CN116144682B

CN116144682B - Application of raffinose synthase gene SpRAFS1 of feather needle grass in promoting plant root development

Info

Publication number: CN116144682B
Application number: CN202310289990.4A
Authority: CN
Inventors: 李鸿彬; 李榕; 王斐; 杨丹
Original assignee: Shihezi University
Current assignee: Shihezi University
Priority date: 2023-03-23
Filing date: 2023-03-23
Publication date: 2024-04-12
Anticipated expiration: 2043-03-23
Also published as: CN116144682A

Abstract

The invention discloses an application of a feather needle raffinose synthase gene SpRAFS1 in promoting plant root development, and belongs to the technical field of genetic engineering. The nucleotide sequence of the raffinose synthase gene SpRAFS1 of the feather needle grass is shown as SEQ ID NO:1, the amino acid sequence of the encoded protein is shown as SEQ ID NO: 2. The invention clones to obtain the feather needle raffinose synthase SpRAFS1 gene, and successfully constructs and converts the plant expression vector, researches the function of the gene in transgenic plants, and experiments prove that the feather needle raffinose synthase SpRAFS1 gene is excessively expressed in the transgenic plants, so that the root system growth of the plants is more vigorous, and the gene has the function of improving the plant root system development; the method has important significance for revealing stress resistance mechanism of the feather seedling and enriching molecular biological theory of plant root system development.

Description

Application of raffinose synthase gene SpRAFS1 of feather needle grass in promoting plant root development

Technical Field

The invention relates to the technical field of genetic engineering, in particular to application of a feather needle grass raffinose synthase gene SpRAFS1 in promoting plant root development.

Background

In recent years, environmental problems have become one of the main factors affecting plant growth and development, and poor environments mainly include drought, waterlogging, high temperature, low temperature, salinization, and the like. Adverse stress affects crop growth and physiological changes, and can lead to crop yield reduction and quality reduction.

The root system is taken as one of important organs of crops, the strong and developed root system is the basis of the growth and development of the crops, and the root system plays the following roles in the growth process of the crops: (1) The transmission function is that the root system can absorb nutrition and moisture and transmit the nutrition and moisture to the overground part, and organic substances generated by the overground part are also transmitted to the root system. (2) The storage function is that the crops can transport the nutrients to the roots for storage in the growth process, and when the nutrients are insufficient at the overground parts, the nutrients can be transported from the roots to keep the normal growth of the crops. (3) The supporting function, the root has the fixing function, the root system can stabilize crops in soil and support the overground part from the adverse effect of external environment. (4) The root system can synthesize various necessary amino acids, alkaloids and the like for the growth of crops, and can secrete various compounds, organic acids and the like to cope with the influence of external environment changes and promote the absorption of crops to nutrients (Mei Siwei and the like.2018). Plant roots employ various mechanisms to increase the ability to acquire resources and withstand abiotic stresses (White et al, 2013a, b), including root hair generation, development of appropriate root structures, cultivation of beneficial symbiotic relationships, and improvement of physical and biological soil conditions in the rhizosphere (White et al, 2013 b).

The feather needle grass (shipagrostispennata) has the morphological characteristics that the stalks are upright, clustered, smooth and hairless; the plant height is 30-70 cm. Feather seedling is produced on sand, dunes and sand ridges with the altitude of 400-1600 m in desert, and county cities such as Haba river, boolean, urst, spermary river, sarium, marnas, hu Tu Ji, changji, miquan, qigong and Tarim basin are produced; there is also a distribution in the middle asia and europe. Feather seedling is a perennial herb plant, has a very root system structure, resists early stage, wind erosion and sand burial, and is a sand-fixing plant which is not available for fixing quicksand (buying and lifting.1990). The feather needle grass has a very rich root system structure, not only has downward elongated roots (up to 90 cm), but also has developed horizontal root systems (the number of the roots can reach more than 80 and the length can reach 4-5 m) which extend laterally, and the feather needle grass is radially distributed in a sand layer of 40 cm. The root hairs are distributed almost over the entire lateral root, are interlaced with each other and tightly bind the sand particles to the root surface, so that the sand particles are not successfully eluted by washing with distilled water. The white fine roots partially exposed on the sand surface do not have root sheaths due to long-time wind and sun exposure, but do not show wilting.

The raffinose oligosaccharides (raffinose oligosaccharides) consist of a series of 6-glucosyl groups (alpha-1, 6-Galn-Suc, 1.ltoreq.n.ltoreq.3) of alpha-1, 6-galactose linked to sucrose (sucroses, suc), mainly consisting of raffinose (containing 1 galactosyl group), stachyose (containing 2 galactosyl groups) and acteose (containing 3 galactosyl groups), which are widely distributed in different tissue sites of plants. Recent studies indicate that raffinose oligosaccharide metabolism plays an important role in plant growth and development and adverse stress reaction. Biosynthesis of raffinose oligosaccharides begins with synthesis of raffinose, which is catalyzed by Sucrose Synthase (SS) to produce sucrose from fructose and UDP-glucose, then catalyzed by raffinose synthase to transfer galactosyl from galactosyl inositol to sucrose to produce raffinose, and catalyzed by stachyose synthase to transfer galactosyl to raffinose to produce stachyose, and then catalyzed by calycosin synthase to produce calycosin (Peterbauer et al, 2001). Inositol galactose synthase (GolS, galactinol synthase, EC2.4.1.123) and raffinose synthase (RAFS, raffinose synthase, EC2.4.1.82) are key enzymes in the raffinose biosynthetic pathway. GolS functions to catalyze the synthesis of inositol (Gol, galctol) from inositol (inonositol) and UDP-Galactose (UDP-Gal, UDP-galctose); the function of RAFS is to catalyze the synthesis of Raffinose (RAFS) from inositol galactose (Gol, galactanol) and Sucrose (Suc, sucrosi) (Sengupta et al 2015).

The invention carries out transcriptional analysis on the feather needle grass to find that the raffinose synthase gene is closely related to the development of the roots of the feather needle grass. The plant expression vector of the gene is constructed, and the gene is introduced into arabidopsis thaliana, so that the gene is proved to promote plant root development, and the gene has important significance for revealing stress resistance mechanism of the feather seedling and enriching molecular biology theory of plant root development.

Disclosure of Invention

The invention aims to provide an application of a feather needle grass raffinose synthase gene SpRAFS1 in promoting plant root development, so as to solve the problems in the prior art.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides an application of a feather needle raffinose synthase gene SpRAFS1 in promoting plant root development, wherein the nucleotide sequence of the feather needle raffinose synthase gene SpRAFS1 is shown in SEQ ID NO: 1.

Further, the amino acid sequence of the protein encoded by the raffinose synthase gene SpRAFS1 of the feather needle grass is shown as SEQ ID NO: 2.

Further, the promotion of plant root development is specifically: introducing the feather needle grass raffinose synthase gene SpRAFS1 into plants, increasing the quantity and length of fibrous roots, the length of main roots and promoting root growth vigor of the plants.

Further, the plant comprises arabidopsis thaliana.

The invention also provides a primer for cloning the full length of the raffinose synthase gene SpRAFS1 of the feather needle grass, which comprises the nucleotide sequence shown in SEQ ID NO:3 and the forward primer shown in SEQ ID NO: 4.

The invention also provides a recombinant vector of the raffinose synthase gene SpRAFS1 of the feather needle grass, which contains the raffinose synthase gene SpRAFS1 of the feather needle grass.

The invention also provides a recombinant bacterium comprising the recombinant vector.

The invention also provides an application of the recombinant vector or the recombinant bacterium in improving plant root systems.

The invention discloses the following technical effects:

the invention clones to obtain the SpRAFS1 gene of the feather needle grass, successfully constructs and transforms a plant expression vector, and discovers that the same growing environment and time are the most vigorous in the development of main roots and lateral roots of the Arabidopsis thaliana transferred by the SpRAFS1 gene through carrying out phenotype observation analysis on four plants of wild WT, the SpRAFS1 gene, the Atrafs1 mutant and the Sprafs 1-transferred Atrafs1, the growth of the fibrous roots of the Arabidopsis thaliana transferred by the Sprafs1 mutant is almost not obvious, and the development of the fibrous roots of the Arabidopsis thaliana transferred by the Sprafs1 is better than that of the Atrafs1 mutant. Experimental results prove that the SpRAFS1 gene of the feather needle grass is overexpressed in transgenic plants, so that the root system growth of the plants is more vigorous, and the gene has the function of improving the root system development of the plants. The method has important significance for revealing stress resistance mechanism of the feather seedling and enriching molecular biological theory of plant root system development.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram showing PCR amplification of raffinose synthase SpRAFS1 gene; wherein M: marker d2000;1-6: spRAFS1 gene amplification band;

FIG. 2 is a 35S SpRAFS1 double cleavage verification chart; wherein M: marker III, 1:1300,2: spRAFS1,3:1300,4:1300-SpRAFS1,5:1300-SpRAFS1;

FIG. 3 is a PCR map of 35S:: spRAFS1 transformed agro-rod monoclonal bacteria; wherein M: marker III, 1-3: bacterial liquid PCR strips;

FIG. 4 is a 35S: spRAFS1 transformed wild type Arabidopsis PCR detection result, wherein M: marker III, 1-7: extracting PCR bands from transgenic Arabidopsis thaliana DNA;

FIG. 5 is 35S: PCR detection result of the SpRAFS1 transformed atlafs 1 mutant Arabidopsis thaliana, wherein M: marker III, 1-13:35S, extracting PCR results by converting the SpRAFS1 into the arabidopsis thaliana DNA of the Atrafs1 mutant;

FIG. 6 is a root development diagram of four Arabidopsis thaliana for WT, spRAFS1, atrafs1, and SpRAFS1 to Atrafs 1;

FIG. 7 is a graph showing the statistics of the number of fibrous roots of four Arabidopsis thaliana, WT, spRAFS1, atrafs1, and SpRAFS 1-to-Atrafs 1;

FIG. 8 is a graph showing the statistical results of the fibril length of WT, spRAFS1, atrafs1 and four Arabidopsis thaliana from SpRAFS1 to Atrafs 1;

FIG. 9 is a main root length statistic result of four Arabidopsis thaliana from WT, spRAFS1, atrafs1, and SpRAFS1 to Atrafs 1;

FIG. 10 is a plasmid map of plant expression vector pBI 1300.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, 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 invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The pMD-19T cloning vector and the LAtaq enzyme are purchased from TaKaRa BioCo; RNA extraction kits were purchased from Omega company; cDNA first strand reverse transcription kit, T4 ligase, rapid restriction endonuclease purchased from Fermentas BioCo; related reagents such as an RNA reverse transcription kit, LATaqDNAPolylnerase, T4-DNALigase and the like are purchased from Dalianbao biological company; related enzymes such as XbaI and XmaI are purchased from Fermentas company; 2 XTaqPCRMaster Mix, DNA gel recovery kit, plasmid extraction microcheck were purchased from TIANGEN company; kanamycin, gentamicin, MES, acetosyringone and MgCl used for test ₂ And chemical reagents such as culture medium preparation are all made into domestic analytical purity and are purchased from Shanghai biological engineering company. The synthesis of the primers used for PCR and DNA sequencing were performed by Shanghai Biotechnology Co., ltd.

Example 1: cultivation and planting of feather needle grass

Drying the feather seedling seeds collected in the field, selecting full seeds, removing caryopsis peel, and soaking in 0.05% GA3 solution for 24h. Filling sand brought back from the field into a flowerpot paved with two layers of newspapers, pouring tap water, pressing out a sand pit with the depth of about 1cm at the bottom end of tweezers, clamping seeds soaked by GA3 into the sand pit by using the tweezers, covering with a proper amount of sand, finally culturing in an environment of 37 ℃, observing germination conditions, and collecting tissue samples for standby. The feather needle grass material for subsequent RNA extraction was successfully obtained.

Example 2: extraction of total RNA of feather needle grass and synthesis of cDNA

Total RNA of the sample of Toddha's feather is extracted by referring to the instructions of the kit for extracting total RNA of the plant of Omega company, and cDNA is synthesized by using the reverse transcription kit after detecting the integrity by agarose gel electrophoresis of 1.0%.

Example 3: cloning of the RafS1 Gene of RafS Gene of Raspy grass

Based on the earlier research in the laboratory, with reference to the result of transcriptome sequencing, primers SpRAFS1-F whose sequence is 5'-ATGCCAGACATGCTAAATTGGTT-3' (SEQ ID NO: 3) and SpRAFS1-R whose sequence is 5'-GTAATCTACTGAAACCGTCCATAGG-3' (SEQ ID NO: 4) were designed, and PCR amplification was performed using cDNA of the Tornado de as a template, with the following reaction system: cDNA (50 ng/. Mu.L) 1. Mu.L, 2 XTaqPCRMastermix 10. Mu.L, primer SpRafS 1-F0.5. Mu.L, primer SpRAFS 1-R0.5. Mu.L, ddH ₂ O8. Mu.L, total 20. Mu.L. The amplification procedure was: 95 ℃ for 5min;95 ℃ for 30s,57 ℃ for 30s,72 ℃ for 2min,35 cycles; 72 ℃ for 10min; preserving at 4 ℃. After detecting the amplified product by agarose gel electrophoresis at a concentration of 1% (FIG. 1), the target band was recovered. Are sent to Shanghai Bioengineering Co., ltd for sequencing. Sequencing results of the raffinose synthase gene SpRafS1 of the feather needle grass are shown as SEQ ID NO:1, the amino acid sequence of the encoded protein is shown as SEQ ID NO: 2.

The sequencing results are consistent with the transcriptome sequencing results, which indicates that the SpRAFS1 gene cloning was successful.

Example 4: construction of SpRAFS1 Gene plant expression vector

The pBI1300 vector (purchased from vast plasmid platform: www.miaolingbio.com) containing the 35S promoter was digested with two enzymes to obtain a vector fragment, and the target gene fragment and the vector fragment were recovered. In vitro ligation is carried out on the target gene fragment and the carrier fragment, and the ligation reaction conditions are as follows: connecting at constant temperature of 16 ℃ for 6 hours; the plasmid map of the pBI1300 vector is detailed in FIG. 10. The identified correct recombinant plasmids were designated 35S:: spRAFS1, respectively. The recombinant plasmid was identified by cleavage with both KpnI and XbaI and then visualized by agarose gel electrophoresis, FIG. 2 is a 35S: spRAFS1 double cleavage proof map (M: marker III, 1:1300,2: spRAFS1,3:1300,4:1300-SpRAFS1,5:1300-SpRAFS 1.).

Experimental results: the SpRAFS1 gene plant expression vector is successfully constructed.

Example 5: transformation of Agrobacterium

Operating within an ultra clean bench: the plant expression vector which has been identified is transferred into Agrobacterium GV1301, the transformation procedure is as follows:

(1) Taking 5 mu l of the identified plasmid, adding the plasmid into 100 mu l of agrobacterium competent cells, uniformly mixing, and carrying out ice bath for 20min;

(2) Placing in liquid nitrogen for 3-5min;

(3) Heat shock at 37deg.C for 5min, and ice bath for 2min;

(4) Adding 600 μl of fresh LB culture medium without any antibiotics, mixing, and shake culturing at 28deg.C and 200rpm for 4-5 hr;

(5) Centrifuging at 5000rpm for 5min;

(6) The supernatant was discarded, and 100. Mu.l of the bacterial liquid was spread on LB solid medium containing Rif (100 mg/L), gen (50 mg/L) and Kan (50 mg/L), and cultured in a constant temperature incubator at 28℃for 2d.

And (5) selecting a monoclonal in a flat plate and carrying out PCR identification correctly to obtain the target gene which is successfully transferred into agrobacterium. FIG. 3 is a PCR map of 35 S:SpRAFS 1 transformed agro-rod monoclonal bacteria, M: marker III, 1-3: bacterial liquid PCR bands. Experimental results: the agrobacterium transformed with the SpRAFS1 gene was obtained.

Example 6: flower dropping method for dip dyeing of arabidopsis thaliana

(1) When the bolting height of the planted wild arabidopsis reaches about 3-4cm, cutting off top-grown inflorescences, stimulating the growth of axillary inflorescences, taking care to avoid damaging the axillary inflorescences, and carrying out transformation when the axillary inflorescences grow out, wherein the taught flowers and pods are removed before the first transformation so as to reduce the workload during screening;

(2) Preparation of a dip dyeing liquid: activating the identified agrobacterium containing the target gene in LB liquid containing the three antibodies (50 mg/LKan, 50mg/LGen and 20 mg/LRif), shake culturing overnight at 28 ℃, inoculating the overnight cultured bacteria into 100mL of LB liquid containing the three antibodies according to 1:100, performing expansion culture for about 6-8h, and obtaining a bacterial liquid OD ₆₀₀ When 1.5-2.5 is reached, the bacterial liquid is taken out, centrifuged at 5000rpm for 5min at 4 ℃, the supernatant is poured off, and the liquid is subjected to 1/2MS (5-6% sucrose+0.02% Silwet L-77) to suspend and precipitate, so that OD is obtained ₆₀₀ Reaching about 0.8;

(3) The dyeing solution is sucked by a 200 mu l pipettor, carefully dripped on the flower buds and not dripped on the leaves as much as possible, and the dyed arabidopsis thaliana is subjected to dark culture for 24 hours and then is subjected to normal culture, and is subjected to dip dyeing twice a week approximately for 5-6 times. After the dip dyeing is finished, the arabidopsis thaliana is cultivated under long illumination, and seeds are collected after pods turn yellow, and a later experiment is carried out.

(4) Collected seeds were sown in plates containing three antibodies (50 mg/LKan, 50mg/LGen, 20 mg/LRif), the continued growth of successfully transformed seedlings was green and the failed transformed seedlings were yellow and dead. Positive plants were initially screened for successful transformation using phenotypes.

In this embodiment, arabidopsis thaliana is selected as the transgenic plant material, and other plants may be selected as the transgenic material.

Example 7: identification of transgenic Arabidopsis thaliana

The collected and dyed arabidopsis seeds are sown on a 1/2MS (containing 50 mg/LKan) solid culture medium on a sterile operation table according to a conventional planting method, after the arabidopsis seeds grow for two to three weeks in a phytotron, the arabidopsis seedlings which are not transformed successfully gradually whiten and die, and the arabidopsis seedlings which are transformed successfully normally grow. Transferring the normally grown arabidopsis thaliana to culture soil for continuous culture, extracting the DNA of the arabidopsis thaliana subjected to primary screening, and performing PCR identification (figure 4) to obtain a positive plant, thereby ensuring the follow-up experiment.

Example 8:

acquisition of the Arabidopsis thaliana material by converting the Atrafs1 mutant into the Sprafs1 mutant:

searching the corresponding gene ID of the SpRAFS1 gene (AT 1G 55740) in an Arabidopsis Tair website (https:// www.arabidopsis.org/index. Jsp), and searching the corresponding mutant Arabidopsis SALK_090247C through a mutant purchasing website (https:// www.arashare.cn/index /), so as to complete the purchase of the Arabidopsis mutant Atrafs1 seed. Referring to the SpRAFS1 transgenic Arabidopsis experimental step, the SpRAFS1 transgenic Atrafs1 mutant gene is obtained and the Arabidopsis is complemented.

3 seeds of each of four types of arabidopsis materials obtained by earlier experiments, namely WT wild type, spRAFS1 transgene, atrafs1 mutant and SpRAFS 1-to-Atrafs 1 mutant gene complement type, are selected and evenly spread on a 1/2MS culture medium without antibiotics, and the plates are vertically placed and placed in a dark environment at 21 ℃ for 16h for cultivation. And (5) repeatedly planting for a plurality of times to obtain corresponding root system data.

FIG. 5 is 35S: PCR detection result of the SpRAFS1 transformed atlafs 1 mutant Arabidopsis thaliana, wherein M: marker III, 1-13:35S, spRAFS1 transformation of the Arabidopsis thaliana mutant DNA extraction PCR results.

Performing sterile seeding culture on four kinds of Arabidopsis thaliana (WT, spRAFS1, atrafs1 and SpRAFS 1-to-Atrafs 1) under the same condition, transferring to the same plate for vertical culture for 10-15 days after germination, and observing root growth difference, wherein the results are shown in FIG. 6; the number of the four Arabidopsis fibrous roots in the graph 6 is statistically analyzed, at least 3 plates are statistically analyzed, and the repeated 3 times are carried out, and the result is shown in the graph 7; statistical analysis was performed on the length of the four arabidopsis fibrous roots in fig. 6, and the statistical analysis was repeated 3 times for at least 3 plates, and the results are shown in fig. 8; the four Arabidopsis main root lengths in FIG. 6 were statistically analyzed, and the statistical analysis was repeated 3 times for at least 3 plates, and the results are shown in FIG. 9.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. Feather needle grass raffinose synthase geneSpRAFS1Use of the gene for raffinose synthase of the species Mentha spicata for promoting root development in plantsSpRAFS1The amino acid sequence of the encoded protein is shown in SEQ ID NO:2 is shown in the figure; the plant is Arabidopsis thaliana.

2. The use according to claim 1, wherein the promotion of plant root development is to increase the number and length of fibrous roots and main root length of the plant.