CN116855509A - Grass drought-enduring gene LmMYB1 and application thereof - Google Patents

Abstract

The application provides a grass drought-enduring gene LmMYB1, belonging to the technical field of plant genetic engineering, wherein the CDS nucleotide sequence of the LmMYB1 is shown as SEQ ID NO. 1. The over-expression of LmMYB1 can improve the resistance of ryegrass with multiple flowers under drought stress, so that the yield of ryegrass with multiple flowers in winter and spring is effectively improved. The application also provides application of the grass drought-enduring gene LmMYB1 in ryegrass genetic breeding.

Description

Grass drought-enduring gene LmMYB1 and application thereof

Technical Field

The application belongs to the technical field of plant genetic engineering, and particularly relates to a grass drought-enduring gene LmMYB1 and application thereof.

Background

Lolium multiflorum (Lolium multiflorum lam.) is also known as Italian ryegrass, annual ryegrass, belonging to the genus Lolium (Gramineae) of Gramineae, with high nutritive value and good palatability, and is one of the best-known pastures in the world, mainly distributed in temperate and subtropical regions. Ryegrass is one of cold-season grasses with the widest cultivation and utilization area in southern areas of China, is used as a family grass seed produced by grass eating animal husbandry in winter and spring, and is a main source for feed supply of cattle, sheep, rabbits, fishes and winter and spring Ji Qinglu.

Drought, which is an environmental factor widely existing in nature, especially seasonal drought in winter and spring, has become one of the most critical factors restricting the formation of ryegrass yield. In order to cope with drought stress and improve the pasture production capacity, various breeding schemes such as conventional cross breeding and molecular auxiliary breeding are adopted at present. These methods have long cycle and low efficiency, and are difficult to cope with changeable natural environments. Therefore, there is an urgent need to further explore resistance-related genes, understand stress response mechanisms and potential mechanisms of molecular pathways, and thereby accelerate pasture breeding by using over-expression and other genetic engineering methods.

Disclosure of Invention

In order to solve the problem of insufficient drought tolerance of ryegrass, the application provides a grass drought tolerance gene LmMYB1, and the overexpression of LmMYB1 can improve the resistance of ryegrass under drought stress, so that the yield of ryegrass in winter and spring is effectively improved.

The application also provides application of the grass drought-enduring gene LmMYB1 in ryegrass genetic breeding.

The application is realized by the following technical scheme:

the application provides a grass drought-enduring gene LmMYB1, wherein the CDS nucleotide sequence of the LmMYB1 is shown as SEQ ID NO. 1.

Based on the same inventive concept, the application also provides application of the grass drought-enduring gene LmMYB1 in ryegrass genetic breeding.

Based on the same inventive concept, the application provides a coding protein of a grass drought-enduring gene LmMYB1, and the amino acid sequence of the coding protein is shown as SEQ ID NO. 2.

Specifically, the amino acid sequence of the encoded protein is as follows:

MGRSPCCEKAHTNKGAWTKEEDDRLTAYIKAHGEGCWRSLPKAAGLLRCGKSCRLRWINYLRPDLKRGNFSEEEDELIIKLHSLLGNKWSLIAGRLPGRTDNEIKNYWNTHIRRKLMSRGIDPVTHRSINEQHGSNITISFEADAAAREDNKGAVFRRDEPTKVAITQANHHQMEWGQGKPLKCPDLNLDLCISPPIQEEKPVVKREAGVGVCFSCSLGGLPKSTDCKCSSFLGFRTAMLDFRSLEMK。

based on the same inventive concept, the application also provides application of the coding protein of the grass drought-enduring gene LmMYB1 in ryegrass genetic breeding.

Based on the same inventive concept, the application provides a method for improving drought tolerance of ryegrass, which comprises the following steps:

ligating the LmMYB1 gene to a vector and then transforming agrobacterium tumefaciens;

infecting the calluses of ryegrass multiflora with agrobacterium tumefaciens containing the LmMYB1 gene, co-culturing, and screening to obtain resistant calluses;

and (3) carrying out plant regeneration culture on the resistant callus to obtain the ryegrass resistance seedling.

Further, the transformation of agrobacterium tumefaciens after the LmMYB1 gene is connected to a vector specifically comprises the following steps:

the LmMYB1 gene is connected into a PHG vector under the control of a CaMV 35S promoter, and the obtained PHG-LmMYB1 is further transformed into agrobacterium tumefaciens GV3101.

Further, the method comprises the steps of infecting the calluses of ryegrass multiflorum with agrobacterium tumefaciens containing the LmMYB1 gene, co-culturing, and screening to obtain resistant calluses, wherein the method specifically comprises the following steps:

inducing ryegrass explants to produce embryogenic callus;

preparing an infection bacterial liquid by using agrobacterium tumefaciens containing the LmMYB1 gene as a raw material;

punching holes at different positions of the embryogenic callus, soaking the embryogenic callus in the infectious microbe liquid for infection, and placing the embryogenic callus in a co-culture medium for co-culture after the infection is finished;

placing the co-cultured embryogenic callus in a screening culture medium for screening culture to obtain the resistant callus.

Further, the plant regeneration culture is carried out on the resistant callus to obtain the ryegrass resistant seedling, which specifically comprises the following steps:

transferring the resistant callus to a differentiation culture medium for plant regeneration culture, and transferring to a rooting culture medium for rooting culture to obtain the ryegrass resistant seedling.

Further, after the ryegrass resistant seedlings are obtained, hardening the ryegrass resistant seedlings, and then transplanting.

Further, the method comprises the steps of punching holes at different positions of the embryogenic callus, immersing the embryogenic callus in the infectious microbe liquid for infection, and placing the embryogenic callus in a co-culture medium for co-culture after the infection is finished, and specifically comprises the following steps:

punching holes at different positions of the embryogenic callus, soaking the embryogenic callus in the infectious microbe liquid for infection for 25min, and placing the embryogenic callus in a co-culture medium after infection is finished, and co-culturing in a dark environment for 3d.

One or more technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

1. according to the grass drought-enduring gene LmMYB1, the drought tolerance of transgenic arabidopsis can be improved by over-expressing the LmMYB1 gene, the drought tolerance of arabidopsis is improved by participating in a cell wall biological process and reducing the stomatal density, and the LmMYB1 is further over-expressed in ryegrass, so that the result shows that the LmMYB1 can also improve the resistance of ryegrass with flowers under drought stress, and the yield of ryegrass with flowers in winter and spring is effectively improved.

2. The application of the grass drought-enduring gene LmMYB1 in ryegrass genetic breeding provided by the application applies the LmMYB1 gene to ryegrass genetic breeding in a genetic engineering manner, and the LmMYB1 gene is overexpressed in a ryegrass transgenic strain, so that the resistance of ryegrass in drought stress can be remarkably improved, and the yield of ryegrass in winter and spring can be further improved.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, 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 of LmMYB1 protein multiple sequence alignment and phylogenetic tree construction.

FIG. 2 is a graph of qRT-PCR analysis of LmMYB1 response to drought and ABA stress.

FIG. 3 is a positive validation graph of transgenic Arabidopsis: (A) DNA detection of T1 generation strain of transgenic Arabidopsis plant, the target gene is hygromycin phosphotransferase (HPT, 589 bp); (B) qRT-PCR verifies the expression level of LmMYB1 in different strains of transgenic Arabidopsis.

FIG. 4 is a phenotypic analysis of wild-type and transgenic Arabidopsis under abiotic stress: (A) Phenotype of WT and transgenic lines in 1/2MS dishes with 5. Mu.M ABA, 200mM mannitol and 100mM NaCl added; (B) (C) root length and fresh weight of aerial parts of WT and transgenic lines under different conditions. n=15, p <0.05, p <0.01, p <0.001, p <0.0001.

FIG. 5 shows the results of an drought tolerance test for increasing Arabidopsis thaliana over-expression of LmMYB 1: (A) Phenotype of WT and transgenic lines (OE 1, OE4, OE 9) under drought stress; (B) survival rate of WT and (OE 1, OE4, OE 9) after rehydration; (C-G) drought tolerance-related physiological parameters; data are presented as mean and standard deviation of four independent experiments, asterisks represent the difference between WT and OE4, OE9 (< 0.05, < 0.01).

FIG. 6 shows the results of an Arabidopsis thaliana stomatal density reduction test by overexpression of LmMYB 1: (A-C) representative plots of WT and transgenic lines (OE 1, OE 4) stomatal density; (D) Air pore density statistics for WT and transgenic lines (OE 1, OE 4); asterisks represent the difference between WT and OE (×p <0.01, ×p < 0.001).

FIG. 7 shows the results of an LmMYB1 transgenic ryegrass potting natural drought stress test: (a) a 14d postpotted natural drought stress phenotype; (B) - (D) drought stress response related physiological indicators (Fv/Fm, electrolyte leakage rate, health index).

Detailed Description

The advantages and various effects of the present application will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the application, not to limit the application.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, 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 case of conflict, the present specification will control.

Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present application are commercially available or may be prepared by existing methods.

The whole idea of the application is as follows:

in order to cope with drought stress and improve the production capacity of ryegrass, various breeding schemes such as conventional crossbreeding and molecular auxiliary breeding are adopted at present. These methods have long cycle and low efficiency, and are difficult to cope with changeable natural environments. Therefore, there is an urgent need to further explore resistance-related genes, understand stress response mechanisms and potential mechanisms of molecular pathways, and thereby accelerate pasture breeding by using over-expression and other genetic engineering methods.

The plant responds to drought stress in a variety of ways. In order to cope with oxidative stress caused by drought stress, its own enzyme scavenging system will be used for defense, and CAT, POD and SOD are important antioxidants in enzyme scavenging systems, under drought stress they increase in various forms, effectively reducing damage caused by free radicals to plants. On the other hand, the variation of leaf structure and morphology also plays an important role in coping with drought, and the stomata of plants make them more adaptable to varied climates and environments.

Based on the gene, the application explores and screens the resistance related genes from the angles of leaf structure, shape, air hole change and the like when the plant is in response to drought environment, thereby improving the drought tolerance of ryegrass. We found in the lolium multiflorum transcriptome data that LmMYB1 had higher expression levels in root and leaf tissues of lolium multiflorum and was up-regulated by induction of drought stress, suggesting that LmMYB1 may be involved in lolium multiflorum response to drought stress. According to the application, the LmMYB1 gene is overexpressed in arabidopsis, the drought tolerance is effectively improved, physiological and molecular mechanisms of the LmMYB1 gene under drought stress are further explored, and the result shows that the overexpression of the LmMYB1 improves the drought tolerance of transgenic arabidopsis by participating in a cell wall biological process and reducing the stomatal density. Further, lmMYB1 is overexpressed in ryegrass, and the result shows that LmMYB1 can also improve the resistance of ryegrass under drought stress.

The application relates to a grass drought-enduring gene LmMYB1 and application thereof, which are described in detail below with reference to examples and experimental data.

Example 1

The present example performs genetic transformation and drought resistance evaluation of Arabidopsis thaliana.

LmMYB1 Multi-sequence alignment and bioinformatics analysis

The LmMYB1 gene sequence is extracted from the transcriptome data of ryegrass, and the exact sequence is obtained after cloning. Meanwhile, other homologous protein sequences were downloaded from National Center for Biotechnology Information (NCBI), and DNAMAN software was used to analyze the amino acid sequences under default parameters. The phylogenetic tree analysis was performed using MEGA11 software, and the results are shown in fig. 1.

FIG. 1 shows the multi-sequence alignment of LmMYB1 proteins and the construction of a phylogenetic tree, wherein FIG. (A) shows the comparison of the homology of MYB proteins with LmMYB1 proteins of ryegrass in other plants, and the conserved regions of amino acid sequences are marked with red and black boxes. Panel (B) is phylogenetic tree construction and analysis of LmMYB1 and other plant MYB proteins.

2. Genetic transformation of Arabidopsis thaliana

For transformation of arabidopsis, we cloned the CDS of LmMYB1 from cDNA of ryegrass leaves and ligated into PHG vector under the control of CaMV 35S promoter, PHG-LmMYB1 further transformed agrobacterium tumefaciens GV3101. Arabidopsis thaliana transformation was performed using the Arabidopsis thaliana col-0 floral dip method. Positive transgenic plants were screened by hygromycin to obtain at least 9 independent transgenic plants (i.e., OE1 to OE 9) until T3 generation for further analysis.

The screened homozygous Arabidopsis lines are identified, and the positive lines are amplified by PCR to screen the target gene hygromycin phosphotransferase (HPT, 589 bp), and the results show that all OE 1-OE 9 are successfully transferred into LmMYB1 genes (figure 3A). qRT-PCR analysis is carried out on the extracted seedling leaves of the obtained arabidopsis thaliana LmMYB1-OE strain, and differences of expression amounts among different strains are compared, so that the result shows that the transcription level of the LmMYB1 in all strains is obviously improved compared with that of the wild type arabidopsis thaliana, the result shows that the LmMYB1 has been successfully transferred into the wild type arabidopsis thaliana, and the LmMYB1 genes are over-expressed in over-expression strains, wherein the expression amounts of the LmMYB1 in OE1, OE4 and OE9 are relatively higher, and the candidate strains are selected as candidate strains for deep experiments (FIG. 3B).

3. Drought resistance evaluation of transgenic arabidopsis thaliana

Seeds of different lines were inoculated in 1/2MS medium for vertical cultivation. When the root length was about 1cm, 1/2MS, 1/2 MS+5. Mu.M ABA, 1/2MS+200mM mannitol, 1/2MS+100mM NaCl treated medium were transferred under sterile conditions, respectively. The phenotype after stress was observed and photographed, and the seedling overground weight and root length were measured.

As shown in FIG. 4, lmMYB1 can significantly improve the resistance of Arabidopsis under 5. Mu.M ABA, 200mM mannitol and 100mM NaCl stress. The results show that on a normal plate, the wild type and the over-expressed strain have no significant difference in root length and overground part weight, while under the stress of 5 mu M ABA, the root length of the over-expressed strain is longer, and the strong adaptability is shown; simulation of drought stress with 200mM mannitol found that the overexpressed strain was significantly better than the wild-type, both in root length and in aerial part weight; the over-expressed strain has a greater weight of the aerial parts and an increased salt stress resistance under 100mM NaCl stress. From plate experiments, lmMYB1 can increase the resistance of plants to abiotic stress, especially drought stress.

The WT line and the OE line (OE 1, OE4 and OE 9) grow identically after germination for 12d on a 1/2MS medium, and are transplanted into a flowerpot for cultivation (the substrate is peat soil, perlite and vermiculite, the volume ratio is 8:1:1). Watering is stopped after the culture for 3 weeks under normal conditions, and phenotype observation and photographing are carried out when natural drought occurs. And (5) after rehydrating for 1 week, observing again, photographing again, and counting the survival rate.

And measuring drought stress related physiological indexes such as antioxidant enzyme (POD, SOD, CAT), malondialdehyde (MDA) content, electrolyte Leakage (EL) and the like before and after drought stress so as to determine the responses of different wild types and over-expression systems to the drought stress.

As shown in fig. 5, the growth conditions of the wild type and the over-expressed strain are not significantly different before the drought stress starts (fig. 5A), the soil drought experiment is started after water cut, the overground part of the wild type arabidopsis thaliana withers to be more rapid and yellow under the drought stress, and the over-expressed strain shows higher resistance; the antioxidant enzyme (POD, CAT, SOD) activity of Arabidopsis LmMYB1-OE was up-regulated more than the wild-type after drought stress (FIGS. 5D-5F); the MDA and EL of Arabidopsis thaliana LmMYB1-OE were significantly lower than the wild-type under drought stress (FIGS. 5C, 5G), indicating that LmMYB1-OE had a higher effect of maintaining cell homeostasis. After rehydration, both the wild type and the overexpressed lines had different degrees of resuscitation, but the survival rate of the overexpressed lines was significantly higher (fig. 5B). In conclusion, the results show that LmMYB1 can significantly improve drought tolerance of Arabidopsis thaliana.

4. Pore density measurement

The leaf of Arabidopsis thaliana was sampled at 30d, and the pore density was observed. The sheared leaf was rapidly fixed in 2.5% glutaraldehyde (pH 7.2). And 3 plants of the largest leaf of each strain are taken and stored in a refrigerator at 4 ℃. The leaves are taken out from glutaraldehyde fixing solution, the epidermis of the leaves is wiped clean by absorbent cotton stained with alcohol, a thin layer of nail polish is smeared on the epidermis, after the leaves are dried, nail polish films are stripped by forceps, and the leaves are placed on a glass slide to be naturally stretched, and are observed under a microscope. One piece was made for each sample. 10 fields were randomly selected from the sections, observed under an optical microscope (10×40), photographed, counted, and the average value calculated.

As a result, as shown in FIG. 6, stomata of the wild-type and overexpressing strains were observed by means of an optical microscope. Three biological replicates were set up for wild-type and overexpressing strains, each replicate selecting rosette leaves of the same location to make fixed sections, and 10 field photographs were randomly collected for statistical stomatal density under (10×40) fields of view for each section. The results showed that the average stomatal density per field of WT, OE1, OE4 was 6.267, 5.200, 4.933, the overexpressed strain was significantly reduced by 17.02% (n=30, p=0.0026) and 21.29% (n=30, p=0.0002) compared to the wild-type arabidopsis leaf stomatal density (fig. 6D). The results further demonstrate that LmMYB1 may increase drought tolerance in arabidopsis by reducing stomatal density.

Example 2

The genetic transformation and drought resistance evaluation of ryegrass with flowers were performed in this example.

1. Genetic transformation of ryegrass

1.1 pretreatment of explants

Seeds of ryegrass have glumes, contain endophytes, and are a serious source of contamination when used as explants to induce callus. To circumvent this problem, seed glumes were post-removed and sterilized prior to testing. The glume-removed seed disinfection procedure is as follows: sterilizing with 75% ethanol for 2min, washing with double distilled water for 6 times, soaking with 0.1% mercuric chloride for 20min, and washing with double distilled water for 9 times.

1.2 callus induction and subculture

The sterilized bare seeds are put into a callus induction culture medium, and callus induction is carried out under the dark culture condition at 26 ℃. After 40d, transferring the induced callus to a subculture medium for 14d for 2 times under the same condition as the callus induction.

1.3 preparation of Agrobacterium solution

The single colony of Agrobacterium was selected by streaking a resistant plate and cultured overnight in a resistant liquid LB medium (28 ℃,220 ℃)

r/min); then the strain is amplified and cultured until the OD600 = 1.6-1.8; centrifuging at 5000r/min for 5min, removing supernatant, adding infection liquid to suspend the supernatant, and repeating the operation twice; and finally suspending the submerged bacteria by using an infection liquid, wherein the OD600 = 0.6-0.8, and placing the submerged bacteria into a 28 ℃ incubator for culturing for 1-2 hours, and carrying out ice bath on ice for later use.

1.4 infection of callus

Taking out embryogenic callus which is vigorous in development on a subculture medium and is in a pale yellow and granular hard state, pricking holes at different parts of the callus before infection, soaking the embryogenic callus in prepared infectious bacteria liquid for 25min, transferring the infected callus to sterile filter paper, absorbing excessive bacteria liquid, spreading the embryogenic callus on a co-culture medium, and culturing the embryogenic callus in a dark environment at 28 ℃ for 3d.

1.5 callus screening

In an ultra-clean workbench, placing the co-cultured ryegrass callus into a sterile culture flask, washing the co-cultured ryegrass callus with a cleaning solution (200 mg/L of sterile water of timentin) for 3-4 times, transferring the co-cultured ryegrass callus onto sterile absorbent paper, absorbing liquid on the surface of the callus, spreading the co-cultured ryegrass callus onto a screening culture medium, and culturing the co-cultured ryegrass callus in the incubator at the dark temperature of 25 ℃.

1.6 differentiation culture

After 30d of screening culture, the resistant callus is transferred to a differentiation medium for plant regeneration culture.

1.7 rooting culture

The adventitious buds develop to about 3cm and can be transferred to a rooting culture medium for rooting culture of plants.

1.8 seedling hardening and transplanting

After rooting culture for a period of time, when the regenerated plants grow to about 7cm, opening the bottle cap, hardening seedlings in a greenhouse (20 ℃ +/-5 ℃), then removing the seedlings, carefully cleaning root culture medium residues, transplanting the seedlings into a small basin containing nutrient soil (turfy soil: vermiculite: perlite=8:1:2), and watering the seedlings with root-fixing water.

In this example, genetic transformation of Lolium multiflorum all recombinant vectors were identical to the genetic transformation of' 2. Arabidopsis thaliana in example 1. The required medium formulation is as follows.

Callus induction medium: basic culture medium MS, final concentration of 2,4-D is 5mg/L, final concentration of hydrolyzed casein is 300mg/L, and pH is adjusted to 5.8; subculture medium: basic culture medium MS, final concentration of 2,4-D is 5mg/L, final concentration of acid hydrolyzed casein is 300mg/L, and pH is adjusted to 5.8; agrobacterium suspension medium: basic culture medium MS,2,4-D final concentration 2mg/L, acetosyringone final concentration 150 mu mol/L, pH value adjusted to 5.8; co-culture medium: basic culture medium MS, acetosyringone final concentration 150 mu mol/L, pH is adjusted to 5.8; screening the culture medium: basic culture medium MS, hygromycin final concentration 40mg/L, pH to 5.8; differentiation medium: basic culture medium MS,6-BA final concentration 2mg/L, naphthylacetic acid final concentration 0.5mg/L, sialon final concentration 0.1mg/L, pH value adjusted to 5.8; rooting medium: basic culture medium MS, final concentration of naphthylacetic acid is 0.5mg/L, final concentration of indoleacetic acid is 0.5mg/L, and pH is adjusted to 5.8.

2. Drought resistance evaluation of transgenic ryegrass

Extracting and screening the RNA of the LmMYB1-OE positive plant of the ryegrass, and carrying out reverse transcription to synthesize cDNA, and carrying out qRT-PCR identification. And further analyzing the expression quantity of LmMYB1 between different transgenic plants, and determining OE54 and OE60 as subsequent test plants. And (3) for drought resistance evaluation of transgenic plants, when the ryegrass with flowers in the pot plant enters a tillering stage, selecting wild type and over-expressed LmMYB1-OE ryegrass plants with consistent growth vigor, stopping watering, and carrying out natural drought.

For LmMYB1 expression profiling, wild-type and overexpressed LmMYB1-OE of ' Chuannong No. 1' ryegrass were grown in the university of Sichuan agriculture in a growthroom (E, 103℃51'42", N,30℃42'19 '). The daily cycle of the growth chamber was 14/10h and 22/20℃respectively. The illumination is 750 mu mol m ^-2 ·s ^-1 . The air humidity was 70%. The Lolium multiflorum seeds of Chuannong No. one are sterilized and placed in square flowerpots with the height of 6cm, the length of 25cm and the width of 6cm for cultivation. The seeds are sterilized by 1% sodium hypochlorite solution for 10min, washed by deionized water for 3-5 times, and 150 seeds are uniformly sown in each square basin. Water was used before germination and 1/2 Hoagland solution was used after germination. When the number of the plant leaves reaches 3-4, stress treatment is carried out. Drought stress was simulated using 15% PEG-6000 and 30. Mu. Mol ABA was added for treatment. Leaves were harvested 0h, 3h, 6h, 12h and 24h after drought and ABA stress. All tissues were immediately stored in liquid nitrogen and then at-80 ℃.

The results are shown in fig. 2, and LmMYB1 was up-regulated by drought stress induction under drought stress, 3.57-fold up-regulated at 12h (fig. 2A); lmMYB1 was also up-regulated by ABA induction under ABA stress, 3.21-fold up-regulated at 24h (FIG. 2B), suggesting that LmMYB1 may be closely related to ryegrass drought and ABA stress response.

As shown in fig. 7, natural drought stress was applied to the LmMYB1 transgenic ryegrass OE54, OE60, after 14d of natural drought. The result shows that the wild type gradually withers and turns yellow, the photosynthetic efficiency (Fv/Fm) and the health index (Plant health index) are obviously reduced, and the electrolyte leakage rate is also continuously increased, so that the drought has a larger influence on the wild type ryegrass; the anti-sightedness transgenic plant is stable in relative growth vigor, which shows that LmMYB1 can maintain the activity of photosynthesis related paths and obviously improve the drought tolerance of ryegrass.

The LmMYB1 nucleotide sequence obtained by the application is as follows.

>LmMYB1

ATGGGGAGGTCGCCGTGCTGCGAGAAGGCGCACACCAACAAGGGCGCGTGGACTAAGGAGGAGGACGACCGGCTCACTGCCTACATCAAGGCCCCTGGCGAGGGCTGCTGGCGCTCGCTGCCCGCCAAGGCGGCGGGACTCCTCCGTTGCGGCAAGAGCTGCCGCCTCCGGTGGATCAACTACCTGCGGCCCGACCTCAAGCGCGGCAACTTCAGCGAGGAGGAGGACGAACTCATCATCAAGCTCCACAGCCTCCTGGGCAACAAATGGTCCCTGATCGCCGGGAGACTGCCGGGGAGGACGGACAACGAG

ATCAAGAACTACTGGAACACGCACATCAGGCGGAAGCTGATGAGCCGGGGGATAGACCCGGTGACACACCGGTCGATC

AACGAGCAGCACGGGTCCAACATAACCATATCATTCGAGGCTGCGGCTGCGGCGGCAAGGGAGGACAACAAGGGCGCC

GTGTTCCGGCGAGACGAGCCCAAGATGGCCCCGGCGGCGATCACCCACCAGATGGAGTGGGGCCAGGGGAAGCCGCTC

AAGTGCCCGGACCTGAACCTGGACCTCTGCATCAGCCCCCCGATCCAAGAGGAGAAGCCCGTAGTGAAGCGTGAGGCT

GGCGTCGGCGTCTGCTTCAGCTGCAGCCTGGGCGGCCTCCCCAAGAGCACCGACTGCAAGTGCAGCAGCTTCCTCGGC

TTCCGGACCGCCATGCTCGACTTCAGAAGCCTCGAGATGAAATGA

The multiple sequence alignment showed that LmMYB1 has two typical MYB conserved domains R, the R2R3-MYB transcription factor (FIG. 1A). The phylogenetic tree is found that MYB genes similar to other plants are compared, and the homology relationship between the MYB genes and ZmMYB31, taMYB29, atMYB29 and AtMYB47 is closer. Presumably LmMYB1 may have similar increases to these genes

Plant stress tolerance.

Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present application have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the application.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (10)

1. A grass drought-enduring gene LmMYB1 is characterized in that the CDS nucleotide sequence of the LmMYB1 is shown as SEQ ID NO. 1.

2. The use of a grass drought-enduring gene LmMYB1 according to claim 1 in genetic breeding of ryegrass.

3. The encoded protein of the grass drought-enduring gene LmMYB1 is characterized in that the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.

4. Use of a protein coding for the drought-enduring gene LmMYB1 of grass according to claim 3 for genetic breeding of ryegrass.

5. A method of improving drought tolerance of ryegrass, the method comprising:

ligating the LmMYB1 gene to a vector and then transforming agrobacterium tumefaciens;

infecting the calluses of ryegrass multiflora with agrobacterium tumefaciens containing the LmMYB1 gene, co-culturing, and screening to obtain resistant calluses;

and (3) carrying out plant regeneration culture on the resistant callus to obtain the ryegrass resistance seedling.

6. The method for improving drought tolerance of ryegrass as claimed in claim 5, wherein said transforming agrobacterium tumefaciens after ligating the LmMYB1 gene to the vector comprises:

the LmMYB1 gene is connected into a PHG vector under the control of a CaMV 35S promoter, and the obtained PHG-LmMYB1 is further transformed into agrobacterium tumefaciens GV3101.

7. The method for improving drought tolerance of ryegrass according to claim 5, wherein the step of infecting the calli of ryegrass with agrobacterium tumefaciens containing the LmMYB1 gene, and performing screening after co-culture to obtain resistant calli comprises the following steps:

inducing ryegrass explants to produce embryogenic callus;

preparing an infection bacterial liquid by using agrobacterium tumefaciens containing the LmMYB1 gene as a raw material;

punching holes at different positions of the embryogenic callus, soaking the embryogenic callus in the infectious microbe liquid for infection, and placing the embryogenic callus in a co-culture medium for co-culture after the infection is finished;

placing the co-cultured embryogenic callus in a screening culture medium for screening culture to obtain the resistant callus.

8. The method for improving drought tolerance of ryegrass as claimed in claim 5, wherein said plant regeneration culture of said resistant calli is carried out to obtain ryegrass resistant seedlings, comprising:

transferring the resistant callus to a differentiation culture medium for plant regeneration culture, and transferring to a rooting culture medium for rooting culture to obtain the ryegrass resistant seedling.

9. The method for improving drought tolerance of ryegrass as claimed in claim 5, wherein after obtaining ryegrass resistant seedlings, hardening seedlings and transplanting.

10. The method for improving drought tolerance of ryegrass as claimed in claim 7, wherein the steps of punching holes at different positions of the embryogenic callus, immersing the embryogenic callus in the infectious microbe liquid for infection, and culturing the embryogenic callus in a co-culture medium after infection is finished, specifically comprising the steps of:

punching holes at different positions of the embryogenic callus, soaking the embryogenic callus in the infectious microbe liquid for infection for 25min, and placing the embryogenic callus in a co-culture medium after infection is finished, and co-culturing in a dark environment for 3d.