Tubular protein GhTLP66 and application of coding gene of tubular protein GhTLP66 in regulation of plant salt and drought response

Abstract

The invention relates to a cotton tubular protein coding gene GhTLP66. The gene sequence of the cotton tubular protein coding gene GhTLP66 comprises 1218 nucleotide sequences. According to phenotype analysis and physiological index analysis of two overexpression transgenic lines of GhTLP66, it is found that the two overexpression lines are too sensitive to salt and drought, a regulation method for regulating the stress resistance of plants is provided, and a theoretical basis is provided for improvement of the stress resistance of the plants.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to application of tubular protein GhTLP66 and a coding gene thereof in regulating plant salt and drought responses. Background Art

[0002] Soil salinization and drought are two important abiotic stresses that affect plant growth and are important factors limiting crop yields.

[0003] Cotton is an important fiber crop and oil crop, as well as a raw material for textiles and fine chemicals. It plays an important role in industry, agriculture and human life. Cotton is drought-resistant and salt-alkali-tolerant, and is a pioneer crop for improving saline-alkali land. Although cotton is relatively tolerant to salt-alkali and drought, it is still subject to salt and drought stress at the critical stage of cotton development, which severely restricts the growth and development of cotton.

[0004] Identifying genes involved in salt tolerance and drought response in cotton is of great significance for improving cotton yield and effective land utilization. Existing technologies have shown that members of the Tubby-like protein (TLPs) family are a large multigene family that plays an important role in plant response to abiotic stress. At present, 105 TLPs have been identified in cotton genes, but the functions of only a few TLPs have been revealed. The functions of other GhTLPs genes in cotton in responding to abiotic stress need to be further analyzed. Summary of the invention

[0005] The present invention aims to provide an application of a gene encoding a cotton tubular-like protein (GhTLP66), wherein the gene sequence comprises 1218 nucleotide sequences as shown in SEQ ID No. 1:

[0006] This application analyzes the phenotypic and physiological indicators of two overexpressing transgenic strains of the tubular protein GhTLP66 and finds that the two overexpressing strains are overly sensitive to salt and drought. Based on this, this study provides a method for regulating plant stress resistance.

[0007] The technical solution provided by the present invention is as follows: The present invention provides the use of tubular protein GhTLP66 and / or a gene encoding tubular protein GhTLP66 and / or a recombinant vector containing a DNA molecule encoding tubular protein GhTLP66 and / or a transgenic cell line containing tubular protein GhTLP66 and / or a recombinant bacterium of tubular protein GhTLP66 in regulating plant stress resistance.

[0008] In the present invention, the regulation of plant stress resistance is to regulate the resistance to salt and drought by changing the expression amount of the gene encoding the tubular protein GhTLP66.

[0009] In the present invention, the plant is cotton In the present invention, the coding gene of the tubular protein GhTLP66 is shown in SEQ ID No.1; the amino acid sequence of the tubular protein GhTLP66 is shown in SEQ ID No.2: MSFKSIIQDMKGEFGSISRKGFDVKFGYGMRSRSHRVVQDSSVPVDVFKQSCWANMPPELLRDVLMRIEASESTWPPRKNVVVCAGVCRNWREIMKEIVKT PEISCKLTFPISLKQPGPRDSLLQCYIKRNRSNQTYYLYLGLNQASNDDGKFLLAARKCRRPTCTDYIISLNCNDVSKGSSTYIGKLRSNFLGTKFTVYDAQ PPNAGAKVTKSCSTRLINMKQVSPRVPAGNYPVAHISYELNVLGSRGPRRMHCVMDAIPASSIEPGGVAPTQTEFLHSNLDTFPSLLFFRSKSTRAESFQSGPLSDQKDGMLVLRNKSPRWHEQLQCWCLNFNGRVTVASVKNFQLVASPENAAAGQEHENVILQFGKVGKDVFTMDYQYPISAFQAFAICLSSFDTKIACE.

[0010] The present invention also provides a kit for regulating plant stress resistance, which includes the above-mentioned tubular protein GhTLP66 and / or the encoding gene of tubular protein GhTLP66 and / or a recombinant vector containing a DNA molecule encoding tubular protein GhTLP66 and / or a transgenic cell line containing tubular protein GhTLP66 and / or a recombinant bacterium of tubular protein GhTLP66.

[0011] The present invention also provides a method for regulating plant stress resistance, the method comprising: over-expressing a gene encoding tubular protein GhTLP66 to weaken the salt resistance and drought resistance of the plant.

[0012] In the present invention, the specific method for regulating plant stress resistance is: introducing the coding gene of tubular protein GhTLP66 into the target plant, improving the expression or activity of tubular protein GhTLP66 in the target plant, and obtaining a transgenic plant. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a schematic diagram of the partial structure of the pWMV067 vector;

[0014] Figure 2 The result diagram of transcriptional expression of 8 GhTLP66 overexpression strains; Figure 3 WT, GhTLP66-OX#1 and GhTLP66-OX#2 Pictures after 3 days; Figure 4 For WT, GhTLP66-OX#1 and GhTL66P-OX#2 Conductivity picture after salt treatment, ** represents p <0.01; Figure 5 For WT, GhTLP66-OX#1 and GhTLP66-OX#2 Leaf Na after salt treatment + / K + Content determination result picture, ** represents p <0.01; Figure 6 15% PEG6000 treatment of WT, GhTLP66-OX#1 and GhTLP66-OX#2 3 h pictures; Figure 7 For WT, GhTLP66-OX#1 and GhTLP66-OX#2 Conductivity results after 15% PEG6000 treatment, ** represents p <0.01. DETAILED DESCRIPTION

[0015] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0016] Example The method for determining GhTLP66 as a gene for regulating plant stress resistance comprises the following steps: (1) Cloning and overexpression vector construction of cotton TLP66 CDS (2) Obtaining cotton GhTLP66 overexpressing transgenic lines; (3) Two GhTLP66 overexpressing transgenic cotton lines were treated with salt and simulated drought and their phenotypic and physiological parameters were analyzed after treatment; Experimental Materials: The wild-type material used in this experiment was Zhong Cotton Research Institute No. 24 (hereinafter referred to as Zhong 24), and the structure of the vector pWMV067 is as follows Figure 1 as shown).

[0017] The leaves of cotton strain 24 were obtained, and total RNA was extracted using the FastPure Universal Plant Total RNA Isolation Kit (Novozyme RC411-01). The RNA was reverse transcribed into cDNA using the reverse transcription kit PrimeScript™ II 1st StrandcDNA Synthesis Kit (Baori Medical Biotechnology Co., Ltd., 6210A).

[0018] Primers were designed based on the CDS sequence provided by the Cotton Omics Database website (http: / / cotton.zju.edu.cn / 2.search_gene_locus.php, gene ID: GH_D11G1259): TLP66-35S-F 5'-CACGGGGGACGTCGACAATGTCGTTTAAGAGCATTATCCAGG -3', SEQ IDNo.3 (containing SalI restriction site); TLP66-35S-R 5'-ACGGATACATGGATCCCTCACAAGCGATCTTAGTATCG -3', SEQ ID No. 4 (containing a BamHI restriction site).

[0019] Using the cDNA of 24 varieties of upland cotton as a template, the coding region sequence of cotton TLP66 gene was amplified using high-fidelity enzyme PrimeSTAR GXL DNA Polymerase (Bao Ri Yi Biotechnology Co., Ltd., R050Q). The PCR reaction system is as follows: 5× PrimeSTAR GXL Buffer 10 μl, 1× dNTP Mixture (2.5 mM each) 4 μl, 10 μM forward primer 0.5 μl, 10 μM reverse primer 0.5 μl, cDNA 1 μl, PrimeSTAR GXL DNA Polymerase 2 μl, sterile water to 50 μl. PCR amplification program: 98℃ 3 min; 30 cycles: 98℃ 10 s; 55℃ 15 s; 68℃ 1 min 20 s; 68℃ 10 min. Take 4 μl PCR product and detect the expected target band by 1% agarose gel electrophoresis. After product purification and recovery, the TLP66 gene was seamlessly connected to the plant expression vector pWMV067 digested with SalI and BamHI using ClonExpress Ultra One Step Cloning Kit (Novozyme C115-02). The homologous recombination ligation system is as follows: 2×ClonExpress Mix: 2.5 μL, PCR product 1 μL, pWMV067 linearized vector 1.5 uL after enzyme digestion and recovery, a total of 5 μL. Mix the above system, react at 50℃ for 20 min, then add 5 μL of ligation product to E. coli DH5α competent cells, heat shock transform E. coli, and plate culture overnight on LB plates containing 50 μg / ml kanamycin. The next day, the clones that were positive after PCR verification were sent to Shanghai Sangon Biotechnology Technology Service Co., Ltd. for sequencing. The plasmid of the clone that was sequenced correctly was named pWMV067-TLP66, and the plasmid (-20℃) and bacterial solution (bacterial solution: 50% glycerol = 1:1, -80℃) were stored for later use.

[0020] The pWMV067-TLP66 and pWMV067 empty vectors obtained above were respectively transformed into Agrobacterium strain LBA4404. The cotton genetic transformation method was referred to Zhang Chaojun's doctoral thesis (Establishment and genetic analysis of cotton petiole efficient regeneration system, CNKI), and the empty vector and pWMV067-TLP66 were transformed into medium 24 to obtain positive transgenic cotton materials. Genomic DNA was extracted from pWMV067-TLP66 T0 regenerated cotton plants and their T1 and T2 offspring cotton plants, and specific primers were used to pWMV067—F 5'-TCATTGCGATAAAGGAAAGG-3', SEQ ID No. 5; 66R 5'-CACGAGGAGAAACTTGTTTCAT-3', SEQ ID No. 6.

[0021] PCR verification showed that all of them contained a transgenic specific target band of about 931 bp, indicating that GhTULP66 transgenic cotton was successfully obtained. The enzymes used in PCR were purchased from Norwegian. The PCR reaction system included 2 × Rapid Taq Master Mix 10 μl, 10 μM forward primer 0.5 μl, 10 μM reverse primer 0.5 μl, cDNA 1 μl, PrimeSTAR GXL DNA Polymerase 2 μl, and sterile water to 20 μl. PCR reaction program: 95℃ 3 min; 30 cycles: 95℃ 15 s; 55℃ 15 s; 72℃ 20 s; 72℃ 5 min.

[0022] The positive material of the empty vector was named WT. RNA was extracted from the obtained transgenic and WT positive plants, and the expression level of transcription was detected. The detection primers were: TLP66-RT-F 55'- CTTCAATGGACGGGTAACAGT -3', SEQ ID No.7, TLP66-RT-R 5'-CATACCTGTTTGCATATCACTCAC-3', SEQ ID No.8, and the quantitative kit used was PrimeScript™ One Step RT-PCR Kit Ver.2 (RR05A) of Baoriyi Biotechnology Co., Ltd. The lines with no separation of genotype and expression level from T2 to T3 generation were homozygous lines, and the cDNA of homozygous lines was obtained. The positive seedlings transformed with empty vector were used as controls. The expression levels of TLP66 in the obtained positive lines increased to 20, 41, 13, 29, 39, 34, 45 and 52 times that of the wild type, respectively ( Figure 2 ).

[0023] Salt treatment: WT, GhTLP66-OX1 and GhTLP66-OX2 seeds were sterilized with 10% NaClO and soaked in sterile water for 24 hours until the seeds turned white. The materials with consistent germination were selected and rolled into filter paper. The filter paper was placed vertically in a 5 L measuring cup containing distilled water and cultured in the dark. On the third day, the materials with consistent growth were transferred to the hydroponic box, and the water was changed every three days until the two cotyledons were fully expanded (two-leaf stage). Each material was divided into two equal parts, one part was transferred to the hydroponic solution as a control, and the other part was transferred to the hydroponic solution containing 150 mM NaCl. On the third day of salt treatment, the GhTLP66 overexpression line showed obvious wilting phenotype in the leaves, which was recorded by taking photos and samples were taken for the determination of physiological indicators. Determination of relative conductivity: Use a 1 cm hole puncher to take 10 leaves with a diameter of 1 cm and place them in 10 ml of distilled water. Place them in a shaker at 200 rpm for 6 hours, measure the conductivity E1, and then sterilize them in an autoclave at 121 degrees for 20 minutes. Cool them to room temperature and measure the conductivity E2. E1 / E2 is the relative conductivity. + / K + Determination: Weigh 0.2 g of dried sample into a polytetrafluoroethylene digestion inner tank, add 5 mL of nitric acid and soak overnight. Cover the inner cover, tighten the stainless steel outer cover, put it into a constant temperature drying oven, keep it at 80℃ for 2 h, keep it at 120℃ for 2 h, and then increase it to 160℃ for 4 h. Cool it naturally to room temperature in the oven, open it and heat it to drive out the acid until it is almost dry, wash the digestion solution into a 25 mL volumetric flask, wash the inner tank and inner cover 3 times with a small amount of nitric acid solution (1%), combine the washing solution into the volumetric flask and make up to the scale with 1% nitric acid, mix well and set aside. Perform a reagent blank test at the same time. The content of each element in the test solution was determined by the machine (NexlON ® 1000 ICP-MS Simulated drought treatment: WT, GhTLP66-OX1 and GhTLP66-OX2 The seeds were disinfected, soaked and rolled. When they grew to the two-leaf stage in the hydroponic box, half of the materials were used as controls and the other half were transferred to a hydroponic box containing 15% PEG6000. After 3 hours of treatment, the leaves of the overexpression lines showed obvious wilting phenotypes. Photos were taken and samples were taken for the determination of physiological indicators. The determination of conductivity is as above. The proline content was determined using a proline content determination kit (Angel Gene, YH1231).

[0024] Application Examples Genotype analysis, expression level analysis and phenotype analysis were performed using two overexpression strains of the GhTLP66 gene.

[0025] The expression levels of GhTLP66 in the two overexpression lines were 20 and 41 times that of WT, respectively ( Figure 2); After 3 days of 150 mM NaCl treatment at the two-leaf stage, the leaf conductivity of the GhTLP66-OX strain was significantly higher than that of the WT ( Figure 3~4 ) (WT: 11.7, overexpression lines: 14.8, 21.88), and Na + / K + The concentration ratio was significantly higher than that of WT ( Figure 5 )(WT: 0.3, overexpression line: 0.91, 1.02), indicating that GhTLP66 regulates cotton salt tolerance; after 3 h of 15% PEG6000 simulated drought treatment at the two-leaf stage, the wilting degree of GhTLP66-OX was much greater than that of WT ( Figure 6 ), the leaf conductivity of GhTLP66-OX strain was significantly higher than that of WT (WT: 20.8, overexpression strain: 24.8, 39.9) ( Figure 7 ), indicating that GhTLP66 regulates cotton drought resistance.

[0026] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of tubular protein GhTLP66 and / or a gene encoding tubular protein GhTLP66 and / or a recombinant vector containing a DNA molecule encoding tubular protein GhTLP66 and / or a transgenic cell line containing tubular protein GhTLP66 and / or a recombinant bacterium of tubular protein GhTLP66 in regulating plant stress resistance; The coding gene of the tubular protein GhTLP66 is shown in SEQ ID No. 1; The amino acid sequence of the tubular protein GhTLP66 is shown in SEQ ID No.

2.

2. The use according to claim 1, characterized in that: The regulating plant stress resistance is to regulate the resistance to salt and drought by changing the expression amount of the gene encoding the tubular protein GhTLP66.

3. The use according to any one of claims 1 to 2, characterized in that: The plant is cotton.

4. A kit for regulating plant stress resistance, characterized in that: The kit comprises the tubular protein GhTLP66 and / or the encoding gene of tubular protein GhTLP66 as described in claim 1 and / or a recombinant vector containing a DNA molecule encoding tubular protein GhTLP66 and / or a transgenic cell line containing tubular protein GhTLP66 and / or a recombinant bacterium of tubular protein GhTLP66.

5. A method for regulating plant stress resistance, characterized in that: The method comprises: over-expressing the coding gene of tubular protein GhTLP66 to weaken the salt and drought resistance of the plant; The coding gene of the tubular protein GhTLP66 is shown in SEQ ID No. 1; The amino acid sequence of the tubular protein GhTLP66 is shown in SEQ ID No.2.

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