CN112391405A

CN112391405A - Application of tea tree hexokinase CsHXK3 gene in regulation of plant growth and development and enhancement of cold resistance

Info

Publication number: CN112391405A
Application number: CN202011284066.XA
Authority: CN
Inventors: 李娜娜; 王璐; 张可欣; 王新超; 杨亚军
Original assignee: Tea Research Institute Chinese Academy of Agricultural Sciences
Current assignee: Tea Research Institute Chinese Academy of Agricultural Sciences
Priority date: 2020-11-17
Filing date: 2020-11-17
Publication date: 2021-02-23
Anticipated expiration: 2040-11-17
Also published as: CN112391405B

Abstract

The application of tea tree hexokinase CsHXK3 gene in regulating plant growth and enhancing cold resistance belongs to the field of biological gene engineering technology. The invention comprises the application of the tea tree hexokinase CsHXK3 gene in regulating the growth and development of plants and enhancing the cold resistance; a method for regulating growth and development of Arabidopsis thaliana and enhancing cold resistance by using tea tree hexokinase CsHXK3 gene is provided. The invention verifies the biological function of the CsHXK3 gene for the first time through an overexpression experiment, and can provide gene resources for breeding new varieties of tea trees. The invention provides an important gene resource which can generally regulate the growth and development of plants and the cold resistance, and provides an excellent candidate gene for cultivating cold-resistant plant varieties.

Description

Application of tea tree hexokinase CsHXK3 gene in regulation of plant growth and development and enhancement of cold resistance

Technical Field

The invention belongs to the technical field of biological gene engineering, and particularly relates to an application of a tea tree hexokinase CsHXK3 gene in regulation and control of plant growth and development and enhancement of cold resistance.

Background

Tea tree [ 2 ]Camellia sinensis (L.) O. Kuntze]Is an important economic leaf crop in China. The tea tree is native to low-latitude areas such as clouds, nobility and Sichuan, forms the physiological characteristics of preferring warm and cold, and the low-temperature freezing injury in winter and the frost in spring which falls into the spring cold seriously threaten the growth, germination and geographical distribution of the tea trees. The sizes of leaves (super-large leaf type, medium leaf type and small leaf type) of tea varieties imply the strength of cold resistance, and the smaller the leaf is, the stronger the resistance to low-temperature freeze injury is. The lowest temperature at which middle and small leaf species of tea can survive is-14 ℃ to-16 ℃, while large leaf species of tea are only-6 ℃. The cold resistance of tea trees is closely related to the yield of tea leaves and the economic benefit of the tea industry. Therefore, the research on important genes participating in low-temperature resistance in tea trees clarifies the regulation and control mechanism of low-temperature response of the tea trees, breeds tea tree varieties with strong cold resistance and has an important promotion effect on the economic development of the tea industry.

Soluble sugars in plants, such as glucose and fructose (hexose), not only participate in the metabolism of the body as carbon and energy substances, but also serve as important signal molecules to regulate the growth and development of plants and the response to adversity stress. Plant Hexokinase (hxhk) is a bifunctional protein with critical roles in phosphorylating hexoses and mediating sugar signaling. Hexose in the source bank tissue is phosphorylated by HXK to form hexose-6-phosphate, and then participates in processes such as glycolysis, respiration, anabolism and the like, so that energy and metabolic substrates are provided for the life activities of plants; in addition, the HXK can be used as sugar sensing protein to participate in sugar signal transduction, sense signals such as illumination, nutrients and stress, and regulate and control gene expression and growth and development of plants. The CsHXK3 gene sequence related by the invention is disclosed in NCBI (access number: KX 078245), and previous researches show that CsHXK3 belongs to HXKL protein lacking hexose phosphorylation activity, the gene expression of the protein is rapidly induced in response to low-temperature stress, but the function of the protein is not reported at all.

The tea tree is a perennial woody plant, adopts conventional breeding methods such as hybridization, radiation mutagenesis and the like, and has the characteristics of complex breeding program, long breeding period and low breeding efficiency. In addition, the transgenic technology system of tea plant is not mature, and Arabidopsis thaliana is a model plant in the field of plant biology research. Therefore, the CsHXK3 gene can be over-expressed in Arabidopsis by an agrobacterium-mediated transgenic technology, and the biological functions of the CsHXK3 gene under the conditions of plant growth and development and low-temperature stress are further researched. The cloning and function research of the hexokinase CsHXK3 gene provides valuable gene resources for cultivating new excellent tea varieties.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to design and provide the application of the tea tree hexokinase CsHXK3 gene in regulating the growth and development of plants and enhancing the cold resistance. According to the invention, the gene is overexpressed in arabidopsis thaliana to carry out biological function research, and the CsHXK3 gene is proved to regulate the growth and development of arabidopsis thaliana and enhance the low-temperature resistance of arabidopsis thaliana by mediating sugar concentration signals in a plant body, so that a gene resource is provided for cultivating a new variety of cold-resistant plants, and the gene has extremely high potential application value.

The application of the tea tree hexokinase CsHXK3 gene in regulating the growth and development of plants and enhancing the cold resistance.

The application of the tea tree hexokinase CsHXK3 gene in regulating the growth and development of plants and enhancing the cold resistance is characterized in that the plants comprise woody plants and herbaceous plants.

The application of the tea tree hexokinase CsHXK3 gene in regulating and controlling the growth and development of plants and enhancing the cold resistance is characterized in that the nucleotide sequence of the tea tree hexokinase CsHXK3 gene is shown as SEQ ID number 1, and the amino acid sequence of the tea tree hexokinase CsHXK3 gene coding protein is shown as SEQ ID number 2.

The application of the tea tree hexokinase CsHXK3 gene in regulating and controlling the growth and development of plants and enhancing the cold resistance is characterized by comprising the following steps:

1) allowing the plant to comprise the CsHXK3 gene;

or 2) overexpressing the CsHXK3 gene in the plant.

The application of the tea tree hexokinase CsHXK3 gene in regulating and controlling the growth and development of plants and enhancing the cold resistance is characterized in that the mode 2) the CsHXK3 gene is transferred into the plants by adopting an agrobacterium-mediated method to obtain the transgenic plants with the CsHXK3 gene over-expression.

A method for regulating growth and development of arabidopsis thaliana and enhancing cold resistance by utilizing tea tree hexokinase CsHXK3 gene is characterized by comprising the following steps:

(1) extracting total RNA of tea tree leaves, carrying out reverse transcription to obtain cDNA, designing an upstream primer F and a downstream primer R, carrying out PCR amplification by taking the tea tree cDNA as a template to obtain a CsHXK3 gene amplification product, and connecting the amplification product to a plant expression vector PH7FWG2 with a 35S promoter to obtain a recombinant expression vector;

(2) and transforming the recombinant expression vector to agrobacterium, infecting an arabidopsis inflorescence by using the transformed agrobacterium to obtain a transgenic arabidopsis strain.

The method for regulating growth and development of arabidopsis thaliana and enhancing cold resistance by utilizing the tea tree hexokinase CsHXK3 gene is characterized in that the nucleotide sequence of the upstream primer F is shown as SEQ ID number 3, and the nucleotide sequence of the downstream primer R is shown as SEQ ID No. 4.

The biological function of the CsHXK3 gene is verified for the first time through an overexpression experiment. The CsHXK3 gene is over-expressed in Arabidopsis by transgenic technology, and the obtained CsHXK3 transgenic plant has the phenotype of plant type, small leaf, reduced fresh weight of aerial parts and enhanced cold resistance. Therefore, the cloning and the functional verification of the CsHXK3 gene of the tea tree can provide gene resources for the breeding of new varieties of tea trees. The invention provides an important gene resource which can generally regulate the growth and development of plants and the cold resistance, and provides an excellent candidate gene for cultivating cold-resistant plant varieties.

Drawings

FIG. 1 is a diagram showing the detection of the expression of the CsHXK3 gene of over-expressed strains OE-1, OE-2 and OE-3 in example 2 of the present invention;

FIG. 2 is a photograph of growth germination of seeds homozygous for the overexpression lines OE-1, OE-2 and OE-3 in example 3 of the present invention on 1/2 MS medium containing 1.5% glucose (A), 6.0% glucose (B), 1.5% fructose (C), 6.0% fructose (D), and statistics of survival of seeds on media containing different concentrations of glucose (E) and fructose (F);

FIG. 3 is a photograph of normal 23 days at ambient temperature growth of the over-expressing lines OE-1, OE-2 and OE-3 in example 3 of the present invention (A) and fresh weight statistics of aerial parts of each 2 plants (n = 8; B);

FIG. 4 shows photographs (A) of over-expressed strains OE-1, OE-2 and OE-3 of example 4 of the present invention grown normally at room temperature for 21 days and (B) of low temperature treatment at 4 ℃ for 6 hours, as well as conductivity (C), soluble sugar content (D), glucose content (E) and fructose content (F) under normal culture and low temperature stress.

Detailed Description

The invention is further described in the following description with reference to the figures and specific examples to facilitate a better understanding thereof, but without limiting the scope of the invention. Unless otherwise stated, the examples are carried out according to the customary experimental methods (cf. Sambrook J. & Russell D. W., Molecular cloning: a laboratory manual, 2001) or according to the manufacturer's instructions.

pENTR in the examples^TMthe/D-TOPO is a common cloning vector and can be purchased from the market; pH7FWG2 is a commonly used expression vector, commercially available; the arabidopsis wild type variety is Columbia ecotype; agrobacterium GV3101 is a commonly used strain, and is preserved in most molecular biology laboratories.

The main reagents in the following examples are: pENTR^TMa/D-TOPO cloning vector kit, an LR reaction kit, a plant total RNA extraction TRIzol reagent and the like, which are purchased from Invitrogen company; high fidelity PrimeSTAR^®HS DNA Polymerase enzyme, dNTP, reverse transcription kit and the like, purchased from TaKaRa company; a plasmid extraction kit and an agarose gel recovery kit, purchased from Axygen corporation; hygromycin, SYBR green, etc., available from Roche; agarose, kanamycin, spectinomycin, rifampin, and the likePurchased from Sigma. The various other chemicals used in the examples were all imported or homemade analytical grade reagents.

In the examples, the primers to be synthesized and the vector to be sequenced were both served by the Shanghai Huajin Biotechnology Co., Ltd.

Example 1: cloning of tea tree CsHXK3 gene and construction of overexpression vector

According to a CsHXK3 sequence published in an NCBI database, a nucleotide sequence of the CsHXK3 sequence is shown as SEQ ID number 1, an amino acid sequence of encoded protein is shown as SEQ ID number 2, upstream and downstream primers F and R are designed, tea leaf cDNA is used as a template, and a CsHXK3 gene is obtained through PCR amplification. The amplified sequence was ligated into pENTR^TMthe/D-TOPO vector is finally connected to a plant expression vector with the pH of 7FWG2 through an LR reaction.

The upstream primer F used: 5'-CACCATGGGGAAGGTAGGGTTG-3', shown as SEQ ID number 3; a downstream primer R: 5'-TAGCAACTGTACGGTATCCACATT-3', as shown in SEQ ID number 4.

Example 2: construction and screening of CsHXK3 gene overexpression plant

The vector successfully constructed in example 1 was transformed into agrobacterium GV3101 strain, and then transformed into wild arabidopsis by the floral dip method, and seeds of transgenic arabidopsis T0 generation were harvested. The screening resistance gene carried by the pH7FWG2 vector is hygromycin, so, hygromycin is used for carrying out positive screening on transgenic seedlings of Arabidopsis, T1 generation positive seedlings with hygromycin resistance are subjected to single plant harvest, seeds of T1 generation are subjected to hygromycin resistance screening, the separation ratio is identified, and resistant seedlings are selected: lines of non-resistant plantlets, approximately 3:1, indicate that in this line the vector sequence carrying the CsHXK3 gene is inserted in single copy form into the plant genome. And (3) removing seedlings with hygromycin resistance from T2 generations of the strains, harvesting the strains individually, screening for hygromycin resistance, and if the seedlings are not separated, indicating that the transgenic strains are homozygotes, wherein the homozygotes of the T3 generations can be used for subsequent breeding and physiological experiments.

And screening to obtain over-expression strains OE-1, OE-2 and OE-3. Extracting total RNA of wild type OE-1, OE-2 and OE-3, carrying out reverse transcription to synthesize cDNA, detecting the expression level of CsHXK3 in over-expressed strains OE-1, OE-2 and OE-3 by using a qRT-PCR method, and finding that the CsHXK3 genes are all highly expressed in OE-1, OE-2 and OE-3 and the expression quantity is increased in sequence as shown in figure 1.

Example 3: growth phenotype of plants overexpressing CsHXK3

Wild type, OE-1, OE-2 and OE-3 Arabidopsis seeds were sown on 1/2 MS solid medium of 1.5% glucose and 6.0% glucose and 1.5% fructose and 6.0% fructose with the seed germination growth states shown in FIGS. 2A, B, C and D. Wild type seeds were able to germinate healthily on both 1.5% and 6.0% glucose medium with no significant difference in survival (fig. 2E), while three over-expressed lines showed less significant survival than 1.5% glucose on 6.0% glucose medium (fig. 2E). Likewise, the seed survival rate of the three over-expressed strains on 6.0% fructose containing medium was significantly lower than 1.5% fructose compared to wild type (fig. 2F). The result shows that the CsHXK3 can sense the sugar concentration signal to participate in the regulation of the growth and the germination of the plant seeds.

Sowing wild type, OE-1, OE-2 and OE-3 Arabidopsis seeds into seedling growing blocks for growing, and photographing and recording when the seeds are cultured for 23 days, as shown in figure 3A; fresh aerial weight measurements were performed (8 biological replicates per 2 strains as one biological replicate) as shown in FIG. 3B. As a result, three over-expressed plants all showed a phenotype of reduced leaf area and fresh weight of aerial parts compared with the wild type, wherein lines OE-2 and OE-3 with high over-expression amount are significantly reduced.

Example 4: low temperature resistance of over-expressed CsHXK3 plant

Wild type, OE-1, OE-2 and OE-3 Arabidopsis seeds are sowed into seedling blocks for growth, and 2 seedlings are planted in each seedling block. Culturing at normal temperature for 21 days, and showing the growth state in FIG. 4A; after 6 hours of 4 ℃ cold treatment, the over-expressed lines were found to have significant phenotypic difference with wild type at low temperature, i.e. wild type showed complete leaf wilting and collapse, and the over-expressed lines were leaf strong, as shown in FIG. 4B.

The relative conductivity of the plants is detected, and the relative conductivity of 3 overexpression lines is remarkably lower than that of the wild type after the plants are treated at normal temperature, 4 ℃ for 3 days at low temperature and 0 ℃ for 8 hours, as shown in figure 4℃, the fact that the overexpression CsHXK3 gene in the plants is favorable for the stability of cell membranes is shown.

Under the condition of normal temperature, the contents of glucose and fructose in OE-2 strains and OE-3 strains are both significantly higher than that of wild strains, as shown in FIGS. 4E and F. After 7 days of low temperature treatment at 4 ℃, the soluble total sugar, glucose and fructose contents in 3 over-expression strains were increased to different degrees compared with the wild type, wherein the sugar contents in the OE-2 strain were all increased significantly, as shown in FIGS. 4D, E and F. Therefore, under the low-temperature stress, the content of soluble sugar components in plant cells can be increased by improving the expression quantity of the CsHXK3 gene.

The cold resistance of the plant is positively regulated and controlled by the CsHXK3 gene through mediating the sugar concentration signal in the plant tissue by integrating the phenotype and the physiological index change of the plant. It should be noted that the above-mentioned description only describes a few specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and some modifications can be made on the basis of the present invention. Accordingly, all changes that can be made by one skilled in the art without departing from the spirit of the invention are to be considered within the scope of the invention.

Sequence listing

<110> institute of tea leaf of Chinese academy of agricultural sciences

<120> application of tea tree hexokinase CsHXK3 gene in regulation of plant growth and development and enhancement of cold resistance

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<213> Tea tree hexokinase CsHXK3 gene (Tea tree hexokinase CsHXK3 gene)

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atggggaagg tagggttggg gttggcggtg gggtgcgcag tggcgacgtg cgcaatcgcg 60

gcggtgatgg tggggaggag ggtgaggagg cagcggaggt ggaggagagc ggtggcggtt 120

ttggaggagt tcgaggaagc gtgctctacg tcggttttgc gtctgagaca ggtagttgat 180

gctatggcgg ttgagatgca cgcgggttta gcctctgaag gtggtagtaa gctcaagatg 240

cttctcacct acgtcgataa acttcccaat gggagtgaga agggaaccta ttacgcgcta 300

gatcttggag gtactaattt cagggtcttg cggcttcagc ttgtgggtga taggtctgcc 360

attcctgatc ctgatgttga aaggaaaccg attcctctaa atctaaaggc cagcacaggc 420

gaggaactct ttgatttgat tgcttcatca ctaaaagagt ttatagaaaa gaaagaagga 480

gttcatgaac tatcagcagt caaaagaagg gaacttggat ttacattctc gttccctgtg 540

aagcaattgt ctgtctcgtc aggcattcta atcaaatgga cgaaaggttt taccattgaa 600

gacatggttg gaagagatgt ttctgaatgc ttacaacaaa caatgtctaa aaaaggcgta 660

aatatggggg tagcagcact tgtaaatgat actgtgggaa cattagcact tggacattat 720

gatgacgaag acacagttgc tgcagtgata attgggactg gtacaaatgc ctgctatttg 780

gagcgggcag atgctattat taaatgtcaa ggcttgctta ctacttcagg aggcatggta 840

gttaacatgg aatggggaaa tttctggtca tctcatctgc caagaacatc ttacgatact 900

gatttagatg ccgatagcct gaacccaaat gatcaaggtt ttgagaaaat gatatcagct 960

atgtatctgg gtgacattgt aagaagagtg cttctcagga tgtcacagga gtctgatatt 1020

tttggacctg tagcttctaa actatcagtg ccttctattt taagtacaga acatatggct 1080

gctatgcatg aggatgattc ccccgatttg aaggaagtag tcagaatttt aaatgatgtc 1140

ctagagattc ccggtgtccc tctaaaagtt cggaagcttg ttgtgaaggt atgtgatgtg 1200

gtgactcgca gggcagcccg attggcagct tccggaattg ttggcatctt gaagaagatt 1260

ggtcgagatg ggagtggtgg cattacaagt ggaagaatca aaagtggaag tcatagtaag 1320

atcagaagaa cagttgtggc gattgaggga ggtttgtata ccggttattt gatgttcaga 1380

gaatacttga atgaagccat gaccgaaatc ttgggcgaag atgtctccgc acatgtcatt 1440

cttaggatca cgaaagatgg accaggcatt ggatcagcac tcctggccgc attgtattcg 1500

tcttccaatg tggataccgt acagttgcta tag 1533

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<213> tea tree hexokinase CsHXK3 gene encoding protein (The tea tree hexokinase CsHXK3 gene encodes a protein)

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Cys Ala Ile Ala Ala Val Met Val Gly Arg Arg Val Arg Arg Gln Arg

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Arg Trp Arg Arg Ala Val Ala Val Leu Glu Glu Phe Glu Glu Ala Cys

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Ser Thr Ser Val Leu Arg Leu Arg Gln Val Val Asp Ala Met Ala Val

50 55 60

Glu Met His Ala Gly Leu Ala Ser Glu Gly Gly Ser Lys Leu Lys Met

65 70 75 80

Leu Leu Thr Tyr Val Asp Lys Leu Pro Asn Gly Ser Glu Lys Gly Thr

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Tyr Tyr Ala Leu Asp Leu Gly Gly Thr Asn Phe Arg Val Leu Arg Leu

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Gln Leu Val Gly Asp Arg Ser Ala Ile Pro Asp Pro Asp Val Glu Arg

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Lys Pro Ile Pro Leu Asn Leu Lys Ala Ser Thr Gly Glu Glu Leu Phe

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Asp Leu Ile Ala Ser Ser Leu Lys Glu Phe Ile Glu Lys Lys Glu Gly

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Val His Glu Leu Ser Ala Val Lys Arg Arg Glu Leu Gly Phe Thr Phe

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Ser Phe Pro Val Lys Gln Leu Ser Val Ser Ser Gly Ile Leu Ile Lys

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Trp Thr Lys Gly Phe Thr Ile Glu Asp Met Val Gly Arg Asp Val Ser

195 200 205

Glu Cys Leu Gln Gln Thr Met Ser Lys Lys Gly Val Asn Met Gly Val

210 215 220

Ala Ala Leu Val Asn Asp Thr Val Gly Thr Leu Ala Leu Gly His Tyr

225 230 235 240

Asp Asp Glu Asp Thr Val Ala Ala Val Ile Ile Gly Thr Gly Thr Asn

245 250 255

Ala Cys Tyr Leu Glu Arg Ala Asp Ala Ile Ile Lys Cys Gln Gly Leu

260 265 270

Leu Thr Thr Ser Gly Gly Met Val Val Asn Met Glu Trp Gly Asn Phe

275 280 285

Trp Ser Ser His Leu Pro Arg Thr Ser Tyr Asp Thr Asp Leu Asp Ala

290 295 300

Asp Ser Leu Asn Pro Asn Asp Gln Gly Phe Glu Lys Met Ile Ser Ala

305 310 315 320

Met Tyr Leu Gly Asp Ile Val Arg Arg Val Leu Leu Arg Met Ser Gln

325 330 335

Glu Ser Asp Ile Phe Gly Pro Val Ala Ser Lys Leu Ser Val Pro Ser

340 345 350

Ile Leu Ser Thr Glu His Met Ala Ala Met His Glu Asp Asp Ser Pro

355 360 365

Asp Leu Lys Glu Val Val Arg Ile Leu Asn Asp Val Leu Glu Ile Pro

370 375 380

Gly Val Pro Leu Lys Val Arg Lys Leu Val Val Lys Val Cys Asp Val

385 390 395 400

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

405 410 415

Leu Lys Lys Ile Gly Arg Asp Gly Ser Gly Gly Ile Thr Ser Gly Arg

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Ile Lys Ser Gly Ser His Ser Lys Ile Arg Arg Thr Val Val Ala Ile

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

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Leu Arg Ile Thr Lys Asp Gly Pro Gly Ile Gly Ser Ala Leu Leu Ala

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Ala Leu Tyr Ser Ser Ser Asn Val Asp Thr Val Gln Leu Leu

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<212> DNA

<213> upstream primer F (Upestream primer F)

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caccatgggg aaggtagggt tg 22

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<213> downstream primer R (Downstem primer R)

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Claims

1. The application of the tea tree hexokinase CsHXK3 gene in regulating the growth and development of plants and enhancing the cold resistance.

2. The use of the tea tree hexokinase CsHXK3 gene as defined in claim 1 for regulating the growth and development of plants and enhancing cold resistance, wherein said plants include woody plants and herbaceous plants.

3. The application of the tea tree hexokinase CsHXK3 gene in regulating the growth and development of plants and enhancing the cold resistance as claimed in claim 1, wherein the nucleotide sequence of the tea tree hexokinase CsHXK3 gene is shown as SEQ ID number 1, and the amino acid sequence of the protein encoded by the tea tree hexokinase CsHXK3 gene is shown as SEQ ID number 2.

4. The application of the tea tree hexokinase CsHXK3 gene in regulating the growth and development of plants and enhancing the cold resistance of the plants as claimed in claim 1, which is characterized by comprising the following modes:

1) allowing the plant to comprise the CsHXK3 gene;

or 2) overexpressing the CsHXK3 gene in the plant.

5. The application of the tea tree hexokinase CsHXK3 gene in regulating the growth and development of plants and enhancing the cold resistance of plants as claimed in claim 4, wherein the mode 2) is to transfer the CsHXK3 gene into the plants by an agrobacterium-mediated method to obtain transgenic plants with over-expressed CsHXK3 gene.

6. A method for regulating growth and development of arabidopsis thaliana and enhancing cold resistance by utilizing tea tree hexokinase CsHXK3 gene is characterized by comprising the following steps:

7. The method for regulating growth and development of arabidopsis thaliana and enhancing cold resistance by utilizing the tea tree hexokinase CsHXK3 gene as claimed in claim 6, wherein the nucleotide sequence of the upstream primer F is shown as SEQ ID number 3, and the nucleotide sequence of the downstream primer R is shown as SEQ ID No. 4.