caggcttccc cttgcgtt 18

<210> 35

<211> 347

<212> DNA

<213> Artificial Sequence

<400> 35

atggatctcc tcttcttgga gaaggccctc ctgggccttt tcgtggcggt ggtactagcc 60

atcaccatct ctaagcttcg tggcaagcgg ttcaagctcc ctcctggacc attacccgtg 120

ccggtgttcg gcaactggct ccaagtgggt gatgacttga accaccgcaa cttgacagat 180

ttggccaaga aatacggtga catattttta cttcgaatgg gacagcgtaa tctagtggtg 240

gtgtcttcac ctgagctagc caaagaggtg ctccactcgc agggagtgga gttcggctca 300

agaactagga acgtagtgtt tgatatattc acgggtaagg gtcaaga 347

Claims (9)

1. A coding gene GbC4H of cinnamic acid-4-hydroxylase from cotton has a nucleotide sequence shown in SEQ ID No. 2.

2. A recombinant vector into which the coding gene GbC4H according to claim 1 is inserted.

3. A recombinant bacterium comprising the recombinant vector according to claim 2.

4. The use of the coding gene GbC4H of claim 1 or the recombinant vector of claim 2 or the recombinant bacterium of claim 3; the application comprises any one or more of the following applications:

1) regulating and controlling the blight resistance of cotton;

2) regulating and controlling a flavonoid pathway of cotton;

3) regulating and controlling the accumulation of flavonoids in cotton;

4) breeding cotton with blight resistance;

5) constructing transgenic cotton plants with reduced blight resistance.

5. The use according to claim 4, wherein the flavonoids comprise flavonoid antibacterial substances.

6. A method for improving the fusarium wilt resistance of cotton and/or promoting accumulation of flavonoids in cotton comprises the following steps: over-expressing the coding gene GbC4H of claim 1 in cotton.

7. Use of an agent that silences the coding gene of claim 1, GbC4H, in the construction of a transgenic cotton plant with reduced wilt resistance and/or reduced accumulation of flavonoids.

8. The use of claim 7, wherein the agent comprises recombinant Agrobacterium; the recombinant agrobacterium comprises a VIGS silencing vector for encoding a specific fragment of gene GbC 4H; the nucleotide sequence of the specific fragment of the coding gene GbC4H is shown in SEQ ID NO. 35.

9. The use of claim 8, wherein the vector of origin of the VIGS silencing vector is pTRV2 vector.

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