CN103044534A - Related gene of drought resistant medicago sativa as well as encoding protein and application of gene and protein - Google Patents

Abstract

The invention discloses a protein encoded by an alfalfa drought-resistance-related gene, wherein the protein has the amino acid sequence shown in SEQ ID No: 2, or the protein has the amino acid sequence shown in SEQ ID No: 2 through one or An amino acid sequence that still has drought resistance activity after substitution, deletion or addition of several amino acid residues. The invention also relates to the coding gene of the protein, the expression vector and the cell containing the gene, and also relates to a method for cultivating drought-resistant alfalfa and their application. The expression of the alfalfa drought-resistance-related gene (bZIP) in the alfalfa can significantly enhance the drought-resistance performance of the alfalfa. The discovery of this gene provides genetic resources for the research on drought resistance of major crops, and will play an important role in the research on the drought resistance performance of genetic engineering improved plants.

Description

Genes related to drought resistance in alfalfa and their encoded proteins and applications

technical field

The invention relates to an alfalfa drought-resistance-related gene, a protein encoded by the gene, an expression vector and a cell containing the alfalfa drought-resistance-related gene, a method for cultivating drought-resistant alfalfa, and alfalfa Application of drought-resistance-related gene and its coded protein, expression carrier and cell containing the gene in cultivating plants with improved drought-resistance ability.

Background technique

Alfalfa is the most important leguminous forage in my country and even in the world, and is known as the "king of forage". Alfalfa is widely planted in Northwest China, and the climate characteristics of drought and little rain in Northwest China are very unfavorable to the normal growth and development of crops, seriously restricting the development of agriculture and animal husbandry.

Therefore, in-depth research on the drought resistance of alfalfa is of great significance for overcoming the constraints of alfalfa cultivation by natural conditions such as drought and wind erosion in this area, expanding its planting range, and improving productivity.

In the past few decades, the research on the drought resistance of alfalfa has gradually shifted from the research on the identification of drought resistance to the research on improving the drought resistance of alfalfa itself, especially in recent years or in the past ten years. Drought resistance breeding and variety improvement of alfalfa are particularly prominent.

At present, the most effective and commonly used method is the traditional recurrent selection method, which is to produce offspring by selecting plants in the original population, identify the offspring, and then use the excellent offspring to recombine to form a new population to improve the average performance of the population. The pedigree method can then be used to select inbred lines for the new population.

With the development of molecular biology, biotechnology has been widely used in breeding. But compared with other crops (such as corn, sorghum, etc.), there are relatively few studies on alfalfa drought-resistant breeding. Therefore, the molecular biological mechanism related to drought resistance of alfalfa needs further research.

Contents of the invention

The present invention is based on the research on the molecular biology mechanism related to alfalfa drought resistance, on the one hand, it provides alfalfa drought resistance related gene and the protein encoded by the gene, on the other hand, it also provides an expression vector containing the alfalfa drought resistance related gene and Cells, and methods for breeding drought-resistant alfalfa, also provide their applications.

The invention provides a protein encoded by an alfalfa drought-resistance-related gene, wherein the protein has the amino acid sequence shown in SEQ ID No: 2, or the protein has the amino acid sequence shown in SEQ ID No: 2 through one or more An amino acid sequence that still has drought resistance activity after substitution, deletion or addition of amino acid residues.

The present invention also provides an alfalfa drought-resistance-related gene, wherein the gene has the nucleotide sequence shown in SEQ ID No: 1, or the gene has the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No: 2 .

The present invention also provides an expression vector, wherein the expression vector contains the gene related to alfalfa drought resistance provided by the present invention.

The present invention also provides a transgenic cell, wherein the transgenic cell contains the gene related to alfalfa drought resistance provided by the present invention.

The present invention also provides a method for cultivating drought-resistant alfalfa, wherein the method includes introducing the alfalfa drought-resistance-related gene provided by the present invention into alfalfa cells to obtain alfalfa cells and transgenic plants with improved drought resistance.

The present invention also provides the application of the alfalfa drought-resistance-related gene provided by the present invention, the encoded protein thereof, the expression vector containing the gene, and the transgenic cell containing the gene in cultivating plants with improved drought resistance.

The alfalfa drought-resistance-related gene (bZIP) provided by the present invention was induced and expressed in alfalfa by drought stress, and the inventors of the present invention introduced the alfalfa drought-resistance-related gene (bZIP) into alfalfa through transgenic technology, and the results showed that alfalfa The expression of a drought-resistance-related gene (bZIP) in alfalfa can significantly enhance the drought-resistance performance of alfalfa. The drought-resistant gene cloned from the alfalfa of the present invention provides genetic resources for the drought-resistant research of main crops (especially the alfalfa), and will play an important role in the research on the drought-resistant performance of plants improved by genetic engineering.

Description of drawings

Figure 1 shows the analysis results of real-time quantitative PCR of the bZIP gene (SEQ ID NO: 1), and the results show that the expression level of the bZIP gene can be induced and increased by drought stress.

Fig. 2 shows the structure of the pCAMBIA1302 vector inserted with the bZIP gene used for cell localization in Example 2.

Fig. 3 is a map of subcellular localization observed when an empty expression vector not containing the bZIP gene of interest is transferred into onion epidermal cells.

Fig. 4 is that the expression vector containing ZIP gene (SEQ ID NO: 1) is transferred in the onion epidermis cell, the subcellular localization figure that observes.

Fig. 5 shows the structure of the pCAMBIA1302 vector inserted with bZIP gene used for Agrobacterium-mediated bZIP gene transformation of alfalfa plants in Example 3.

Detailed ways

The present invention provides a protein encoded by alfalfa drought resistance-related genes, wherein the protein has the amino acid sequence shown in SEQ ID No: 2, or the protein has the amino acid sequence shown in SEQ ID No: 2 through one or An amino acid sequence that still has drought resistance activity after substitution, deletion or addition of several amino acid residues. Preferably, the protein has the amino acid sequence shown in SEQ ID No: 2.

Correspondingly, the present invention also provides a kind of gene (bZIP) related to alfalfa drought resistance, wherein, the gene has the nucleotide sequence shown in SEQ ID No: 1, or the gene has the amino acid sequence shown in SEQ ID No: 2 The nucleotide sequence of the sequence. Preferably, the gene has the nucleotide sequence shown in SEQ ID No: 1.

The alfalfa drought resistance-related gene (bZIP) provided by the present invention is obtained by the inventors by constructing a cDNA library specifically expressed under alfalfa drought stress and screening the cDNA library.

The method for constructing the cDNA library includes: using 30% (w/v) PEG8000 to simulate drought treatment for 12 hours on alfalfa (Medicago sativa L.cv. Baoding alfalfa, Beijing China Animal Science and Technology Co., Ltd.), The plants were harvested and placed at -80°C as the experimental group. Plants without drought treatment served as the control group. A cDNA library specifically expressed in alfalfa under drought stress was constructed using the PCR-Selected cDNA Subtraction Kit (Clontech, Mountain View, CA, USA).

During the construction of the library, 525 single colonies were obtained and all were sequenced. After removing carrier sequences and redundant sequences, 130 EST sequences were finally obtained. These sequences range in size from 250bp to 1000bp. Among them, the EST sequence with a length of 567bp attracted the attention of the experimenters, and the EST sequence was cloned by 5'RACE and 3'RACE respectively using the SMARTer ^TM RACE cDNA Amplification Kit (Clontech, USA), and finally the gene was obtained Full length sequence (SEQ ID NO: 2). Among them, the method of gene cloning is a routine experimental operation in the field of molecular biology, and the specific method is as follows:

A. Prepare basic liquid: 2.0μl 5×first strand buffer, 1.0μl DTT (20mM), 1.0μl ldNTP Mix (10mM);

B. Prepare cDNA for 5'RACE: 2.75 μl RNA, 1.0 μl 5'-CDS primer A (ACGCGACGGTTTCAACATCCCTCTC);

Prepare cDNA for 3’RACE: 3.75 μl RNA, 1.0 μl 3’-CDS Primer A (GAGCTGATGCTGTGGCTGCTGGTTG);

C. Place the prepared liquid at 72°C for 3 minutes, then cool at 42°C for 2 minutes.

D. Add 1 μl of SMARTer IIA oligo to the 5'RACE cDNA in B.

E. Prepare the cDNA reaction solution of 5'RACE and 3'RACE: 4.0μl of the buffer obtained in step A, 0.25μl of RNase inhibitor (40U/μl), 1.0μl of SMARTScribe reverse transcriptase (100U).

F. Add the liquid in step E to step C to complete the preparation of the cDNA synthesis reaction solution for 3'RACE; add the liquid in step E to step D to complete the preparation of the cDNA synthesis reaction solution for 5'RACE.

G. Place the prepared reaction solution at 42°C for 90 minutes, and finally at 70°C for 10 minutes to complete the cDNA synthesis.

Among them, the 3'RACE reaction system is: 94°C for 30 seconds, 68°C for 30 seconds, 72°C for 3 minutes, a total of 30 cycles. The 5'RACE reaction system is: 94°C for 30 seconds, 65°C for 30 seconds, 72°C for 3 minutes, a total of 40 cycles.

Get the PCR product and perform electrophoresis detection on 1.0 (W/V)% agarose gel, recover the target band, connect the recovered product to the pEGM T-Easy carrier, transform Escherichia coli DH5α competent cells, extract the plasmid and sequence it, Sequencing results show that the gene has the nucleotide sequence (1781bp) shown in SEQ ID: No: 1.

The present invention also provides an expression vector, wherein the expression vector contains a gene with the following nucleotide sequence: the nucleotide sequence shown in SEQ ID No: 1, or the amino acid sequence shown in SEQ ID No: 2 the nucleotide sequence. The gene provided by the present invention can be constructed into an expression vector by existing methods, and any enhanced promoter or inducible promoter can be added before its transcription start nucleotide. In order to facilitate the identification and screening of transgenic plant cells or plants, the vectors used can be processed, such as adding plant selectable markers (such as BAR gene, GUS gene, luciferase gene, etc.) or antibiotic markers with resistance (such as hygromycin, kanamycin, etc.).

The present invention also provides a transgenic cell, wherein the transgenic cell contains a gene with the following nucleotide sequence: the nucleotide sequence shown in SEQ ID No: 1, or the amino acid sequence shown in SEQ ID No: 2 the nucleotide sequence. The cells may be monocot cells, or dicotyledon cells, such as rice, wheat, corn, cucumber, tomato or alfalfa cells. Alfalfa cells are preferred, and alfalfa cells are more preferred.

The present invention also provides a method for cultivating drought-resistant alfalfa, wherein the method includes introducing a base having a nucleotide shown in SEQ ID No: 1 into alfalfa cells to obtain alfalfa cells with improved drought resistance and transgenic plants.

According to the present invention, the method that the gene provided by the present invention will be introduced into alfalfa cells is a conventional method in the field of genetic engineering, such as by Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conduction , Agrobacterium-mediated and other conventional biological methods to transform plant cells or tissues, and then cultivate the transformed plant cells into plants.

The present invention also provides the application of the alfalfa drought-resistance-related gene provided by the present invention, the encoded protein thereof, the expression vector containing the gene, and the transgenic cell containing the gene in cultivating plants with improved drought resistance. Preferably, the plant is Medicago sativa.

Example 1

Real-time quantitative PCR analysis of bZIP gene

In order to further study the expression of the bZIP gene (gene shown in SEQ ID NO: 1) obtained from the suppression subtractive hybridization library, the method for real-time quantitative PCR was used to analyze, and the specific steps were as follows:

Taking 50-day-old alfalfa plants (Medicago sativa L.cv. Baoding alfalfa, Beijing China Animal Science and Technology Co., Ltd.) as experimental materials, they were treated with 30% (w/v) PEG 8000 for 0, 15, 30 Minutes and 1, 6, 12, 24 hours, total RNA was extracted. 5 μg of total RNA was used for the preparation of the first-strand cDNA, and the specific steps were as follows: First, 0.5 μl 50 μM oligo(dT) (Invitrogen), 1 μl 10 mM dNTP Mix and 5 μl DEPC-treated water were added, heat-shocked at 65°C for 5 minutes, and put Cool on ice for 1 minute; then, add 4 μl 5×First Strand Buffer, 2 μl 0.1M dithiothreitol (DTT), 1 μl 40U/μl RNaseOUT (Invitrogen) and 200USuperScript II Reverse Transcriptase (Invitrogen), and place at 25°C for 5 minutes, 50 60 minutes at 70°C and 15 minutes at 70°C. The synthesized cDNA was stored at -20°C.

The primers used for real-time quantitative PCR are:

Primer 1: 5'-3': ACCAAGACTGAAAAGCCTTC

Primer 2: 5'-3': TTCTCCATCAGTGGTCGGTG.

A SYBR green reporter assay kit was used for real-time quantitative PCR reactions. 18S rRNA of Medicagotruncatula was used as internal standard. Three replicate experiments were performed for each gene. The reaction system was as follows: 2 μl 10×PCR buffer, 2 μl 25 mM Mg ²⁺ , 0.5 μl 25 mM dNTP, 0.5 μl 10 μM forward primer, 0.5 μl 10 μM reverse primer, 1 μl 20×SYBR Green MasterMix (Invitrogen), 0.2 μl 5U/μl Taq ( Invitrogen, 11304-029), 1 μl cDNA, and finally the total volume was made up to 20 μl with ultrapure water. The reaction conditions were as follows: dissolve DNA at 95°C for 2 minutes, and then perform 40 cycles of amplification reaction, 95°C for 10 seconds, 60°C for 30 seconds, and 70°C for 45 seconds. Finally, the 2 ^-ΔΔCt statistical method was used to analyze the PCR results. The result is shown in Figure 1.

As can be seen from the results in Figure 1, the expression level of the bZIP gene (SEQ ID NO: 1) can be induced and increased by drought stress.

Example 2

Subcellular localization of bZIP gene

Construction of plant expression vector: 1) RNA extraction (Trizol method extraction), 2) reverse transcription (M-MLV, promega company), 3) PCR amplification: use the reverse-transcribed cDNA as a template, and use Taq enzyme to do PCR , the designed restriction sites (NcoI and SpeI) were ligated into the PCR product, 4) the PCR product and the vector were subjected to restriction treatment, 5) the restriction product was connected, and the vector structure was shown in Figure 2.

Transform Escherichia coli for detection, and use a gene gun to transfer the empty expression vector that does not contain the bZIP gene of interest into onion epidermal cells to observe its subcellular location (as shown in Figure 3); Imported into onion epidermal cells, observed its subcellular localization (as shown in Figure 4).

In Fig. 3, it can be seen from the comparison of A and B that the empty expression vector that does not contain the bZIP gene of interest is expressed in all parts of the cell; and as can be seen from the comparison of A and B in Fig. 4, the expression vector containing the ZIP gene ( The expression vector of SEQ ID NO: 1) is only expressed in the nucleus.

Example 3

Agrobacterium-mediated transformation of alfalfa plants with bZIP gene

1. Materials and Reagents

1. Plant material

The tested alfalfa variety was alfalfa (Medicago sativa L.cv. Baoding alfalfa, Beijing China Animal Science and Technology Co., Ltd.).

The cotyledons of sterile seedlings germinated for 7 days were the recipient materials for genetic transformation.

2. Agrobacterium strains and plasmid vectors

The Agrobacterium strain used is Agrobacterium tumefaciens: GV3103 (Beijing Tianenze Gene Technology Co., Ltd.), Agrobacterium culture medium:

Plasmid vector: pCAMBIA1302 (purchased from Shanghai Xipiji Biotechnology Co., Ltd.). Insert the bZIP gene into pCAMBIA1302, the specific steps are as follows:

1) RNA extraction (Trizol method extraction), 2) reverse transcription (M-MLV, promega company), 3) PCR amplification: using the reverse-transcribed cDNA as a template, amplify the bZIP gene sequence (primers: 5 '-3': GGGGCTC CCATGG ATGGGAAATAGTGACGAAGAGAAATC Nco I and 5'-3': CGCGGCTC TGATCA ACCAGCAGCCACAGCATCAGCTCTAGSpe I), 3) PCR amplification products were detected by electrophoresis on 1% (W/V) agarose gel, and recovered PCR amplified product of the target size, and pCAMBIA1302 vector and PCR product were subjected to double enzyme digestion with Nco I and Spe I, 4) After the enzyme digestion reaction was completed, electrophoresis was detected with 1.0% (W/V) agarose gel, Recover the fragment containing the target band, connect it by T4 DNA ligase, 5) transfer the ligated product into competent Escherichia coli DH5α, and obtain the pCAMBIA1302 vector inserted with the bZIP gene, which contains the CaMV35s promoter, bZIP gene (SEQ ID NO: 1) and a hygromycin resistance screening gene, the transgenic plants obtained can be preliminarily identified by hygromycin screening during genetic transformation. The structure of the plasmid vector containing the target fragment is shown in Figure 3.

The pCAMBIA1302 vector inserted with the bZIP gene is introduced into Agrobacterium tumefaciens: GV3103, and the specific steps are as follows:

1) Agrobacterium tumefaciens: GV3103 stored at -70°C was streaked on a plate containing 50 μg/ml streptomycin, and cultured at 28°C.

2) Pick a single colony and inoculate it in 5ml YM liquid medium, shake and culture at 220rpm 28°C for 12-16hr.

3) Take 2ml of bacterial liquid and transfer it to 100ml of YM liquid medium, shake at 220rpm at 28°C until OD600=0.5.

4) Transfer to a sterile centrifuge tube, centrifuge at 5000rpm for 5min, and remove the supernatant.

5) Add 10ml of pre-cooled 0.1M CaCl2 solution, gently suspend the cells, and place on ice for 20min. Centrifuge at 5000 rpm at 4°C for 5 min, and remove the supernatant.

6) Add 4ml of pre-cooled 0.1M CaCl2 solution containing 15% glycerol and gently suspend.

7) The Agrobacterium suspension was divided into sterile Eppendorf tubes, and 200 μl of each tube was frozen and stored at -70°C.

8) Take about 1 μg of plasmid DNA and add it to 200ml GV3103 competent cells, mix well, put in ice bath for 30 minutes, and place at -70°C for 10 minutes.

9) Water bath at 37°C for 5min or 42°C for 1min, then ice bath for 2min, add 800ml of YM liquid medium at 28°C, shake at 175rpm for 3hrs, and spread on the YM plate containing 50μg/ml Kanamycin. Incubate at 28°C until a single colony is formed.

The plasmid of the Agrobacterium with the bZIP gene introduced was extracted and sequenced, and the result showed that the nucleotide sequence of the introduced gene was consistent with SEQ ID NO: 1, indicating that the expression vector containing the target gene bZIP was not lost.

2. Experimental method

1. Culture of Agrobacterium

Agrobacterium with bZIP gene introduced was plated on YMB solid medium containing 50 mg/L kanamycin and 50 mg/L rifampicin, placed in an incubator, and cultured at 28°C. Two days later, a single colony was picked from the plate and inoculated in 20ml YMB liquid medium containing 50mg/L kanamycin and 50mg/L rifampicin, cultured at 180rpm at 28°C. Use the shaken bacterial solution to scratch the plate and incubate at 28°C. After a single colony grows, store the plate at 4°C.

2. Transformation of bZIP gene

Pick a single colony on the plate, inoculate it in 20ml of YMB liquid medium containing 50mg/L kanamycin and 50mg/L rifampicin, and cultivate it on a constant temperature shaker at 28°C and 180rpm. Two days later, the bacterial strain was recovered, and the bacterial solution was poured into a 10ml centrifuge tube, centrifuged at 4000rpm for 10min. Pour off the supernatant, and resuspend the bacterial cells with the modified SH liquid medium without antibiotics, so that the OD ₆₀₀ value of the bacterial liquid is 0.6-0.8, and set aside. 4. Preparation of explants: cut the cotyledons of alfalfa with a size of 4-5 days from the aseptic seedlings with a knife, and cut them into small pieces with a length of 3-4mm. The cut explants were placed in the improved SH liquid culture In the base, prevent from drying out and set aside. After the preparation of the explants is completed, the explants soaked in the improved SH liquid medium are poured into the pre-sterilized filter head, and the explants are recovered. The recovered explants were placed in the prepared bacterial solution for Agrobacterium infection. Every once in a while, shake a few times to help the Agrobacterium adhere to the explant. After dipping for 15 minutes, pour it on a sterile filter head and recover the explants. Put the explants on sterile filter paper, and blot the bacterial fluid outside the explants. The explants were placed on the filter paper in the co-cultivation medium without cephalosporin, the petri dish was sealed with Parafilm, placed in a dark place, and cultured at 28°C for 4 days.

After the explants infected by Agrobacterium tumefaciens were placed on the co-cultivation medium for 4 days in the dark, they were transferred to a culture medium containing 2mg/L 2,4-D, 0.2mg/L KT, 30mg/L hygromycin and On the improved SH solid medium of 300mg/L Cef, callus was induced; after 20 days of culture, the induced callus was transferred to a medium containing 0.2mg/L KT, 30mg/L hygromycin and 250mg/L On the UM medium of Cef, embryoid bodies are induced; when the embryoid bodies mature (about 30 days of culture), the embryoid bodies are transplanted to 1/2MS medium to induce rooting, thereby completing plant regeneration.

The composition of the medium in each step of the regeneration process is as follows:

Co-cultivation medium: improved SH medium + 2mg/L 2,4-D + 0.2mg/L KT;

Callus induction medium: improved SH medium + 2mg/L 2,4-D + 0.2mg/L KT + 300mg/LCef + 30mg/L Hyg;

Embryoid induction medium: UM medium + 0.6mg/L KT + 250mg/L Cef + 30mg/LHyg;

Rooting medium: 1/2MS medium + 250mg/L Cef + 30mg/L Hyg.

Comparative example 1

Obtaining of empty vector control plants

Agrobacterium was transformed with plasmid pCAMBIA1302 to obtain recombinant Agrobacterium, and alfalfa was transformed with recombinant Agrobacterium to obtain a control plant transformed with an empty vector. The method was as in Example 2.

Example 4

The alfalfa of the alfalfa transformed into the empty vector obtained in 30 comparative examples 1 and the alfalfa obtained in the embodiment 3 of 30 strains and inserted into the alfalfa of the pCAMBIA1302 carrier of the bZIP gene are carried out to carry out the drought resistance experiment, and specific method is as follows: 1) the plant is obtained from The soil was taken out, and the soil on the roots was washed with water; 2) The washed plants were placed in Hoagland Complete plant liquid growth solution containing 30% (w/v) PEG8000 for simulated drought treatment for 2 days.

The results showed that all the alfalfa transfected with the pCAMBIA1302 empty vector died, while 28 alfalfa transfected with the pCAMBIA1302 vector inserted with the bZIP gene survived, and most of the leaves remained green, and the plants grew well.

It can be seen that the expression of the alfalfa drought-resistance-related gene (bZIP) provided by the present invention in alfalfa can significantly enhance the drought-resistance performance of alfalfa. The discovery of this gene provides genetic resources for the research on drought resistance of major crops (especially alfalfa), and will play an important role in the research on the drought resistance performance of genetic engineering improved plants.

Claims (10)

1. A protein encoded by alfalfa drought resistance-related genes, characterized in that the protein has the amino acid sequence shown in SEQ ID No: 2, or the protein has the amino acid sequence shown in SEQ ID No: 2 through one or An amino acid sequence that still has drought resistance activity after substitution, deletion or addition of several amino acid residues. 2. The protein according to claim 1, wherein the protein has the amino acid sequence shown in SEQ ID No: 2. 3. An alfalfa drought-resistance-related gene, characterized in that the gene has a nucleotide sequence shown in SEQ ID No: 1, or the gene has a nucleotide sequence encoding the amino acid sequence shown in SEQ ID No: 2 . 4. The gene according to claim 3, wherein the gene has the nucleotide sequence shown in SEQ ID No: 1. 5. An expression vector, characterized in that the expression vector contains the gene according to claim 3. 6. A transgenic cell, characterized in that the transgenic cell contains the gene of claim 3. 7. The transgenic cell according to claim 6, wherein the transgenic cell is an alfalfa transgenic cell. 8. A method for cultivating drought-resistant alfalfa, characterized in that the method comprises introducing the gene of claim 3 into alfalfa cells to obtain alfalfa cells and transgenic plants with improved drought resistance. 9. Application of the protein according to claim 1, the gene according to claim 3, the expression vector according to claim 5, and the transgenic cell according to claim 6 in cultivating plants with improved drought resistance. 10. The use according to claim 9, wherein the plant is Medicago sativa.

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