CN110078808B

CN110078808B - Cotton GhKNAT7-A03 protein and coding gene and application thereof

Info

Publication number: CN110078808B
Application number: CN201910406102.6A
Authority: CN
Inventors: 喻树迅; 马强; 魏恒玲; 王寒涛; 马亮
Original assignee: Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Priority date: 2019-05-16
Filing date: 2019-05-16
Publication date: 2021-10-01
Anticipated expiration: 2039-05-16
Also published as: CN110078808A

Abstract

The invention discloses a cotton GhKNAT7-A03 protein, and a coding gene and application thereof. The invention protects the application of GhKNAT7-A03 protein or related biomaterials thereof in regulating and controlling the strength of plant fibers. The secondary cell wall of fibers between bundles of arabidopsis stems over-expressing GhKNAT7-A03 gene is thinned, and lignin synthesis is hindered. In the development of cotton fibers, GhKNAT7-A03 can also inhibit the synthesis of lignin and promote the synthesis of cellulose to regulate the synthesis of secondary cell walls of the fibers, thereby influencing the formation of the strength of the cotton fibers. The invention has important significance for cotton breeding.

Description

Cotton GhKNAT7-A03 protein and coding gene and application thereof

Technical Field

The invention relates to cotton GhKNAT7-A03 protein and a coding gene and application thereof.

Background

Cotton is an important economic crop, and its fiber is an important natural fiber and an important raw material in textile industry. With the further development of the textile industry, new textile technologies have increasingly high requirements on the quality of the fibers. Therefore, the research on the cotton fiber development and the related gene expression regulation has important biological theoretical significance and practical application value.

Cotton fiber is a single cell fiber formed by differentiation and development of ovule epidermal cells. All ovule epidermal cells have the potential to differentiate into cotton fibers, but only 30% eventually can form fibers. The development process can be divided into 4 stages of fiber initiation, fiber elongation, secondary wall thickening and dehydration maturation. These 4 phases are a complex process of many gene interactions, internetworking, and co-expression regulation. The development stages of the fibers do not have obvious boundaries, namely the initial differentiation and the elongation of the cotton fibers have mutually overlapped parts, and the number and the length of the fibers are greatly influenced by the development stages; the two periods of fiber elongation (primary wall synthesis) and secondary wall thickening partially overlap, and are closely related to the formation of fiber quality, and in the secondary wall thickening period, the synthesis of cellulose at this stage has a large influence on properties such as fiber strength and micronaire value.

Disclosure of Invention

The invention aims to provide cotton GhKNAT7-A03 protein and a coding gene and application thereof.

In a first aspect, the invention claims the use of GhKNAT7-a03 protein or its related biomaterials for modulating plant fiber strength;

the related biological material is a nucleic acid molecule capable of expressing the GhKNAT7-A03 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the GhKNAT7-A03 protein is any one of the following proteins:

(A1) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

(A2) the protein which is derived from the sequence 1 and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1 in the sequence table;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

The GhKNAT7-A03 protein is derived from cotton.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

In the above protein, the tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, etc.

In a second aspect, the invention claims the application of GhKNAT7-A03 protein or related biomaterials in the following (A) or (B) or (C):

(A) regulating and controlling the thickness of the secondary wall of the plant interbeam fiber cells;

(B) regulating and controlling the expression level of the plant cellulose synthetic gene;

(C) regulating and controlling the expression level of plant lignin synthesis genes;

the related biological material is a nucleic acid molecule capable of expressing the GhKNAT7-A03 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule; the GhKNAT7-A03 protein is any one of the proteins shown in the (A1) to (A4) in the specification.

In the first aspect, the regulating the strength of the plant fiber may be embodied as: the activity and/or expression level of the GhKNAT7-A03 protein or the coding gene thereof in the plant is improved, and the strength of plant fibers is increased.

In a second aspect, the modulating the thickness of the secondary wall of the plant interbeam fibroblast may be embodied as: the activity and/or expression of the GhKNAT7-A03 protein or the coding gene thereof in the plant is increased, and the thickness of the secondary wall of the plant intertuberculus fiber cells is reduced.

In the second aspect, the regulation and control of the expression level of the plant cellulose synthesis gene may be embodied as: the activity and/or expression level of the GhKNAT7-A03 protein or the coding gene thereof in the plant is improved, and the expression level of the plant cellulose synthesis gene is increased.

In the second aspect, the regulation of the expression level of the plant lignin synthesis gene may be embodied as: the activity and/or expression level of the GhKNAT7-A03 protein or the coding gene thereof in the plant is improved, and the expression level of a plant lignin synthesis gene is reduced.

In the application, when the plant is arabidopsis thaliana, the expression level of the plant lignin synthesis gene is specifically the expression level of the lignin synthesis gene AtPAL1 gene and AtCOMT gene.

In the above application, when the plant is cotton, the regulation may specifically occur during the secondary wall thickening period of cotton fibers. When the plant is cotton, the expression level of the plant cellulose synthesis gene is specifically the expression level of the cellulose synthesis genes GhCESA4 and GhCESA8 during the secondary wall thickening period (20, 30DPA) of cotton fibers. When the plant is cotton, the expression quantity of the plant lignin synthesis gene is specifically the expression quantity of the lignin synthesis gene GhCOMT1 gene and GhCAD5 gene in the secondary wall thickening period (20, 30DPA) of cotton fiber.

In a third aspect, the invention claims any of the following methods:

(B1) a method for producing a plant variety having improved fiber strength, which comprises the step of increasing the expression level and/or activity of GhKNAT7-A03 protein in a recipient plant;

(B2) a method for breeding a plant variety with reduced thickness of the secondary wall of plant intertubercular fibroblasts, comprising the step of increasing the expression level and/or activity of GhKNAT7-A03 protein in a recipient plant;

(B3) a method for producing a plant variety having an increased expression level of a plant cellulose synthesis gene, comprising the step of increasing the expression level and/or activity of GhKNAT7-A03 protein in a recipient plant;

(B4) a method for producing a plant variety having a reduced expression level of a lignin synthesis gene, comprising the step of increasing the expression level and/or activity of GhKNAT7-A03 protein in a recipient plant;

the GhKNAT7-A03 protein is any one of the proteins shown in the (A1) to (A4) in the specification.

Further, the present invention claims any of the following methods:

(C1) a method of breeding a transgenic plant comprising the steps of: introducing a nucleic acid molecule capable of expressing GhKNAT7-A03 protein into a receptor plant to obtain a transgenic plant; the transgenic plant fiber strength is greater than that of the recipient plant;

(C2) a method of breeding a transgenic plant comprising the steps of: introducing a nucleic acid molecule capable of expressing GhKNAT7-A03 protein into a receptor plant to obtain a transgenic plant; the thickness of the secondary wall of the intertillary fiber cells of the transgenic plant is smaller than that of the receptor plant;

(C3) a method of breeding a transgenic plant comprising the steps of: introducing a nucleic acid molecule capable of expressing GhKNAT7-A03 protein into a receptor plant to obtain a transgenic plant; the expression quantity of the cellulose synthetic gene of the transgenic plant is greater than that of a receptor plant;

(C4) a method of breeding a transgenic plant comprising the steps of: introducing a nucleic acid molecule capable of expressing GhKNAT7-A03 protein into a receptor plant to obtain a transgenic plant; the expression quantity of the lignin synthesis gene of the transgenic plant is less than that of a receptor plant;

The 'introduction of a nucleic acid molecule capable of expressing the GhKNAT7-A03 protein' into a recipient plant is realized by introducing a recombinant expression vector containing a coding gene of the GhKNAT7-A03 protein into the recipient plant.

The recombinant expression vector containing the coding gene of the GhKNAT7-A03 protein can be constructed by using the existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions.

When a recombinant plant expression vector is constructed using the gene encoding the GhKNAT7-a03 protein, any one of an enhanced promoter or a constitutive promoter (e.g., cauliflower mosaic virus (CAMV)35S promoter, Ubiquitin promoter from maize (Ubiquitin)) or a tissue-specific expression promoter (e.g., seed-specific expression promoter) may be added before the transcription initiation nucleotide, and they may be used alone or in combination with other plant promoters. In addition, when a plant expression vector is constructed using the gene encoding the GhKNAT7-a03 protein, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codons or initiation codons of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, luciferase gene, etc.), an antibiotic marker having resistance (gentamicin marker, kanamycin marker, etc.), or a chemical-resistant marker gene (e.g., herbicide-resistant gene), etc., which can be expressed in plants.

In the invention, the recombinant expression vector can be specifically a recombinant expression vector obtained by replacing a fragment between BamH I and Sac I enzyme cutting sites of an over-expression vector pBI121 with a double-stranded DNA molecule shown as a sequence 2 in a sequence table.

In the above method, the recombinant expression vector carrying the coding gene of the GhKNAT7-a03 protein is introduced into the recipient plant, and specifically may be: plant cells or tissues are transformed by conventional biological methods using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and the transformed plant tissues are grown into plants.

Transformed cells, tissues or plants are understood to comprise not only the end product of the transformation process, but also transgenic progeny thereof.

The coding gene of the GhKNAT7-A03 protein is a DNA molecule as described in any one of the following items:

(D1) a DNA molecule shown in a sequence 2 of a sequence table;

(D2) a DNA molecule which hybridizes with the DNA molecule defined in (D1) under stringent conditions and encodes the GhKNAT7-A03 protein;

(D3) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of identity with the DNA sequence limited by (D1) or (D2) and codes the GhKNAT7-A03 protein.

In the above genes, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In the above method, when the plant is Arabidopsis thaliana, the change in the expression level of the lignin synthesis gene of the plant is specifically a change in the expression levels of the lignin synthesis gene AtPAL1 gene and AtCOMT gene.

In the above method, when the plant is cotton, the change in the expression level of the plant cellulose synthesis gene may specifically occur during the secondary wall thickening period (20, 30DPA) of cotton fibers. When the plant is cotton, the change of the expression level of the plant cellulose synthesis gene is specifically the change of the expression levels of the cellulose synthesis genes GhCESA4 and GhCESA8 during the secondary wall thickening period (20, 30DPA) of cotton fibers. When the plant is cotton, the change of the expression level of the plant lignin synthesis gene is specifically the change of the expression level of the cotton fiber secondary wall thickening period (20, 30DPA) lignin synthesis gene GhCOMT1 gene and GhCAD5 gene.

In a fourth method aspect, the invention claims the GhKNAT7-A03 protein or its related biological material, or the use of any of the above methods in plant breeding.

The breeding aims to breed plants with high fiber strength.

The plant may be specifically characterized by a lower thickness of the secondary wall of interbeam fibroblasts or a higher expression of cellulose synthesis genes or a lower expression of lignin synthesis genes.

When the plant is arabidopsis thaliana, the expression quantity of the plant lignin synthesis gene is specifically the expression quantity of the lignin synthesis gene AtPAL1 gene and AtCOMT gene.

When the plant is cotton, the expression level of the plant cellulose synthesis gene is specifically the expression level of the cellulose synthesis genes GhCESA4 and GhCESA8 during the secondary wall thickening period (20, 30DPA) of cotton fibers. When the plant is cotton, the expression quantity of the plant lignin synthesis gene is specifically the expression quantity of the lignin synthesis gene GhCOMT1 gene and GhCAD5 gene in the secondary wall thickening period (20, 30DPA) of cotton fiber.

In the above aspects, the plant may be (C1) or (C2) or (C3):

(C1) a dicot or monocot;

(C2) malvaceae plant or Cruciferae plant;

(C3) cotton or arabidopsis thaliana.

The cotton can be upland cotton TM-1, reclaimed N27-3 or Suyou 6108.

The Arabidopsis thaliana can be Columbia ecotype Arabidopsis thaliana (Col-0).

The cotton GhKNAT7-A03 gene is cloned from upland cotton, and the expression mode finds that the gene is dominantly expressed in the secondary cell wall thickening period (20-30DPA, days post antisense) of cotton fibers; constructing an overexpression vector of the gene, transforming Arabidopsis by a flower dipping method, and finding out thinning of secondary cell walls of fibers among stalk bundles of an over-expressed strain of the Arabidopsis by histochemical staining, wherein the thinning is mainly caused by the obstruction of lignin synthesis; in upland cotton materials with different fiber strengths, the expression condition of the gene in the thickening stage of the secondary cell walls of the fibers is detected, the expression level of the gene in the materials with high fiber strength is higher than that of the materials with low fiber strength, the expression trend of the gene is the same as that of cellulose synthases GhCESA4 and GhCESA8, and the expression trend of the gene is opposite to that of lignin synthesis-related genes GhCAD5 and GhCOMT1, so that the GhKNAT7-A03 can also inhibit the synthesis of lignin in the development of cotton fibers, and unlike Arabidopsis, the GhKNAT7-A03 in the cotton fibers can also promote the synthesis of the cellulose to regulate and control the synthesis of the fiber cell walls so as to influence the formation of the secondary cell walls of the cotton fibers. The invention has important significance for cotton breeding.

Drawings

FIG. 1 is an electrophoretogram of GhKNAT7-A03 fragment.

FIG. 2 shows the result of PCR identification of GhKNAT7-A03 transgenic plants.

FIG. 3 shows statistics of phenotype observation of GhKNAT7-A03 transgenic Arabidopsis thaliana.

FIG. 4 shows the expression level identification of GhKNAT7-A03 in different strains of GhKNAT7-A03 transgenic Arabidopsis thaliana and the expression pattern analysis of genes related to the synthesis of lignin and cellulose.

FIG. 5 is the analysis of the expression pattern of GhKNAT7-A03 gene in cotton material with different fiber strength.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Ultra One Step Cloning Kit: vazyme, cat # s: c115-01.

Overexpression vector pBI 121: beijing Huayuyo Biotech Co., Ltd., Cat No.: DWF 9808.

Agrobacterium tumefaciens LBA 4404: shanghai Diego Biotechnology Limited, Cat number: AC 1030.

KOD FX Neo enzyme: toyobo company, cat #: KFX-201.

Upland cotton TM-1: described in literature "LI F G, FAN GY, LU C R, XIAO G H, ZOU C S, KOHEL RJ, MA ZY, SHANG H H, MA X F, WU J Y, LIANG X M, HUANG G, PERCYR G, LIUK, YANG W H, CHEN W B, DU X M, SHI C C, YUAN YL, YE W, LIU X, ZHANG XY, LIU W Q, WEI H L, WEI S J, ZHU S J, ZHANG H, SUN F M, WANG X F, LIANG J, WANGJ H, HE Q, HUANG L H, CUI J, SONG L, WANG K B, XU X, YU J Z, ZHU Y X, YUN X, YU X, tissue of tissue culture (Gouch) 2, YU J, KU J Z, ZHU Y X, YU Y X, U SX, tissue culture of tissue culture (Gouch) 2, III, G H, KU J, H, KU J, H, KU J, U G H, U: 524- "of the design section; the public is available from the cotton institute of the Chinese academy of agricultural sciences.

Reclamation N27-3: the method is described in the literature, "correlation research on genetic distance and relative advantages of fiber quality traits and F _1 and F _2 expressions of Junjiji, Chenhong, Yu, Linhai, Ningxinzhu, Lijilian, Liu Pinna, Liu Li, Xiangji mountain, Deng Fu Jun. upland cotton [ J ] Cotton science report, 2013,25(02): 142-; the public is available from the cotton institute of the Chinese academy of agricultural sciences.

Suyou 6108: described in the literature, "Sun Junling, Zhou Zhong loy, Jia Yin Hua, Pan Mei, what guard officer, numerous and powerful prints, Wang Li, Du Xiong Ming," fifteen "cotton excellent innovation germplasm introduction [ J ]. Jiangxi cotton, 2011,33(04): 55-57."; the public is available from the cotton institute of the Chinese academy of agricultural sciences.

Columbia ecotype Arabidopsis thaliana (Col-0): beijing Huayuyo Biotech Co., Ltd., Cat No.: NRR 00220.

Example 1, preparation of GhKNAT7-A03 protein and Gene encoding the same

Total RNA from upland cotton TM-1 cotton fibers was extracted and reverse transcribed into cDNA. Through a large number of sequence analysis, expression analysis and functional verification, a DNA coding sequence is found from cDNA, as shown in sequence 2 of the sequence table, and the protein coded by the DNA coding sequence is shown in sequence 1 of the sequence table.

The protein shown in the sequence 1 of the sequence table is named GhKNAT7-A03 protein and consists of 299 amino acid residues. The gene coding the GhKNAT7-A03 protein is named as the GhKNAT7-A03 gene, and the open reading frame is shown as a sequence 2 in a sequence table.

Example 2 construction of recombinant expression vector and obtaining of recombinant Agrobacterium

Construction of recombinant expression vector

1. Total RNA of the cotton fiber of Gossypium hirsutum TM-1 was extracted and reverse-transcribed into cDNA, PCR amplification was performed using cDNA as a template and a primer pair consisting of OE-GhKNAT7-A03F and OE-GhKNAT7-A03R, and about 900bp PCR amplification product (GhKNAT7-A03 fragment) was recovered, as shown in lane 1 of FIG. 1.

OE-GhKNAT7-A03F:5’-GAACACGGGGGACTCTAGAATGCAAGAACCAGGTGGG-3’；

OE-GhKNAT7-A03R:5’-gatcggggaaattcgagctcCTACCTTTTCCGCTTGGA-3’。

2. The over-expression vector pBI121 was digested with restriction enzymes BamH I and Sac I, and the vector backbone of about 14700bp was recovered.

3. The PCR amplification product obtained in the step 1 and the carrier skeleton obtained in the step 2 are adopted

The recombinant plasmid pBI121-GhKNAT7-A03 was obtained by Ultra One Step Cloning Kit ligation.

The PCR amplification product (GhKNAT7-A03 fragment) and the vector backbone were mixed in a molar ratio of 2:1 (Table 1) and reacted at 50 ℃ for 10 min.

TABLE 1 ligation reaction System

Name of reagent	Amount of reagent used
		2x ClonExpress Mix	2μl
Carrier skeleton	0.5μl
		GhKNAT7-A03 fragment	4μl
dd H₂O	up to 10μl

According to the sequencing result, the structure of the recombinant plasmid pBI121-GhKNAT7-A03 is described as follows: the fragment between the BamH I and Sac I cleavage sites of the overexpression vector pBI121 was replaced by a double-stranded DNA molecule shown in sequence 2 of the sequence listing.

II, obtaining recombinant agrobacterium

1. The recombinant plasmid pBI121-GhKNAT7-A03 is introduced into Agrobacterium tumefaciens LBA4404 to obtain recombinant Agrobacterium tumefaciens, which is named recombinant Agrobacterium LBA4404/pBI121-GhKNAT 7-A03.

2. The overexpression vector pBI121 is introduced into agrobacterium tumefaciens LBA4404 to obtain recombinant agrobacterium, and the recombinant agrobacterium is named as recombinant agrobacterium LBA4404/pBI 121.

Example 3 functional verification of GhKNAT7-A03 protein in Arabidopsis thaliana

First, obtaining transgenic plant

Adopting an inflorescence dip-dyeing method to infect Columbia ecotype arabidopsis thaliana (Col-0) with recombinant agrobacterium LBA4404/pBI121-GhKNAT7-A03 to obtain a homozygous transgenic GhKNAT7-A03 gene strain, and the specific steps are as follows:

1. the recombinant agrobacterium LBA4404/pBI121-GhKNAT7-A03 is inoculated in an LB liquid culture medium, and is subjected to shaking culture at 28 ℃ and 180rpm until the OD value of a bacterial liquid is 1.2-1.6, and the bacterial liquid is centrifuged at 3000rpm to collect the bacterial body.

2. Suspending the thallus obtained in step 1 by 1/2MS liquid culture medium containing 5% (mass percentage) of sucrose and 0.03% (volume percentage) of silwet L-77, and adjusting OD₆₀₀An infection suspension was obtained at 0.8.

3. Soaking the Columbia ecotype arabidopsis inflorescence in the infection suspension obtained in the step 2 for 30-50s, wrapping the arabidopsis by using a preservative film, culturing in the dark for 24h, culturing under normal conditions, and harvesting seeds after the seeds are mature.

4. The seeds harvested in the step 3 are sterilized by 0.1% HgCl solution, then are vernalized for 3-4 days at 4 ℃, and are planted on 1/2MS solid culture medium containing 100mg/L kanamycin (agar concentration is 0.6%), positive plants and negative plants are observed in about 10 days, and the plants which can normally grow are possible to be positive plants.

5. And (3) taking the plant leaves which grow normally in the step (4), carrying out PCR amplification by adopting a primer pair consisting of a primer F1 and a primer R1 (the reaction system is shown in table 2, and the reaction program is shown in table 3), setting a positive control which adopts the recombinant agrobacterium liquid in the step (1) as a template, and setting a negative control which adopts sterilized water as the template.

F1：5’-GACGCACAATCCCACTATCC-3’；

R1：5’-CGAACCCTCTCCATTAAAGAT-3’。

TABLE 2 reaction System for PCR

Name of reagent	Amount of reagent used
		2×PCR Buffer	25μl
2mM dNTPs	10μl
		F1(10μM)	1μl
R1(10μM)	1μl
		KOD FX Neo(1U/μl)	1μl
2mm square blade	Proper amount of
		Sterilized distilled water	up to 50μl

TABLE 3 amplification procedure for PCR

And (3) carrying out 1% agarose gel electrophoresis on the PCR amplification product, and if the amplification product of about 680bp is obtained, determining that the plant to be detected is a GhKNAT7-A03 gene-transferred plant. The results of the PCR identification are shown in lanes 1-7 of FIG. 2, lane 8 being a negative control and lane 9 being a positive control.

And (4) screening 7 strains of the GhKNAT7-A03 gene-transferred positive strains according to the electrophoresis result, and harvesting seeds of T0 generations.

And (3) sowing seeds of the T0 generation for passage, and detecting a positive strain of each generation of plants by adopting the method until the plants are propagated to the T3 generation to obtain a homozygous transgenic arabidopsis thaliana strain.

Second, obtaining empty carrier plants

Replacing the recombinant agrobacterium LBA4404/pBI121 with the recombinant agrobacterium LBA4404/pBI121-GhKNAT7-A03, and performing the same steps to obtain the T3 generation homozygous empty vector Arabidopsis thaliana strain.

Third, phenotypic identification

And (3) the plant to be detected: t3 generation homozygous transgenic Arabidopsis lines, T3 generation homozygous empty vector transgenic Arabidopsis lines and wild type Arabidopsis.

1. And performing phenotype observation on the plant to be detected, selecting the plant to be detected which grows vigorously, bolting for two weeks, respectively carrying out bare-handed slicing on the hypocotyl of the plant, then carrying out phloroglucinol solution dyeing on the slices, and carrying out microscope observation.

10 strains of each strain were selected for detection.

The results are shown in FIG. 3. In FIG. 3, a is a phenotypic observation of Wild-type Arabidopsis thaliana (Wild-type); b and c are phenotype observations of transgenic Arabidopsis thaliana 35S: GhKNAT 7-A03; d is phenotype observation of GhKNAT7-A03 stalk fascicular fibers (if); e is the statistical analysis of the secondary wall thickness of the stem fasciculate fiber cells of GhKNAT 7-A03; scale bar 1cm (fig. 3a-c) and 10 μm (fig. 3 d).

TABLE 4 primer information

The result shows that a certain proportion of plants have the phenotype of branch generation or stem bifurcation in the transgenic Arabidopsis thaliana 35S, GhKNAT7-A03, but the wild type does not exist (figure 3a-c), so that the GhKNAT7-A03 is presumed to be capable of regulating the growth and development of Arabidopsis thaliana stems; section staining observation of the hypocotyls of the wild type and the transgenic Arabidopsis 35S (GhKNAT 7-A03) shows that the secondary wall thickness of the fascicular fiber cells of the stem of the transgenic Arabidopsis 35S (GhKNAT 7-A03) is obviously thinner than that of the wild type (FIG. 3d, e). The phenotype of the homozygous empty vector arabidopsis line and the wild type arabidopsis has no obvious difference.

2. Taking the stem of a plant to be tested which grows for 6 weeks, extracting total RNA and carrying out reverse transcription to obtain cDNA, taking the cDNA as a template, and adopting primers shown in table 4 to carry out expression quantity analysis on genes AtPAL1 and AtCOMT related to synthesis of lignin and cellulose in arabidopsis thaliana (taking AtActin2 as an internal reference gene).

The results are shown in FIG. 4. The results show that in the transgenic Arabidopsis (OE-L1, OE-L4, OE-L5) over-expression, the lignin synthesis related gene AtPAL1 and AtCOMT expression are obviously down-regulated. The phenotype of the homozygous empty vector arabidopsis line and the wild type arabidopsis has no obvious difference.

The above results indicate that GhKNAT7-A03 regulates the growth of Arabidopsis secondary cell walls mainly by inhibiting lignin synthesis.

Example 4 analysis of expression patterns of GhKNAT7-A03 in different fiber strength materials

The material to be tested: upland cotton TM-1, reclaimed N27-3 and Suyou 6108.

1. And (5) counting the fiber length and the strength of the mature fiber of the material to be detected.

2. Taking fibers of materials to be detected in different development stages (10DPA, 20DPA and 30DPA), extracting total RNA and performing reverse transcription to obtain cDNA, taking the cDNA as a template, and detecting the expression conditions of the GhKNAT7-A03 gene, the cellulose synthesis related gene GhCESA4 and GhCESA8, the lignin synthesis related gene GhCOMT1 and GhCAPD 5 (taking GhHis3 as an internal reference gene) by using primers shown in Table 5 through real-time fluorescent quantitative PCR amplification.

TABLE 5 primer information

The results are shown in FIG. 5. In fig. 5, a is the fiber length and strength statistics of three fiber quality differential materials; b is the expression quantity of the gene GhKNAT7-A03 in different development periods of the three material fibers; c is the expression quantity of the genes GhCESA4, GhCESA8, GhCOMT1 and GhCAD5 in different development periods of the three material fibers. The results show that the transcription level of the cellulose synthesis related gene has positive correlation with the fiber strength in three upland cotton materials with different fiber qualities, namely, the higher the fiber strength is, the higher the expression level of the secondary wall thickening period (20, 30DPA) GhCESA4 and GhCESA8 is; while lignin synthesis related genes GhCOMT1 and GhCAD5 show opposite trends; the target gene GhKNAT7-A03 is preferentially expressed in the secondary wall thickening period (20-30DPA) and shows similar results with cellulose synthesis related genes, which indicates that the GhKNAT7-A03 can regulate the growth and development of cotton fibers mainly in the secondary wall thickening period by inhibiting the expression of lignin synthesis genes and promoting the expression of cellulose synthesis genes in the growth and development process of the cotton fibers.

Sequence listing

<110> Cotton research institute of Chinese academy of agricultural sciences

<120> cotton GhKNAT7-A03 protein and coding gene and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 299

<212> PRT

<213> Cotton (Gossypium spp)

<400> 1

Met Gln Glu Pro Gly Gly Leu Gly Met Met Ser Ser Gly Gly Gly Ser

1 5 10 15

Glu Thr Ile Gly Gly Leu Ser Ser Gly Glu Val Ser Val Thr Gly Asp

20 25 30

Gln Asn Cys His Leu Lys Ala Glu Ile Ala Thr His Pro Leu Tyr Glu

35 40 45

Gln Leu Leu Ala Ala His Val Ser Cys Leu Arg Val Ala Thr Pro Ile

50 55 60

Asp Gln Leu Ser Leu Ile Asp Ala Gln Leu Ala Glu Ser His Asn Ile

65 70 75 80

Leu Arg Ser Tyr Ala Ser Gln Gln Gln Gly His Ser Leu Ser Pro His

85 90 95

Glu Arg Gln Glu Leu Asp Asn Phe Leu Ala Gln Tyr Leu Ile Val Leu

100 105 110

Cys Thr Phe Lys Glu Gln Leu Gln Gln His Val Arg Val His Ala Val

115 120 125

Glu Ala Val Met Ala Cys Arg Glu Ile Glu Asn Asn Leu Gln Ala Leu

130 135 140

Thr Gly Val Thr Leu Gly Glu Gly Thr Gly Ala Thr Met Ser Asp Asp

145 150 155 160

Glu Asp Glu Leu Gln Met Asp Phe Ser Met Asp Gln Ser Gly Pro Glu

165 170 175

Gly His Asp Leu Met Gly Phe Gly Pro Leu Leu Pro Thr Glu Ser Glu

180 185 190

Arg Ser Leu Met Glu Arg Val Arg Gln Glu Leu Lys Ile Glu Leu Lys

195 200 205

Gln Gly Phe Lys Ser Arg Ile Glu Asp Val Arg Glu Glu Ile Leu Arg

210 215 220

Lys Arg Arg Ala Gly Lys Leu Pro Gly Asp Thr Thr Thr Val Leu Lys

225 230 235 240

Asn Trp Trp Gln Gln His Ser Lys Trp Pro Tyr Pro Thr Glu Asp Asp

245 250 255

Lys Ala Lys Leu Val Glu Glu Thr Gly Leu Gln Leu Lys Gln Ile Asn

260 265 270

Asn Trp Phe Ile Asn Gln Arg Lys Arg Asn Trp His Ser Asn Ser Gln

275 280 285

Ser Val Thr Ser Leu Lys Ser Lys Arg Lys Arg

290 295

<210> 2

<211> 900

<212> DNA

<213> Cotton (Gossypium spp)

<400> 2

atgcaagaac caggtgggtt aggtatgatg agtagtggtg gtggaagtga aacgattgga 60

gggttaagta gcggtgaagt gtcagttacc ggtgaccaga actgtcatct caaggcggag 120

atagctacac atccacttta tgaacaactt ttagcagctc atgtgtcttg tcttcgtgtt 180

gctacaccga ttgatcagtt gtctttgatt gatgcacagt tagcggagtc gcataacatt 240

ttgagatcct atgcttcaca acaacaaggt cactctttat caccacatga aagacaagag 300

cttgataact tcttggcaca atatttgata gtgttgtgta ctttcaaaga acagcttcaa 360

caacatgtca gagtccatgc cgttgaagct gtcatggctt gccgtgaaat tgaaaataac 420

ttacaagcgc tcactggagt aaccttgggt gaaggcacag gtgcaacaat gtcagacgat 480

gaggatgaac ttcaaatgga cttctccatg gatcaatctg ggcccgaagg ccatgattta 540

atgggttttg gtcccttact tcccacagaa tctgaaagat ctttaatgga gagggttcgt 600

caagagctca agattgaact caaacagggt ttcaaatcaa gaattgagga tgtgagggag 660

gaaatattac gcaaaagaag ggctggaaaa ttaccaggtg acaccactac agtgctcaag 720

aactggtggc aacagcactc aaagtggcca tacccaactg aagatgacaa ggccaaactt 780

gtggaggaaa ctggattgca actaaagcaa atcaacaact ggttcatcaa ccaaaggaag 840

cgcaattggc acagcaattc tcagtcggtc acctccttga agtccaagcg gaaaaggtag 900

Claims

The application of GhKNAT7-A03 protein or related biomaterials thereof in regulating and controlling the thickness of the secondary wall of an Arabidopsis intertillary fiber cell:

the related biological material is a nucleic acid molecule capable of expressing the GhKNAT7-A03 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the amino acid sequence of the GhKNAT7-A03 protein is shown as a sequence 1 in a sequence table;

the application is embodied as follows:

the expression level of the GhKNAT7-A03 protein or the coding gene thereof in the Arabidopsis is increased, and the thickness of the secondary wall of the Arabidopsis intertidal fiber cells is reduced.
The application of GhKNAT7-A03 protein or related biomaterials thereof in regulating and controlling the expression level of plant cellulose synthesis genes:

the related biological material is a nucleic acid molecule capable of expressing the GhKNAT7-A03 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the amino acid sequence of the GhKNAT7-A03 protein is shown as a sequence 1 in a sequence table;

the application is embodied as follows:

the expression level of the GhKNAT7-A03 protein or the coding gene thereof in the plant is increased, and the expression level of a plant cellulose synthesis gene is increased;

the plant is cotton;

the plant fiber synthetic gene is GhCESA4 gene and/or GhCESA8 gene.
The application of GhKNAT7-A03 protein or related biological materials thereof in regulating and controlling the expression level of plant lignin synthesis genes:

the related biological material is a nucleic acid molecule capable of expressing the GhKNAT7-A03 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the amino acid sequence of the GhKNAT7-A03 protein is shown as a sequence 1 in a sequence table;

the application is embodied as follows:

the expression level of the GhKNAT7-A03 protein or the coding gene thereof in the plant is increased, and the expression level of a plant lignin synthesis gene is reduced;

when the plant is arabidopsis, the plant lignin synthesis gene is AtPAL1 gene and/or AtCOMT gene;

when the plant is cotton, the plant lignin synthesis gene is a GhCOMT1 gene and/or a GhCAD5 gene.
4. A method of breeding a plant variety having reduced secondary wall thickness of plant intertubercular fibroblasts, comprising the step of increasing the expression level of GhKNAT7-a03 protein in a recipient plant; the amino acid sequence of the GhKNAT7-A03 protein is shown as a sequence 1 in a sequence table; the plant is Arabidopsis thaliana.
5. A method for producing a plant variety having an increased expression level of a plant cellulose synthesis gene, comprising the step of increasing the expression level of GhKNAT7-A03 protein in a recipient plant;

the amino acid sequence of the GhKNAT7-A03 protein is shown as a sequence 1 in a sequence table;

the plant is cotton;

the plant fiber synthetic gene is GhCESA4 gene and/or GhCESA8 gene.
6. A method for producing a plant variety having a reduced expression level of a lignin synthesis gene, comprising the step of increasing the expression level of GhKNAT7-A03 protein in a recipient plant;

when the plant is arabidopsis, the plant lignin synthesis gene is AtPAL1 gene and/or AtCOMT gene;

when the plant is cotton, the plant lignin synthesis gene is GhCOMT1 gene and/or GhCAD5 gene;

the amino acid sequence of the GhKNAT7-A03 protein is shown as a sequence 1 in a sequence table.
7. A method of breeding a transgenic plant comprising the steps of: introducing a nucleic acid molecule capable of expressing GhKNAT7-A03 protein into a receptor plant to obtain a transgenic plant; the thickness of the secondary wall of the intertillary fiber cells of the transgenic plant is smaller than that of the receptor plant; the amino acid sequence of the GhKNAT7-A03 protein is shown as a sequence 1 in a sequence table; the plant is Arabidopsis thaliana.
8. A method of breeding a transgenic plant comprising the steps of: introducing a nucleic acid molecule capable of expressing GhKNAT7-A03 protein into a receptor plant to obtain a transgenic plant; the expression quantity of the cellulose synthetic gene of the transgenic plant is greater than that of a receptor plant; the amino acid sequence of the GhKNAT7-A03 protein is shown as a sequence 1 in a sequence table;

the plant is cotton;

the plant fiber synthetic gene is GhCESA4 gene and/or GhCESA8 gene.
9. A method of breeding a transgenic plant comprising the steps of: introducing a nucleic acid molecule capable of expressing GhKNAT7-A03 protein into a receptor plant to obtain a transgenic plant; the expression quantity of the lignin synthesis gene of the transgenic plant is less than that of a receptor plant;

when the plant is arabidopsis, the plant lignin synthesis gene is AtPAL1 gene and/or AtCOMT gene;

when the plant is cotton, the plant lignin synthesis gene is GhCOMT1 gene and/or GhCAD5 gene;

the amino acid sequence of the GhKNAT7-A03 protein is shown as a sequence 1 in a sequence table.
10. The method of any of claims 7-9, wherein: the 'introduction of a nucleic acid molecule capable of expressing the GhKNAT7-A03 protein' into a recipient plant is realized by introducing a recombinant expression vector containing a coding gene of the GhKNAT7-A03 protein into the recipient plant.
11. The method of claim 10, wherein: the nucleotide sequence of the coding gene of the GhKNAT7-A03 protein is shown as a sequence 2 in a sequence table.