CN115819531B - Application of over-expressed MtWUSCHEL gene in improving leaf area and delaying flowering of leguminous forage - Google Patents
Application of over-expressed MtWUSCHEL gene in improving leaf area and delaying flowering of leguminous forage Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses an application of over-expression of a medicago sativa MtWUSCHEL gene in increasing the leaf area of leguminous forage and delaying flowering; wherein the nucleotide sequence of the alfalfa MtWUSCHEL gene is shown as SEQ ID No. 1. In the invention, a plant over-expression vector is constructed by utilizing a gene MtWUSCHEL under the background of alfalfa, and genetic transformation operation is carried out to obtain an over-expression transgenic plant. Analysis shows that the pasture quality of the over-expression plant is obviously improved, the flowering time of alfalfa is delayed by up to 20 days, and the leaf area can be improved by about 11%. The application of the invention is expected to play an important role in cultivating novel leguminous forage plant varieties, and has great significance in promoting the production of economic crops in the grass industry in China.
Description
Technical Field
The invention relates to application of a transcription factor WUSCHEL (WUS) gene related to stem tip stem cell determination, in particular to application of an over-expression caltrop alfalfa MtWUSCHEL (MtWUS for short) gene in increasing leaf area and delaying flowering of leguminous forage grass (such as alfalfa). Belongs to the technical field of biological gene engineering.
Background
The alfalfa is called as the pasture king, is an excellent leguminous pasture which is the earliest in use and the widest in cultivation in the world, is one of important protein feed sources in animal husbandry production, and has the advantages of strong stress resistance, high yield, good palatability and the like, thus having great production and utilization potential and research value. However, the alfalfa in China has the problems of low yield, poor quality and the like, and can not meet the various requirements of the development of animal husbandry and grassland, so that the yield and quality level of the alfalfa are urgently needed to be improved to meet the market demands.
The protein of alfalfa mainly exists in the leaf, and the leaf can contribute 60% of grass yield at most, so that the leaf is not only an important index of alfalfa growth and development, yield constitution and variety characteristics, but also one of important indexes reflecting the nutritional value of alfalfa forage. In general, the larger the leaf area, the more abundant the protein content, the higher the nutritional value of alfalfa, and the higher the utility value. Therefore, the breeding of new alfalfa varieties with rich leaf quantity, high protein content and high quality is always the target pursued by alfalfa breeding workers.
Flowering is the key to the transition of alfalfa from vegetative to reproductive growth, and reduced grass quality at the flowering stage is a common problem for grass crops, which has a significant impact on alfalfa grass yield. Attempts to delay alfalfa flowering have hoped to improve in several ways: firstly, the nutrition growth stage is prolonged, the accumulation of biomass is increased, and the high quality of pasture is maintained for a longer time; secondly, by delaying flowering, plants can be protected from frost in winter and early spring; thirdly, the field management can be facilitated by prolonging the harvesting time of alfalfa. However, the genetic and genomic basis of alfalfa flowering time and biomass regulation is currently unclear, mainly due to the homopolyploid nature of this species and the lack of adequate genomic resources.
WUS is a member of the WOX (WUSCHEL-RELATED HOMEOBOX) gene family, playing an important regulatory role in critical stages of plant development, including maintenance of stem cell niches and development of Shoot Apical Meristem (SAM) and the like (Clark, 2001;Fletcher,2002;Kieffer et al, 2006; laux et al, 1996). The lack of OsWUS functions in rice results in a reduced tiller number (Xia et al, plant Journal,2020,104,1635-1647). In maize ZmWUS and ZmWUS are highly expressed in Axillary Meristems (AM), involved in branching control and the like (NARDMANN AND WERR, 2006). Among alfalfa, it has been reported that the homologous gene HDL (HEADLESS) of WUS functions as a transcription repressor. Loss of function mutants of HDL are manifested by stunted development of axillary meristems and loss of apical dominance, heart-shaped leaves, etc. (Ikeda et al, 2009; meng et al, 2019). However, the use of the over-expressed Tribulus alfalfa MtWUSCHEL gene to increase leguminous forage (e.g., alfalfa) leaf area and delay flowering has not been reported.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an application of over-expressed alfalfa MtWUSCHEL (MtWUS) gene in increasing the leaf area of leguminous forage and delaying flowering.
The application of the over-expressed medicago sativa MtWUSCHEL (MtWUS) gene in increasing the leaf area of leguminous forage and delaying flowering; wherein the nucleotide sequence of the alfalfa MtWUSCHEL gene is shown as SEQ ID No.1, and the amino acid sequence encoded by the gene is shown as SEQ ID No. 2; the application is realized by cloning MtWUSCHEL gene coding sequences from medicago tribulus through RT-PCR technology, constructing a plant over-expression vector, performing plant transgenic operation to introduce leguminous forage plant cells, and obtaining a leguminous forage transgenic strain of over-expression MtWUSCHEL genes.
In the above application: the leguminous forage is preferably alfalfa or medicago tribulus.
In the above application: the plant over-expression vector is preferably pEarley gate103-MtWUS.
The application of the over-expression medicago sativa MtWUSCHEL gene in improving the forage grass quality of medicago sativa; wherein the nucleotide sequence of the alfalfa MtWUSCHEL gene is shown as SEQ ID No. 1.
The applicant clones MtWUS gene coding sequence from medicago sativa through RT-PCR technology by utilizing primers MtWUS-F (CACCATGGAACAGCCTCAACAACAACAAC) and MtWUS-R (TTAATTAGCATAATCTGGTGACCTACAGC) to construct a plant over-expression vector pEarley gate103-MtWUS, and conducts plant transgenic operation to introduce into medicago sativa plant cells to obtain the medicago sativa transgenic strain over-expressing MtWUS gene. The detection shows that the leaf area of the over-expression MtWUS plant is increased, the quality of pasture is obviously improved, and the flowering time is obviously delayed (about 20 days), so that the data is far higher than that of the existing alfalfa variety.
The beneficial effects of the invention are as follows: the first application of the gene MtWUSCHEL of the medicago sativa in the overexpression of medicago sativa in the compound leaf species remarkably improves the leaf area of the transgenic plant and delays the flowering time (see fig. 1 and 2), and the result shows that the flowering time of the medicago sativa can be delayed by up to 20 days, and the leaf area can be improved by about 11%. The application of the invention is expected to play an important role in cultivating novel leguminous forage plant varieties, which also provides a new theoretical basis and practical basis for crop improvement and has great significance in promoting the production of grass industry economic crops in China.
Drawings
FIG. 1 flowering time analysis of transgenic alfalfa lines overexpressing MtWUS genes.
Wherein A is the phenotype of the whole plant of the WT and MtWUS over-expressed plants, B is the flowering phenotype at the top of the WT plant, C-E is the non-flowering phenotype at the top of different lines of the MtWUS over-expressed plants, F is the statistics of the internode position number of the first flowers of the WT and MtWUS over-expressed plants, and G is the statistics of the number of days of the first flowers of the WT and MtWUS over-expressed plants; WT is wild type, OE-1, OE-3 and OE-5 are MtWUS over-expressed different strains of plants.
The result shows that the wild plant of the alfalfa flowers about 40 days, and the transgenic alfalfa plant which overexpresses MtWUS genes flowers about 60 days, which shows that the flowering time of the alfalfa is obviously delayed after the MtWUS genes are overexpressed.
FIG. 2 leaf phenotype and related index analysis of transgenic alfalfa lines overexpressing MtWUS genes.
Wherein A is the phenotype of the plant leaf of the WT and MtWUS over-expression plants, B is the area statistics of the plant leaf of the WT and MtWUS over-expression plants, C is the aspect ratio statistics of the plant leaf of the WT and MtWUS over-expression plants, D is the crude protein content measurement of the plant leaf of the WT and MtWUS over-expression plants, E is the fresh weight statistics of the plant leaf of the WT and MtWUS over-expression plants, and F is the total chlorophyll, chlorophyll a and chlorophyll B content statistics of the plant leaf of the WT and MtWUS over-expression plants; WT is wild type, OE-1, OE-3 and OE-5 are MtWUS over-expressed different strains of plants.
The results show that: leaves of transgenic alfalfa plants overexpressing MtWUS genes were larger and the leaves were greener in color compared to wild-type. For this purpose, wild plants and MtWUS over-expression plants with consistent growth state are selected, and leaf area, leaf width-to-length ratio, leaf crude protein content, fresh weight and chlorophyll content are counted respectively. The measurement result shows that the leaf area, the leaf width-to-length ratio, the leaf crude protein content, the fresh weight and the chlorophyll content of MtWUS over-expressed plants are obviously higher than those of wild plants, and the MtWUS gene has obvious effect in improving leguminous forage.
Detailed Description
The present invention will be described in detail with reference to the following drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are merely for explaining the present invention, and are not limiting in any way, and any simple modification, equivalent variation and modification of the embodiments according to the technical principles of the present invention are within the scope of the technical solutions of the present invention.
In the examples described below, the experimental methods used, not specifically described, are conventional methods, and reference may be made, for example, to the "molecular cloning Experimental guidelines" (Sambrook and Russell, 2001).
In the examples described below, materials, reagents, strains, vectors and the like used, unless otherwise specified, are all commercially available.
The pEarley gate103 overexpression vector construction described in this example is described in "Keith W Earley,Jeremy R Haag,Olga Pontes,Kristen Opper,Tom Juehne,Keming Song,Craig S Pikaard.2006.Gateway-compatible vectors for plant functional genomics and proteomics.Plant J.".
10 XN 6 large amount of salt mother liquor (1L)：MgSO4·7H2O 1.85g,KNO3 28.3g,(NH4)2SO4 4.63g,CaCl2·2H2O 1.66g,KH2PO4 4g.
1000 XSH trace salt mother liquor (100mL)：MnSO4·H2O 1g,H3BO3 500mg,ZnSO4·7H2O 100mg,KI 100mg,Na2MoO4·2H2O 10mg,CuSO4·5H2O 20mg,CoCl2·6H2O 10mg.
1000 XSH organic mother liquor (100 mL): 500mg of nicotinic acid, 500mg of pyridoxine hydrochloride and 500mg of thiamine hydrochloride.
50X EDFS iron salt mother liquor (500 mL): 3.487g of NaFe.EDTA.
The SH9 medium (1L) has the following formula: 10 XN 6 major salt mother liquor 100mL,1000 XSH trace salt mother liquor 1mL,1000 XSH organic mother liquor 1mL, 50X EDFS ferric salt mother liquor 20mL, inositol 100mg, sucrose 20g, pH adjusted to 5.8. 7g of agar was added to the solid medium.
The formula of SM4 medium (1L) is: MURASHIGE & SKOOG (MS) BASAL MEDIUM (M519) (Phyto Technology LaboratoriesTM) 4.43g,2,4-D stock (10 mg/mL) 0.4mL,6-BAP stock (1 mg/mL) 0.2mL, sucrose 30g, pH adjusted to 5.8. 3g of plant gel was added to the solid medium.
MSBK the formula of the culture medium (1L) is as follows: MURASHIGE & SKOOG (MS) BASAL MEDIUM (M519) (Phyto Technology LaboratoriesTM) 4.43g, kinetin (1 mg/mL) 1mL,6-BAP stock (1 mg/mL) 0.5mL, sucrose 30g, pH adjusted to 5.8. 3g of plant gel was added to the solid medium.
The formula of the 1/2MS culture medium (1L) is as follows: MURASHIGE & SKOOG (MS) BASAL MEDIUM (M519) (Phyto Technology LaboratoriesTM) 2.215g, sucrose 12g, pH adjusted to 5.8. 7g of agar was added to the solid medium.
Example 1 obtaining transgenic alfalfa plants overexpressing the Tribulus alfalfa MtWUS Gene
1. Construction of an over-expression MtWUS vector
1.1 The coding sequence of MtWUS gene shown as SEQ ID No.1 was obtained through NCBI, bioinformatics website. Primers MtWUS-F (CACCATGGAACAGCCTCAACAACAACAAC) and MtWUS-R (TTAATTAGCATAATCTGGTGACCTACAGC) were designed based on the coding sequence. Extracting total RNA of the medicago terrestris by using a TRIzol kit, and reversely transcribing the total RNA into cDNA. The cDNA is used as a template, and the full-length CDS sequence of MtWUS genes is amplified by using an RT-PCR method. The MtWUS gene sequence was ligated into pEarley gate103 vector using Gateway technology, E.coli transformed, positive clones identified and sequenced. The obtained over-expression vector pEarley gate103-MtWUS is transformed into agrobacterium EHA105, positive clones are identified and stored at-80 ℃ for standby.
1.2 Agrobacterium-mediated genetic transformation of alfalfa leaf discs
The Agrobacterium which is preserved at-80 ℃ and obtained in the step 1.1 is streaked and inoculated in a YEP solid medium (containing 100mg/L rifampicin and 50mg/L kanamycin), and is inversely cultured for 2 days at 28 ℃, and the monoclonal is selected for colony PCR identification. The positive single colony is inoculated on YEP liquid culture medium added with rifampicin and kanamycin for overnight culture until the OD 600 is 0.8, the supernatant is discarded, and the bacteria are diluted to OD 600 to be 0.2 by using a conversion solution for later use after centrifugation at 4000rpm for 15min at 28 ℃.
1.3 Preparation of explants
Taking 3 rd to 5 th fully developed complex leaves of alfalfa from top buds as explants, collecting, sterilizing in a super clean bench with bleaching water (active ingredient is sodium hypochlorite 0.5%) containing 0.1% Triton X100 and 30% sodium hypochlorite for 15min, and cleaning with sterile water for 3 times.
1.4 Infection and Co-cultivation of Agrobacterium
And (3) placing the explant obtained in the step (1.3) into the infectious microbe liquid prepared in the step (1.2), vacuumizing to-0.09 MPa, maintaining for 10min, slowly deflating, and gently shaking on a decolorizing shaker for 10min. The explants were placed on sterile filter paper, blotted and placed on co-culture medium and incubated in the dark at 22℃for 3 days.
1.5 Callus induction and differentiation
Transferring the explant to SM4 solid medium containing antibiotics PPT (3 mg/L) and cephalosporin 500mg/L to induce resistant callus, culturing at 22deg.C for 16h light/8 h dark, and subculturing every 2 weeks for 3 times; subsequently transferring the callus to MSBK solid medium containing screening antibiotics PPT (3 mg/L) and cephalosporin (500 mg/L) to induce bud differentiation, culturing at 22 ℃ for 16h in light/8 h in dark for 3 weeks; transferring embryogenic callus to SH9 solid medium containing screening antibiotic PPT (3 mg/L) and cephalosporin 200mg/L, continuously inducing bud differentiation, culturing at 22deg.C in 16h light/8 h dark, and subculturing every 3 weeks until regenerated seedlings are generated; transferring the regenerated seedlings to a 1/2MS solid culture medium for rooting, carrying out subculture every 3-4 weeks, culturing at 22 ℃ for 16h by light/8 h in the dark, and transplanting the rooted seedlings into soil.
1.5 Identification of regenerated alfalfa seedlings
And taking one small leaf of the regenerated seedling to extract genome DNA, and then carrying out PCR detection. PCR amplification was performed using the plasmid pEarley gate103-MtWUS vector as a positive control and primers 35S-F (GCACAATCCCACTATCCTTC) and MtWUS-R (TTAATTAGCATAATCTGGTGACCTACAGC). And determining to obtain transgenic plants over-expressing the alfalfa MtWUS genes.
The enzyme used in PCR is Beijing full gold organismDNA Polymerase (catalog number: AP 111-01), and specific methods of use are described in the specification.
Example 2 statistical analysis of flowering time of transgenic alfalfa plants overexpressing the Tribulus alfalfa MtWUS Gene
Cloning MtWUS genes from medicago sativa, transforming medicago sativa by using agrobacterium EHA105, and finally screening to obtain a transgenic medicago sativa strain which over-expresses the medicago sativa MtWUS genes in the background of the medicago sativa. The phenotype of the transgenic plants was analyzed and the results are shown in FIG. 1.
The results show that the over-expressed plants can significantly delay flowering time. The flowering time of the wild type was around 40 days, whereas the flowering time of the overexpressing plants was around 60 days, delayed by nearly 20 days (fig. 1G). In addition, the internode number at flowering of the overexpressing plants was also significantly increased compared to the wild-type (fig. 1F).
Example 3 analysis of plant type of transgenic alfalfa plants overexpressing the Tribulus alfalfa MtWUS Gene
Cloning MtWUS genes from medicago sativa, transforming medicago sativa by using agrobacterium EHA105, and finally screening to obtain a transgenic medicago sativa strain which over-expresses the medicago sativa MtWUS genes in the background of the medicago sativa. The phenotype of transgenic plants overexpressing the alfalfa MtWUS gene was analyzed and the results are shown in FIG. 2.
The results show that: leaves of transgenic alfalfa plants overexpressing MtWUS gene were enlarged and the leaf color was more green compared to wild-type, i.e., the leaf area of the overexpressed plants was significantly increased compared to wild-type, which resulted in an increase of about 11% in leaf area (fig. 2A-B). For this purpose, wild plants and MtWUS over-expression plants with consistent growth state are selected, and leaf area, leaf width-to-length ratio, leaf crude protein content, fresh weight and chlorophyll content are counted respectively. The measurement results show that the leaf area, the leaf width-to-length ratio, the leaf crude protein content, the fresh weight and the chlorophyll content of MtWUS over-expressed plants are all obviously higher than those of wild type plants (figures 2C-F), which shows that MtWUS gene has obvious effect in improving leguminous forage.
Example 4 analysis of grass quality of alfalfa plants over-expressing the Tribulus alfalfa MtWUS Gene
Cloning MtWUS genes from medicago sativa, transforming medicago sativa by using agrobacterium EHA105, and finally screening to obtain a transgenic medicago sativa strain which over-expresses the medicago sativa MtWUS genes in the background of the medicago sativa. Grass quality was analyzed for transgenic plants overexpressing the Tribulus alfalfa MtWUS gene and the results are shown in Table 1.
And selecting wild type and over-expressed plants with consistent growth states, and respectively measuring various quality related indexes in the wild type and over-expressed plants through near infrared mass spectrometry analysis.
Table 1: quality related index detection result
The result shows that the content of crude protein, crude fat and water-soluble sugar in the over-expressed plant is obviously increased compared with the wild type, and the content of nitrogen, potassium and plant carbon is obviously increased compared with the wild type. This indicates that over-expression MtWUS gene significantly improves the grass quality of alfalfa.
Claims (2)
1. The application of over-expressed medicago sativa MtWUSCHEL gene in increasing the leaf area and delaying flowering of medicago sativa; wherein the nucleotide sequence of the alfalfa MtWUSCHEL gene is shown as SEQ ID No.1, and the amino acid sequence encoded by the gene is shown as SEQ ID No. 2; the application is realized by cloning MtWUSCHEL gene coding sequences from medicago tribulus through RT-PCR technology, constructing a plant over-expression vector pEarley gate103-MtWUS, performing plant transgenic operation, introducing into medicago sativa cells, and obtaining a medicago sativa transgenic strain of the over-expression MtWUSCHEL gene.
2. The application of over-expression of the alfalfa MtWUSCHEL gene in improving the forage quality of the alfalfa; wherein the nucleotide sequence of the alfalfa MtWUSCHEL gene is shown as SEQ ID No. 1.
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