CN114457089B

CN114457089B - Gene for increasing length of tomato root hair and application thereof

Info

Publication number: CN114457089B
Application number: CN202110418850.3A
Authority: CN
Inventors: 李厦; 李恩; 史雪莲; 张彦
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2021-04-19
Filing date: 2021-04-19
Publication date: 2023-06-23
Anticipated expiration: 2041-04-19
Also published as: CN114457089A

Abstract

The invention discloses a gene for increasing the length of tomato root hairs and application thereof, belonging to the technical field of crop molecular genetics. The nucleotide sequence of the gene is shown as SEQ ID NO. 1. Through a series of experiments, the invention discovers that after Solyc04g077960 is excessively expressed in the Ailsa Craig ecological tomatoes by means of a transgenic technology, the length of the root hairs of the tomatoes is obviously increased, the fixation capacity of the tomatoes to soil is effectively improved, and the growth condition of the tomatoes under the condition of poor nutrient substances is obviously improved. The method provided by the invention provides an important molecular marker for breeding work of cultivating new varieties of tomatoes, and provides an important reference for creating new varieties with improved plant biological yield.

Description

Gene for increasing length of tomato root hair and application thereof

Technical Field

The invention relates to the technical field of crop molecular genetics, in particular to a gene for increasing tomato root hair length and application thereof.

Background

Root hair is an important component of plant root system, which is a tissue formed by the bulge of plant root epidermal cells, a special structure of roots (Peterson and Farquhar, 1996). Root hairs play a key role in plants absorbing water and nutrients, plants responding to stress, plants interacting with microorganisms, and the like. Studies have shown that: the long root hairs are beneficial to improving the fixation capacity of plants to soil and improving the absorption of the plants to nutrient substances. Therefore, the deep research on plant root hairs has important theoretical and practical significance for improving the absorption and utilization of crop nutrients and moisture, increasing crop yield and improving crop quality.

Tomato (Solanum lycopersicum l.) is one of the most widely cultivated vegetable crops worldwide, and tomato is also one of the important modes of crop research in plant science. The tomato yield is greatly affected due to the differences of soil quality and nutritional ingredients in different areas. The development of the root hairs of the tomatoes is directly influenced by environmental factors, and the root hairs have important effects on improving the moisture absorption and nutrient substances of the tomatoes and improving the fixation capacity of the tomatoes to soil. However, the molecular mechanisms currently regulating tomato root hair growth are not well known. The elucidation of the molecular mechanism for regulating the growth and development of root hairs is not only helpful for the deep understanding of the development and differentiation rules of plant cells, but also can better serve the production practice. Therefore, by combining genetic engineering with genetic breeding methods, searching for genes for increasing the length of tomato root hairs has important research and application values.

Disclosure of Invention

The invention aims to provide a gene for increasing the length of tomato root hairs and application thereof. The invention discovers and identifies the Solyc04g077960 gene for increasing the length of the root hair of the tomato, and can obviously increase the length of the root hair of the tomato after the Solyc04g077960 gene is excessively expressed in the tomato, thereby effectively improving the fixation capacity of the tomato to soil and obviously improving the growth condition of the tomato under the condition of poor nutrient substances.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in a first aspect of the invention there is provided the use of the gene Solyc04g077960 in any one of (1) to (4) below:

(1) Increasing the length of tomato root hairs;

(2) Improving the fixation capacity of tomatoes to soil;

(3) Improving the growth condition of tomatoes under the condition of being lean in nutrients;

(4) Breeding tomatoes;

the nucleotide sequence of the gene Solyc04g077960 is shown in SEQ ID NO. 1.

Furthermore, the use of recombinant expression vectors, recombinant bacteria and/or transgenic cell lines comprising the above-described gene Solyc04g077960 for increasing the root hair length of tomatoes, improving the fixation capacity of tomatoes to soil and/or improving the growth conditions of tomatoes under nutrient-lean conditions is also within the scope of the invention.

In a second aspect of the invention there is provided the use of a protein encoded by the gene Solyc04g077960 in any one of (1) to (3) below:

(1) Increasing the length of tomato root hairs;

(2) Improving the fixation capacity of tomatoes to soil;

(3) Improving the growth condition of tomatoes under the condition of being lean in nutrient substances.

In the application, the amino acid sequence of the gene Solyc04g077960 coded protein is shown as SEQ ID NO. 2.

In a third aspect of the present invention there is provided a method of increasing the root hair length of tomato comprising the step of increasing the expression level and/or activity of a protein shown in SEQ ID NO.2 in tomato.

In a fourth aspect of the present invention, there is provided a method of growing a transgenic plant having increased root hair length, comprising the steps of:

introducing a gene Solyc04g077960 into a receptor plant, and enabling the gene Solyc04g077960 to be over-expressed in the receptor plant to obtain a transgenic plant; the transgenic plant has an increased root hair length as compared to the recipient plant.

Preferably, the recipient plant is tomato.

Preferably, tomato root hair specific promoter Pro is used _LeEXT1 The constructed expression vector enables the gene Solyc04g077960 to be over-expressed in a receptor plant.

The invention has the beneficial effects that:

the invention discovers for the first time that after Solyc04g077960 is excessively expressed in the Ailsa Craig ecological tomatoes by means of a transgenic technology, the length of the root hairs of the tomatoes is obviously increased, the fixation capacity of the tomatoes to soil is effectively improved, and the growth condition of the tomatoes under the condition of poor nutrient substances is obviously improved. The method provided by the invention provides an important molecular marker for breeding work of cultivating new varieties of tomatoes, and provides an important reference for creating new varieties with improved plant biological yield.

Drawings

FIG. 1 is an analysis of expression patterns of Solyc04g077960 from tomato. Wherein (A) is tomato transcriptome data (the data is derived from Sol Genomics Network, website: https:// solgenomics. Net /), (B) is RT-qPCR results for different tissues of tomato. The different letters in the bar graph represent significant differences (one-way ANOVA, tukey-Kramer test, P < 0.05).

FIG. 2 use of tomato root hair specific promoter Pro _LeEXT1 And (5) analyzing plasmid map and transgenic materials of the constructed expression vector. Wherein (A) is the plasmid map of the expression vector of the transformed tomato, and (B) is the identification result of the exogenous gene DNA level of the transgenic tomato.

FIG. 3 is a picture of root hairs and quantification results of Solyc04g077960 overexpressing transgenic tomato and wild type tomato "Ailsa Craig". Wherein (A) is a root hair picture of wild-type tomato and three transgenic lines (# 1, #15, # 18) with a scale of 1mm, (B) is an RNA level detection result of Solyc04g077960 in wild-type tomato and three transgenic lines (# 1, #15, # 18), and (C) is a root hair length quantification result of wild-type tomato and three transgenic lines (# 1, #15, # 18). The different letters in the bar graph represent significant differences (one-way ANOVA, tukey-Kramer test, P < 0.05).

FIG. 4 is a graph of soil fixation ability and quantification results of Solyc04g077960 overexpressing transgenic tomato and wild type "Ailsa Craig". Wherein (A) is a picture of the soil fixation capacity of wild-type tomato and three transgenic lines (# 1, #15, # 18) with a scale of 1cm, and (B) is a quantification result of the soil fixation capacity of wild-type tomato and three transgenic lines (# 1, #15, # 18). The different letters in the bar graph represent significant differences (one-way ANOVA, tukey-Kramer test, P < 0.05).

FIG. 5 is a graph showing the growth and quantification of Solyc04g077960 overexpressing transgenic tomato under different nutritional conditions than wild type "Ailsa Craig". Wherein (A) is a growth picture of wild tomatoes and three transgenic lines (# 1, #15, # 18) under the condition of sufficient nutrition and the condition of barren nutrition, and the proportion is 2.5cm; (B) Fresh weight results for wild tomato and three transgenic lines (# 1, #15, # 18) under nutritionally adequate conditions for aerial parts of tomato; (C) Fresh weight results for the aerial parts of tomato under nutrient-lean conditions for wild-type tomato and three transgenic lines (# 1, #15, # 18). The different letters in the bar graph represent significant differences (one-way ANOVA, tukey-Kramer test, P < 0.05).

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention are all conventional in the art and are commercially available. The experimental procedure, without specifying the detailed conditions, was carried out according to the conventional experimental procedure or according to the operating instructions recommended by the suppliers.

Example 1:

the transcriptome data of tomato was searched at Sol Genomics Network by bioinformatics means to identify the gene Solyc04g077960 expressed in high amounts in tomato root hairs. RNA of different tissues of tomatoes is extracted, and the result is verified by using RT-qPCR.

The method comprises the following specific steps:

(1) Tomato RNA extraction (the medicine used comes from RNA extraction kit CW 0597S)

Respectively taking materials of different tissues of tomatoes, quickly putting the materials into liquid nitrogen for freezing, grinding the materials into powder, adding 300 mu L RNA Lysis Buffer, and fully grinding the materials again to completely separate protein nucleic acid complexes. At room temperature, 12000g was centrifuged for 1 minute. Transferring the supernatant to a new 1.5mL centrifuge tube, adding the same amount of absolute ethyl alcohol, sucking and beating, and uniformly mixing. Adding the mixed solution of the supernatant and the absolute ethyl alcohol into an adsorption column, centrifuging 12000g for 30 seconds, pouring out waste liquid in a collecting pipe, and putting the adsorption column back into the collecting pipe. 400 mu L RNA Wash Buffer g and 12000g are added into the adsorption column, the mixture is centrifuged for 20 seconds, waste liquid in the collecting pipe is poured out, and the adsorption column is replaced into the collecting pipe. Directly adding 40. Mu.L of DNase I mixture (35. Mu. L DNA Digestion Buffer to 5. Mu.L of DNase I) to the adsorption column ₂ Uniformly mixed), and incubating for 15 minutes at 20-30 ℃. Adding 400 mu L RNA Prep Buffer and 12000g into adsorption column, centrifuging for 30 s, pouring out waste liquid in collecting tube, and suckingThe additional column is replaced in the collection tube. 700 mu L RNA Wash Buffer g and 12000g are added into the adsorption column, the mixture is centrifuged for 30 seconds, waste liquid in the collecting pipe is poured out, and the adsorption column is replaced into the collecting pipe. 400 mu L RNA Wash Buffer g and 12000g were added to the column and centrifuged for 2 minutes to pour the waste liquid from the collection tube. The adsorption column was left at room temperature for several minutes and dried thoroughly. The column was placed in a new RNase-free centrifuge tube, 15. Mu.L DNase/RNase-free Water was added to the middle portion of the column, and after 1 minute at room temperature, it was centrifuged, 12000g was centrifuged for 1 minute, and the obtained RNA was stored in a refrigerator at-80℃or reversed.

(2)RT-qPCR

1. Mu.g of RNA was taken, oligo (dT) primer (100. Mu.M) was added at 0.5. Mu.L, and RNase free H was added ₂ O-supplemented 7.4. Mu.L, incubated at 70℃for 10 minutes, rapidly quenched on ice for more than 2 minutes, slightly centrifuged, and then added with reverse transcription reaction solution (5X M-MLV buffer 2. Mu.L, dNTP mix (50 mM) 0.1. Mu.L, RNase inhibitor (40U/0.5. Mu.L) 0.25. Mu.L, RNase M-MLV (200U/0.5. Mu.L) 0.25. Mu.L) and RNase free H ₂ O was added in 10. Mu.L, reacted at 37℃for 30 minutes, reacted at 50℃for 5 minutes, and reacted at 98℃for 5 minutes, and the obtained cDNA was stored in a-80℃refrigerator or subjected to subsequent experiments. Diluting cDNA sample to 0.5-2 ng/. Mu.L, taking 1. Mu.L in PCR tube, adding reaction liquid (qRT-PCR forward primer (5. Mu.M) 1. Mu.L, qRT-PCR reverse primer (5. Mu.M) 1. Mu.L,

buffer 5. Mu.L), and RNase free H was added ₂ The O was filled in 10. Mu.L and placed in a fluorescent quantitative PCR instrument (Bio-rad CFX 96). The reaction conditions are as follows: pre-denaturation at 95 ℃ for 30 seconds, denaturation at 95 ℃ for 5 seconds, annealing at 58 ℃ for 10 seconds, extension at 72 ℃ for 15 seconds, followed by plate reading, cycling 45 times from step 2, dissolution profile reading from 65 ℃ to 95 ℃ every 0.5 ℃. Each treatment had three biological replicates and three systems replicates. The results were analyzed using Bio-rad CFX management software.

The internal reference quantitative primers are:

F：5’-TTGCTGACCGTATGAGCAAG-3’；(SEQ ID NO.3)

R：5’-GGACAATGGATGGACCAGAC-3’。(SEQ ID NO.4)

the quantitative primers for Solyc04g077960 were:

F：5’-CAACAACAACCTTCCTGATGAG-3’；(SEQ ID NO.5)

R：5’-ATTGATCGCATACCATTTCAGC-3’。(SEQ ID NO.6)

the RT-qPCR results of different tissues of tomatoes are shown in FIG. 1B, and the results show that: the expression level of the gene Solyc04g077960 in tomato roots is obviously higher than that in tomato leaves, flowers and other parts.

Example 2:

use of tomato root hair specific promoter Pro _LeEXT1 Driving Solyc04g077960, constructing an expression vector, taking wild tomato 'Ailsa Craig' as a material, obtaining transgenic tomatoes by a callus infection method, and identifying the transgenic tomatoes by PCR.

The method comprises the following specific steps:

(1) Tomato genome extraction

Fresh leaves of tomato were removed with scissors, placed in a 1.5mL centrifuge tube, and thoroughly ground with a grinding rod. 400. Mu.L of EB buffer was added to the centrifuge tube, mixed well by shaking with a vortex, and centrifuged at 13000rpm for 3 minutes at room temperature. 300. Mu.L of the supernatant was transferred to a 1.5mL centrifuge tube containing 300. Mu.L of isopropyl alcohol, mixed upside down, and left to stand at 4℃for 20 minutes. The supernatant was removed by centrifugation at 13000rpm for 5 minutes at room temperature. Add 500. Mu.L 70% ethanol and mix by inversion. Centrifuge at 13000rpm for 2 minutes at room temperature, remove 70% ethanol. Standing at room temperature for 4-8 hr, adding 75 μl TE for dissolving back after ethanol is completely volatilized, and obtaining tomato genome.

EB buffer formula:

TE formula:

(2) Tomato expression vector construction

The tomato genome is used as a template, the reaction solution is added into a PCR tube according to a reaction system (0.1 mu L of PCR forward primer (10 mu M), 0.1 mu L of PCR reverse primer (10 mu M), 5X Phusion Mix 12.5 mu L of template, 1 mu L of DNase/RNase-free Water and 11.3 mu L of template), after sealing, PCR amplification is carried out, pre-denaturation is carried out at 98 ℃ for 5 minutes, denaturation at 98 ℃ for 30 seconds, annealing at 66 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds (30 seconds/1 Kb), circulation is carried out for 35 times, extension at 72 ℃ for 10 minutes, gel recovery is carried out by a kit method by using 1% agarose gel electrophoresis, and the tomato root hair specific promoter Pro is obtained _LeEXT1 (promoter Pro) _LeEXT1 The sequence of (C) is shown as SEQ ID NO. 7). Cloning of the gene Solyc04g077960, and finally Pro with tomato cDNA as template _LeEXT1 Linking into expression vector by homologous recombination method, linking Solyc04g077960 into expression vector by LR reaction, constructing tomato transgene vector Pro _LeEXT1 : GFP-Solyc04g077960 (shown in FIG. 2A).

Transgenic tomatoes are obtained by a company by a callus infection method, the genome is extracted after the transgenic tomatoes are taken, and the transgenic tomatoes are determined by PCR identification.

Pro _LeEXT1 The cloning primers are as follows:

F：5’-atataagcttAAGCTCTAAGTATGAATTTTTTTAATATCGGAAC-3’；(SEQ ID NO.8)

R：5’-atatggtaccTGAGAAGAATTGGATTCTAAGGCTAGATATATC-3’。(SEQ ID NO.9)

solyc04g077960 clone primers were:

F：5’-CACCatggaacatgttggagctttttttgatg-3’；(SEQ ID NO.10)

R：5’-TCATTGCATATTTGGTAGCATTTTTTGATA-3’。(SEQ ID NO.11)

the transgenic tomato identification primers are as follows:

F：5’-TTGATTTGTCACAACGGAGT-3’；(SEQ ID NO.12)

R：5’-TTGATTTGTCACAACGGAGT-3’。(SEQ ID NO.13)

the result of the exogenous gene DNA level identification of the transgenic tomato is shown in FIG. 2B.

Example 3:

seedlings of wild type tomato and transgenic tomato (transgenic tomato prepared as in example 2) were cultivated on MS medium as follows:

the tomato seeds are sterilized by surface sterilization and then are planted on a 1/2MS solid culture medium containing 0.8 percent (mass fraction) of agar powder, and the interval between the seeds is about 0.8 cm. Culturing in dark at 25deg.C until germination (about 3-4 days), transferring into light culture room, culturing under light at 25deg.C for 16 hr/8 hr darkness, and taking photos with stereo vision after 4 days. Root hair pictures were quantified using Image J software. Roots of wild tomatoes and three transgenic lines (# 1, #15, # 18) were taken, RNA was extracted, inverted, and Solyc04g077960 expression levels of the different lines were detected by RT-qPCR.

As a result, it was found that the length of transgenic tomato root hairs was significantly increased, and the length of different transgenic tomato root hairs was positively correlated with the expression level of Solyc04g077960, further confirming that overexpression of Solyc04g077960 resulted in a longer tomato root hair (fig. 3).

Example 4:

seedlings of wild type tomatoes and transgenic tomatoes (transgenic tomatoes are prepared in example 2) are cultivated in soft soil and after a period of time the fixation capacities of tomatoes to the soil are compared as follows:

seedlings grown in an illumination incubator for 4 days are transplanted into nutrient soil (the ratio of soil to vermiculite is 3:1, and the volume ratio) and the roots are kept perpendicular to the horizontal plane during transplanting. (the reason why the root is perpendicular to the horizontal plane is to remove the error caused by the bending of the root and to facilitate the later pulling-out of the seedling), the seedling was gently pulled out after 3 days of growth in the soil, and the weight (m) ₁ ) Without the weight of the seedlings (m ₂ ) And measuring the length of the root (l). Calculate the weight (m ₀ ) And drawing a chart. The calculation formula is as follows: m is m ₀ ＝(m ₁ -m ₂ )/l。

The results are shown in fig. 4, which shows that: the fixation capacity of the transgenic tomatoes to the soil is obviously improved, and the fixation capacity of different transgenic tomatoes to the soil is positively correlated with the expression level of Solyc04g077960, which shows that the overexpression of Solyc04g077960 effectively improves the soil fixation capacity of tomatoes.

Example 5:

wild-type and transgenic tomatoes (prepared in example 2) were cultivated in nutrient-rich and nutrient-poor medium, comprising the following steps:

transplanting the seedlings growing in the illumination incubator for 4 days into a pot filled with vermiculite (taking care of selecting the seedlings with consistent root length as much as possible), and pouring nutrient solution. The Nutrient rich group (Nutrient-rich) was topped with 1/4 times the Nutrient solution and the Nutrient lean group (Nutrient-pool) was topped with 1/8 times the Nutrient solution. After 15 days of seedling growth, the seedlings were pulled out of the vermiculite medium, the root vermiculite was washed clean, photographed and the fresh weight of the aerial parts was weighed.

The preparation method of the nutrient solution comprises the following steps:

mother liquor formula (g/L):

the formula of the nutrient solution comprises the following steps:

the results are shown in fig. 5, which shows that: the fresh weight of transgenic tomato is significantly higher than that of wild type. Along with the expression level of Solyc04g077960, the fresh weight of the transgenic tomato is also obviously increased, which indicates that the overexpression of Solyc04g077960 is beneficial to improving the growth state of the tomato.

By combining the researches, the invention discovers a gene Solyc04g077960 which is involved in regulating and controlling the elongation of tomato root hairs, improving the fixing ability of tomatoes to soil and improving the growth state of tomatoes under the condition of nutrition deficiency.

The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

SEQUENCE LISTING

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Claims

1. GeneSolyc04g077960Use in any one of the following (1) - (3):

(1) Increasing the length of tomato root hairs;

(2) Improving the fixation capacity of tomatoes to soil;

the geneSolyc04g077960The nucleotide sequence of (2) is shown as SEQ ID NO. 1.

2. Comprising a geneSolyc04g077960The recombinant expression vector, recombinant bacterium and/or transgenic cell line in increasing the root hair length of tomato, improving the fixation capacity of tomato to soil and/or improving the growth condition of tomato under the condition of poor nutrient substances;

the geneSolyc04g077960The nucleotide sequence of (2) is shown as SEQ ID NO. 1.

3. GeneSolyc04g077960Use of the encoded protein in any one of the following (1) - (3):

(1) Increasing the length of tomato root hairs;

(2) Improving the fixation capacity of tomatoes to soil;

geneSolyc04g077960The amino acid sequence of the coded protein is shown as SEQ ID NO. 2.

4. A method for increasing the length of tomato root hairs, comprising the step of increasing the expression level and/or activity of a protein shown by SEQ ID No.2 in tomato.

5. A method of growing a transgenic plant having increased root hair length comprising the steps of:

introduction of the Gene of claim 1 into a recipient plantSolyc04g077960Causing the gene toSolyc04g077960Over-expressing in a recipient plant to obtain a transgenic plant; the transgenic plant has an increased root hair length as compared to the recipient plant;

the recipient plant is tomato.

6. The method of claim 5, wherein a tomato root hair specific promoter is usedPro _LeEXT1 Constructed expression vectors make genesSolyc04g077960Overexpression in recipient plants.