阅读说明：本技术 一种对PALM1基因进行RNAi以提高豆科牧草小叶数量的应用 (Application of RNAi (ribonucleic acid interference) on PALM1 gene to increase number of small leaves of leguminous forage ) 是由 刘海洋 王晓涵 周传恩 于 2021-07-20 设计创作，主要内容包括：本发明公开了一种对PALM1基因进行RNAi以提高豆科牧草小叶数量的应用,其中所述PALM1基因是蒺藜苜蓿三叶发育模式关键基因PALMATA-LIKE PENTAFOLIATA1,其核苷酸序列如SEQ ID No.1所示；所述对PALM1基因进行RNAi的方法是：设计引物,通过RT-PCR技术从紫花苜蓿中克隆PALM1基因；并为了避开该基因保守结构域,在其C端设计RNAi引物,扩增紫花苜蓿PALM1基因中如SEQ ID No.2所示的PALM1-C-RNAi片段,并将该片段转入载体pANDA35HK构建得到RNAi重组载体MsPALM1-RNAi；然后进行遗传转化野生型紫花苜蓿野生型实验,通过愈伤再生的方式获得转基因MsPALM1-RNAi株系,实现提高豆科牧草小叶数量。实验证实转基因植株的小叶数量显著提高,预示本发明实施后将会创造新型豆科牧草植物或后续的牧草品质改良,对我国草业生产具有重大意义和经济价值。(The invention discloses an application of RNAi (ribonucleic acid interference) on a PALM1 gene to improve the number of lobules of leguminous pasture, wherein the PALM1 gene is a key gene PALMATA-LIKE PENTAFOLITA 1 of a trefoil development mode of medicago truncatula, and the nucleotide sequence of the PALMA 1 gene is shown as SEQ ID No. 1; the RNAi method for the PALM1 gene comprises the following steps: designing a primer, and cloning a PALM1 gene from alfalfa by an RT-PCR technology; in order to avoid the conserved domain of the gene, an RNAi primer is designed at the C end of the gene, a PALM1-C-RNAi fragment shown as SEQ ID No.2 in the alfalfa PALM1 gene is amplified, and the fragment is transferred into a vector pANDA35HK to construct an RNAi recombinant vector MPAPLM 1-RNAi; and then carrying out a genetic transformation wild type alfalfa wild type experiment, and obtaining a transgenic MsPALM1-RNAi strain in a callus regeneration mode to improve the number of the small leaves of the leguminous forage. Experiments prove that the number of the lobules of the transgenic plants is remarkably increased, which indicates that the invention can create novel leguminous pasture plants or subsequent pasture quality improvement after implementation, and has great significance and economic value for Chinese grassy production.)

1. The application of RNAi (ribonucleic acid interference) on PALM1 gene to increase the number of small leaves of leguminous forage, which is characterized in that: the PALM1 gene is a key gene PALLMATA-LIKE PENTAFOLITA 1 of the trefoil development pattern of the medicago truncatula, and the nucleotide sequence of the PALM1 gene is shown as SEQ ID No. 1; the RNAi method for the PALM1 gene comprises the following steps: designing primers, wherein the sequence of a forward primer is CTTCCTACAATAGTTCTAGTCCT, the sequence of a reverse primer is TTGGTGTTGGCTTGTTCCC, and cloning a PALM1 gene from alfalfa by using an RT-PCR technology; in order to avoid the gene conserved domain, an RNAi primer is designed at the C end of the gene conserved domain, wherein the forward primer sequence is TGGATTGTGCCTTCTTTACCAT, the reverse primer sequence is CATGACCGTTATCATCAA, a 284bp gene fragment shown as SEQ ID No.2 in the alfalfa PALM1 gene is amplified and named PALM1-C-RNAi, and the fragment is transferred into an RNAi vector pANDA35HK to construct an RNAi recombinant vector which is named MsPALM 1-RNAi; the constructed MsPALM1-RNAi vector is subjected to a genetic transformation wild-type alfalfa wild-type (SY4D) experiment, a transgenic MsPALM1-RNAi strain is obtained in a callus regeneration mode, and the improvement of the number of the small leaves of the leguminous forage is realized.

2. The use of RNAi to the PALM1 gene to increase the number of legume grass fronds according to claim 1, wherein: the pasture of Leguminosae is herba Medicaginis, herba Medicaginis or herba Trifolii Pratentis.

3. A plant expression vector for increasing the number of lobules of leguminous forage is characterized in that: the plant expression vector is MsPALM1-RNAi, which contains a nucleotide sequence shown in SEQ ID No. 2.

4. The plant expression vector for increasing the number of legume grass leaflets of claim 3, wherein: the pasture of Leguminosae is herba Medicaginis, herba Medicaginis or herba Trifolii Pratentis.

Technical Field

The invention relates to an application of genes for improving the quality of leguminous forage, in particular to an application of RNAi (RNA interference) on a key gene PALM1(PALMATA-LIKE PENTAFOLITA 1) of a trefoil development mode of medicago truncatula to improve the number of lobules of leguminous forage (taking medicago sativa as an example), and belongs to the technical field of genetic engineering.

Background

Alfalfa is one of leguminous forage grasses, is the most widely distributed cultivated forage grass in the world at present, and is also the most important perennial high-quality forage grass. With the rapid development of the herbivore animal husbandry and the dairy industry in China, the domestic demand for high-quality alfalfa is increasing day by day. However, the current domestic alfalfa has the problems of low yield, poor quality, unstable goods source and the like, and the shortage situation of the domestic alfalfa becomes an important factor for limiting the development of the Chinese herbivory animal husbandry and the milk industry. Therefore, the improvement of the yield and the quality of the alfalfa is a great urgent need for the development of the grass industry, the animal husbandry and the milk industry in China.

The grass yield of alfalfa is dominated by harvesting leaves and stems, with leaves contributing up to 60% of the grass yield. The leaves are not only important indexes of growth and development, yield composition and variety characteristics of the alfalfa, but also main research objects of cultivation management and pest and disease damage monitoring. Because the leaves store 70 percent of protein of the alfalfa, and the fiber content of the leaves is only 1/3 of the stems, the higher the ratio of the leaves to the stems is, the higher the nutritional value of the alfalfa is, the better the palatability is, and the higher the utilization value is relatively. Therefore, the ratio of leaves to stems (the ratio of the weight of leaves to the weight of stems) is an important index in the alfalfa quality evaluation system. The breeding of new alfalfa varieties with rich leaf quantity and high leaf-stem ratio is always the pursuit target of alfalfa breeding workers. The increased alfalfa leaf trait is used as a morphological marker at present and is considered to have the potential of high yield and high quality.

RNA interference (RNAi), also known as post-transcriptional gene silencing (PTGS), refers to a gene editing technique in which specific homologous double-stranded RNA (dsrna) is introduced into cells to prevent or reduce the level of expression of a target gene.

The leaf development patterns of plants are divided into single leaves and multiple leaves. The single-leaf structure means that only one small leaf is arranged on one petiole, and the multiple-leaf structure means that a plurality of small leaves are arranged on one petiole. The leaves of the medicago truncatula have a compound leaf structure and comprise three small leaves. It has been reported that the trefoil development pattern is determined by PALM1 gene (PALLA-LIKE PENTAFOLITA 1). The inventor utilizes the genome high similarity of medicago truncatula and medicago sativa to clone the PALM1 gene (MspALM1) of the medicago sativa, constructs an RNAi vector to transform wild-type medicago sativa (SY4D), obtains an MspALM1-RNAi transgenic strain through a genetic transformation technology, and researches the regulation and control of the number of lobules of the MspALM1-RNAi in a compound leaf species. Through retrieval, no report is found about the RNAi strain for obtaining the PALM1 gene in alfalfa and about the application of MSPLM 1-RNAi in the control of the number of lobules in compound leaf species.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide the application of RNAi (RNA interference) on a key gene PALM1 (PALMA-LIKE PENTAFOLITA 1) of the clover development mode of the medicago truncatula so as to improve the number of lobules of leguminous pasture (taking alfalfa as an example).

The invention relates to an application of RNAi (ribonucleic acid interference) on PALM1 gene to improve the number of legume grass small leaves, which is characterized in that: the PALM1 gene is a key gene PALLMATA-LIKE PENTAFOLITA 1 of the trefoil development pattern of the medicago truncatula, and the nucleotide sequence of the PALM1 gene is shown as SEQ ID No. 1; the RNAi method for the PALM1 gene comprises the following steps: designing primers, wherein the sequence of a forward primer is CTTCCTACAATAGTTCTAGTCCT, the sequence of a reverse primer is TTGGTGTTGGCTTGTTCCC, and cloning a PALM1 gene from alfalfa by using an RT-PCR technology; in order to avoid the gene conserved domain, an RNAi primer is designed at the C end of the gene conserved domain, wherein the forward primer sequence is TGGATTGTGCCTTCTTTACCAT, the reverse primer sequence is CATGACCGTTATCATCAA, a 284bp gene fragment shown as SEQ ID No.2 in the alfalfa PALM1 gene is amplified and named PALM1-C-RNAi, and the fragment is transferred into an RNAi vector pANDA35HK to construct an RNAi recombinant vector which is named MsPALM 1-RNAi; the constructed MsPALM1-RNAi vector is subjected to a genetic transformation wild-type alfalfa wild-type (SY4D) experiment, a transgenic MsPALM1-RNAi strain is obtained in a callus regeneration mode, and the improvement of the number of the small leaves of the leguminous forage is realized.

In the above application of RNAi to PALM1 gene to increase the number of legume grass leaflets: the leguminous forage is preferably alfalfa, alfalfa or clover.

The invention also discloses a plant expression vector for increasing the number of the lobules of leguminous forage, which is characterized in that: the plant expression vector is MsPALM1-RNAi, which contains a nucleotide sequence shown in SEQ ID No. 2.

Wherein: the leguminous forage is preferably alfalfa, alfalfa or clover.

The experiment proves that: the application of RNAi on the PALM1 gene to improve the number of lobules of leguminous forage provided by the invention has obvious effect, the expression quantity of alfalfa PALMA-LIKE PENTAFOLITA 1 gene after RNAi is obviously reduced compared with the expression quantity of wild type (SY 4D); the number of the phenotypic lobules of the alfalfa transgenic MsPALM1-RNAi strain is changed from three to five, which prompts that the alfalfa PALLA-LIKE PENTAFOLITA 1 gene disclosed by the invention is applied to RNAi operation, the number of the lobules of a plant can be obviously increased, and meanwhile, the number of the lobules of the plant can be obviously increased after the function of the PALLA-LIKE PENTAFOLITA 1 gene is inhibited by RNAi. The method also indicates that the novel leguminous pasture plants can be created after the method is implemented, can be used for subsequent pasture quality improvement, and has great significance and economic value for the Chinese grassy industry production.

Drawings

FIG. 1 shows the determination of the expression level of alfalfa PALMATA-LIKE PENTAFOLITA 1 gene after RNAi.

Wherein: the expression of the three transgenic lines is obviously reduced compared with the wild type (SY 4D).

FIG. 2 phenotypic analysis of alfalfa MsPALM1-RNAi transgenic plants.

The wild type compound leaf structure of the alfalfa comprises three small leaves, and the number of the small leaves of the MsPALM1-RNAi transgenic plant is changed from three to five. This proves that the number of the lobules of the plant is obviously increased after the function of the PALMATA-LIKE PENTAFOLITA 1 gene is inhibited by RNAi.

Detailed Description

The present invention will be described in detail with reference to the following detailed drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are only for explaining the present invention and not for limiting the present invention in any form, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.

In the following examples, materials, plasmids, reagents, kits, and the like used therein were obtained commercially without specific reference.

Example 1 cloning of the alfalfa PALMATA-LIKE PENTAFOLITA 1 Gene

Obtaining a sequence of Arabidopsis thaliana PALMATA-LIKE PENTAFOLITA 1 through a bioinformatics website NCBI, and then performing sequence comparison search by utilizing BLAST to obtain a genome sequence of alfalfa PALMATA-LIKE PENTAFOLITA 1, wherein the nucleotide sequence of the alfalfa PALMATA-LIKE PENTAFOLITA 1 gene is shown in SEQ ID No. 1.

The forward CTTCCTACAATAGTTCTAGTCCT and reverse TTGGTGTTGGCTTGTTCCC primers were designed based on the sequence of SEQ ID No. 1. And (3) extracting the RNA of the alfalfa by using a TRIzol kit. The experimental procedure was as follows:

1) a sample (. ltoreq.100 mg) was immediately taken into a 2ml EP tube with a steel ball and placed in liquid nitrogen and broken up with a grinder. Parameters of the grinding machine: 26-30HZ for 60 seconds, 2) adding 1ml Trizol-RT, and carrying out vortex oscillation on a constant-temperature blending machine at 2000rpm for 3 min; 3) adding 400ul DEPC water, and performing vortex oscillation on a constant-temperature mixing machine at 2000rpm for 3 min; standing for 5min, and centrifuging at 12000rpm for 15 min; 4) transferring the supernatant into a 1.5ml EP tube (duplicate, 600 ul/tube), adding equal volume of isopropanol, gently mixing, standing at room temperature for 15min, centrifuging at 12000rpm for 10min, and discarding the supernatant; 5) adding 800-; and (3) repeatedly washing the precipitate with 75% ethanol again, pouring out the supernatant, performing instantaneous centrifugation, completely sucking the ethanol with a gun tip as much as possible, then placing the precipitate on an ultraclean workbench for 1-2 min, taking out the precipitate until the edge is semitransparent, and paying attention to: excessive drying of the RNA should be avoided, otherwise it is extremely difficult to re-dissolve; 6) adding 70ul DEPC water, flicking, and performing metal bath at 60 deg.C for 10 min; 7) standing at room temperature for 2min, and measuring concentration on ice.

Carrying out reverse transcription on the obtained alfalfa RNA to obtain the Cdnas, wherein the method comprises the following steps:

a reverse transcription kit: transcriptor First Strand cDNA Synthesis Kit

(1) The frozen reagents in the reverse transcription kit were lysed and the reagents were collected by gentle centrifugation to the bottom of the tube, taking care that: the reagent is kept on ice all the time;

(2) placing the PCR tubule without RNase on ice, marking, preparing 20ul reaction system, and reversing RNA amount to 2-3 ug;

(3) putting the reaction system into PCR, reacting for 10min at 65 ℃ to denature the mixture of the template and the primer, and cooling the tube on ice after the reaction is finished;

(4) adding the rest reagent kit reaction reagents into the system:

mixing the above systems carefully, centrifuging instantly to tube bottom, setting PCR program, inactivating reverse transcriptase at 25 deg.C for 10min, 55 deg.C for 30min, and 85 deg.C for 5min, and storing in refrigerator at-20 deg.C after reaction.

The full-length CDS sequence of the PALMATA-LIKE PENTAFOLITA 1 gene is amplified by an RT-PCR method, and the method and the conditions are as follows:

(1) reaction System (50. mu.l)

(2) Reaction procedure

Electrophoresis is carried out, then gel is recovered, and A addition reaction is carried out, wherein the method and the conditions are as follows:

(1) add A reaction System (10. mu.l)

(2) Reaction procedure:

72℃ 30min

16℃ End

then, the pEARLEYGATE201 vector is connected, and the method and the conditions are as follows:

(1) reaction system

(2) The metal bath is at 25 ℃ and the reaction is carried out for 1h

Then, sequence determination is carried out to verify the correctness of the cloned sequence.

Example 2 obtaining of alfalfa PALMATA-LIKE PENTAFOLITA 1 RNAi plants

In order to inhibit the function of endogenous PALM1 gene in alfalfa, the inventor avoids conserved structural domain, designs RNAi primer (forward primer TGGATTGTGCCTTCTTTACCAT and reverse primer CATGACCGTTATCATCAA) at C-terminal of PALM1 gene to amplify 284bp gene segment shown in SEQ ID No.2 in alfalfa PALM1 gene, named PALM1-C-RNAi, and transfers the segment into RNAi vector pANDA35HK to construct RNAi recombinant vector, named MPAPLM 1-RNAi.

The MsPALM1-RNAi vector is subjected to an experiment of genetic transformation of wild-type alfalfa (SY4D), and the method comprises the following steps:

material taking: selecting leaves of alfalfa which do not bloom (not selecting leaves which are too tender, too old and damaged), and selecting robust and unfolded leaves;

taking materials, carrying an ice box, putting the taken leaves into a 50mL centrifuge tube, and placing the centrifuge tube on ice to prevent leaf wilting;

sterilizing the superclean bench by ultraviolet irradiation, transferring the taken leaves into a 50mL centrifuge tube sterilized in advance after the preparation is finished, and performing ddH₂Adding 20% NaClO and one thousandth of Tween-20 into O, washing the material for 15min, shaking the material slightly in the washing process, pouring out the solution after about 5min, washing for one time to 14min, and then using sterilized ddH₂Cleaning with O for 5 times, each time for 1min, and completely cleaning NaClO;

shaking out Agrobacterium (OD)₆₀₀About 0.6-0.8) in a centrifuge tube, centrifuging at 4000rpm for 10min, decanting the supernatant, and resuspending the Agrobacterium to OD with 2-3mL of liquid SM4₆₀₀Is 0.2;

putting the washed leaves into a sterile culture dish in batches, overlapping the leaves with similar sizes, cutting the edges of the leaves by using a blade, cutting the leaves into small blocks, putting the small blocks into a 50mL centrifuge tube, and adding bacterial liquid diluted in proportion;

putting a centrifugal tube containing the leaves into a vacuum pump, and vacuumizing for 10min to reduce cell gaps, so that the bacterial liquid can better invade into the wounds of the leaves, and the conversion efficiency is improved;

placing the centrifuge tube on a horizontal shaking table, and slowly shaking for 10 min;

pouring out the bacterial liquid, and spreading the leaves on an SM4 (added with 1 mlcet) culture medium for a total culture period;

and (3) a later culture stage: after the plant grows on SM4 culture medium (added with 2 mlcet and 200ul of antibiotics according to plant resistance) for 4 weeks, the culture medium is transferred once every two weeks, MSBK culture medium is transferred, MSBK treatment is carried out for two weeks and then the culture medium is replaced to MSS culture medium, PPT can be properly reduced, after the plant takes root, the plant is transferred into 1/2MS culture medium, after a seedling grows out, the plant is transferred into soil to grow in a greenhouse, after the transgenic plant transferred into the greenhouse survives, the transgenic plant is identified, and the RNAi positive transgenic plant is determined.

Wherein, the SM4 culture medium, the MSBK culture medium, the MSS culture medium and the 1/2MS culture medium have the following formulas:

in order to analyze the expression level of MspALM1 in the obtained RNAi positive transgenic plants, the invention collects apical tissues of the RNAi positive transgenic plants, and utilizes qRT-PCR (forward primer GGTTGGACGACACTCATGGAT and reverse primer AGAGTCTTGAGAGGTTTAGTGCAACA) to analyze the expression level of MspALM1, and the used equipment is as follows: BIO-RAD real-time fluorescent quantitative PCR instrument, SYBR green Mix. The method comprises the following steps:

(1) diluting the cDNA to 15 ng/mu l, and making three biological repetitions or three technical repetitions for each sample; the forward primer and the reverse primer are mixed to 2.5 mu m in advance;

(2) real-time PCR System (10. mu.l):

SYBR 5μl

Primers 2μl

cDNA template 3. mu.l

(3) Real-time PCR program:

the results show that the level of endogenous PALM1 inhibition in alfalfa differed among the different transgenic lines, and that MsPALM1 was significantly down-regulated in RNAi-positive transgenic plants compared to the wild type (figure 1).

Example 3 phenotypic analysis of alfalfa MSPALM1-RNAI transgenic plants

The phenotype of the alfalfa MsPALM1-RNAi transgenic plant is analyzed by a conventional method, and the result shows that the wild-type compound leaf structure of the alfalfa comprises three small leaves, and the number of the small leaves of the MsPALM1-RNAi transgenic plant is changed from three to five (figure 2). This demonstrates that the inhibition of PALMATA-LIKE pentaaflata 1 by RNAi can significantly increase leaflet number.

Sequence listing

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