CN118530325A

CN118530325A - BnaDR1 gene, protein and application thereof in controlling drought resistance of brassica napus

Info

Publication number: CN118530325A
Application number: CN202410867995.5A
Authority: CN
Inventors: 方玉洁; 吴涵; 王幼平; 冯珊珊; 杜坤
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2024-07-01
Filing date: 2024-07-01
Publication date: 2024-08-23

Abstract

The invention belongs to the field of biology, and particularly relates to BnaDR genes and proteins and application thereof in controlling drought resistance of brassica napus. The invention separates and uses a DNA fragment containing BnaDR gene, which can endow cabbage type rape with drought resistance enhancing capability. Wherein the nucleotide sequence of the coding region of BnaDR gene is shown in SEQ ID NO. 1 of the sequence table, and the amino acid sequence of the coded protein is shown in SEQ ID NO. 2. The BnaDR gene is separated from the brassica napus, the expression quantity of the BnaDR gene is controlled by a genetic engineering technology, the resistance of the brassica napus to drought stress can be regulated and controlled, and the method has very important significance for cultivating new varieties of the brassica napus with drought resistance improvement.

Description

BnaDR1 gene, protein and application thereof in controlling drought resistance of brassica napus

Technical Field

The invention belongs to the field of cabbage type rape genetic engineering, and particularly relates to BnaDR gene and protein and application thereof in controlling drought resistance of cabbage type rape.

Background

Rape is a heterotetraploid plant obtained by hybridizing cabbage and cabbage, is one of the main sources of world edible vegetable oil, is also the first large oil crop in China, can produce about 450 ten thousand tons of rape oil each year in China, and accounts for 57.2% of the oil production of the oil crop. In the total land area of China, the area occupation ratio of arid areas reaches 22%, the space-time distribution of water resources is unbalanced, the drought stress reduces the soil moisture content to cause adverse effects on physiological processes such as plant respiration, photosynthesis, nutrient transportation, synthesis and the like, rape is very sensitive to water deficiency in the whole growth and development period, and the germination rate of seeds is reduced by drought in the germination period since 2010; plant growth abnormality caused by drought in seedling stage, short plant, small leaf and leaf wilting; drought in bolting stage results in yield reduction and even death of plants, and therefore drought stress is a major environmental factor restricting rape yield. The cultivation and irrigation modes are improved to effectively save water and reduce drought influence, but the improvement of stress resistance of crops is a main way for reducing adverse environmental influence. Cabbage type rape is widely planted in China as the type with the best comprehensive properties among three types of rape, but has poorer drought resistance than cabbage type and mustard type rape. Therefore, the identification of rape drought resistance genes and the research of molecular mechanisms of the genes for regulating and controlling the rape drought resistance are important for the genetic improvement of the cabbage type rape drought resistance. The drought-resistant related genes reported in the rape are very limited at present, and the function research of the drought-resistant genes is not deep enough, so that the genes for controlling the drought resistance of the rape are required to be excavated, and the functions of key drought-resistant genes are required to be analyzed.

Disclosure of Invention

The invention provides BnaDR gene and protein based and application thereof in regulating drought resistance of brassica napus. According to transcriptome data of rape at different time points of normal conditions and drought stress, a cabbage type rape gene which possibly plays an important role in drought stress response is screened out by constructing a rape drought stress response gene regulation network and screening core genes, and is named BnaDR (Brassica napus drought-related 1). The invention separates and uses a DNA fragment containing BnaDR gene, which can endow cabbage type rape with drought resistance enhancing capability. The nucleotide sequence of the BnaDR gene coding region is shown in a sequence table SEQ ID NO. 1, the length of the sequence is 768 bp, the amino acid sequence of the coded protein is shown in a sequence table SEQ ID NO. 2, and the number of the amino acids is 255.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

a BnaDR protein for regulating drought resistance of brassica napus, which is any one of the following (a) or (b):

(a) A protein comprising the amino acid sequence shown in SEQ ID NO. 2;

(b) And (3) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of SEQ ID NO. 2 and is related to the drought resistance regulation of brassica napus and is derived from the SEQ ID NO. 2.

A BnaDR gene for regulating drought resistance of brassica napus, wherein the gene codes for the protein.

A nucleotide sequence of the cabbage type rape drought resistance regulating gene is shown in a sequence table SEQ ID NO. 1.

The coding sequence of BnaDR gene is shown in SEQ ID NO. 1 of the sequence table, the length of the gene sequence is 768 bp, 255 amino acids are coded, and the amino acid sequence is shown in SEQ ID NO. 2.

A gene for regulating drought resistance of cabbage type rape. The gene includes a DNA molecule of any one of the following (a 1) to (a 3):

(a1) A DNA molecule comprising the sequence shown as SEQ ID NO. 1;

(a2) A DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in (a 1) and which encodes a protein associated with the regulation of drought resistance of brassica napus;

(a3) A brassica napus water drought resistance control related DNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the nucleotide sequence defined in (a 1).

It is another object of the present invention to provide an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the aforementioned genes.

The invention also provides application of the gene for regulating and controlling the drought resistance of the brassica napus in transgenic brassica napus.

It is a further object of the present invention to provide the use of (b 1) or (b 2) or (b 3):

(b1) The gene or the expression cassette, the recombinant vector, the recombinant microorganism or the transgenic cell line containing the gene are applied to regulating and controlling the drought resistance of the brassica napus;

(b2) The gene or the expression cassette, the recombinant vector, the recombinant microorganism or the transgenic cell line containing the gene are applied to cultivation of cabbage type rape drought-resistant varieties;

(b3) The gene or the expression cassette, the recombinant vector, the recombinant microorganism or the transgenic cell line containing the gene are applied to regulating and controlling the drought resistance of plants. The plant is cabbage type rape, rice, tobacco, soybean, tomato or wheat.

Further, the application comprises the step of improving the axis length, root length and fresh weight of the embryo under drought stress conditions in the later period of the germination of the brassica napus and the survival rate of the brassica napus under drought stress conditions in the seedling stage.

Further, the use includes increasing the ability of plants to scavenge reactive oxygen species (reactive oxygen species, ROS) under drought stress conditions.

The invention also provides a method for cultivating transgenic plants for improving drought resistance of plants, which improves the content or activity of the nucleotide fragments in target plants to obtain transgenic plants; the drought resistance of the transgenic plants is increased.

A method for cultivating transgenic plants for improving drought resistance of plants, which comprises the steps of cultivating transgenic plants by adopting the method; the transgenic plant contains a cabbage type rape drought resistance regulating gene.

The invention also provides a vector construction method for regulating and controlling the drought resistance gene of the brassica napus, wherein the gene is the gene for regulating and controlling the drought resistance of the brassica napus, and the method comprises the following steps: amplifying cDNA of a cabbage type rape variety Darmor as a template to obtain BnaDR gene fragments, connecting the BnaDR gene fragments to a cloning vector, carrying out PCR (polymerase chain reaction) amplification by using a primer added with a cleavage site in a pMDC vector polyclonal site, and then carrying out enzyme cleavage connection with a corresponding enzyme-cleaved pMDC skeleton vector to finally obtain the BnaDR gene over-expression vector.

The invention also provides a cultivation method of drought-resistant transgenic plants, which is characterized in that the agrobacterium transformation is carried out on the vector constructed by the method, and the genetic transformation of exogenous fragments in brassica napus is realized by using an agrobacterium-mediated method, so that the transgenic plants are obtained.

Further, the genetic transformation method comprises:

Sterilizing cabbage type rape J9712 strain seeds on an ultra-clean workbench with 75% alcohol and 40% sodium hypochlorite (NaClO), cleaning with sterile distilled water, inoculating on M ₀ culture medium, and culturing at 25deg.C in dark place for 5-6 d;

activating the agrobacterium strain BnaDR-pMDC 83 of the seed retention, picking a single colony one day before infection, inoculating the single colony into LB liquid culture medium containing kanamycin (KANAMYCIN, kan) and rifampicin (RIFAMPICIN, RFP) resistance, culturing at 28 ℃ 250 rpm overnight until the concentration of bacterial liquid OD ₆₀₀ =0.8, centrifuging at 5000 rpm at room temperature for 5min to collect bacterial bodies, discarding the supernatant, re-suspending the bacterial bodies with DM liquid, centrifuging again, discarding the supernatant, repeating twice, and re-suspending the bacterial bodies with DM liquid;

Cutting the root and cotyledon of rape seedling with a blade in an ultra clean bench, cutting the hypocotyl into 1.0-1.5 cm segments, temporarily placing in DM liquid, adding the re-suspended agrobacterium liquid into DM liquid containing 1%of AS by volume, infecting 10min, transferring to M ₁ culture medium, and co-culturing at 25deg.C under dark condition for 36-48 h;

Transferring the explant from the M ₁ culture medium to the M ₂ culture medium, culturing for 15-20 days at 25 ℃ under illumination, picking the explant with obvious callus, transferring the explant to the M ₃ culture medium containing 250 mg/L Hyg, and changing the M ₃ culture medium every 15-20 d until green seedlings are differentiated;

Transferring the seedlings to an M ₄ culture medium, and culturing at 25 ℃ under illumination for 2-4 weeks until the seedlings root, wherein the culture medium can be replaced every 15: 15 d;

hardening seedlings, transplanting the seedlings to a big basin or growing in the field after the seedlings are suitable for the bacterial environment and new leaves are grown.

The invention also provides a method for identifying the drought-resistant variety of the brassica napus, extracting genomic RNA of the brassica napus and detecting the expression level of the drought-resistant gene BnaDR of the brassica napus.

Further, the primer sequences are shown as SEQ ID NO. 11 and SEQ ID NO. 12.

The expression vector carrying BnaDR of the present invention can be introduced into plant cells by conventional biotechnological methods such as direct DNA transformation, microinjection, shock transformation, etc., using Ti plasmid, plant viral vector.

The expression vector comprising BnaDR of the present invention may be used to transform hosts (a variety of plants including brassica napus) to develop plant varieties with relatively improved drought resistance. The plant host may also be rice, tobacco, soybean, tomato, wheat, etc.

The DNA amplified fragment containing BnaDR gene is recovered by using DNA recovery kit, and is connected into pMDC83 skeleton carrier by using double enzyme cutting connection method to construct the over-expression carrier of said gene, and is named BnaDR1-pMDC83.

The method comprises the steps of transferring BnaDR to pMDC83 vectors into GV3101 agrobacterium by adopting an electric shock method, transferring BnaDR to pMDC to a brassica napus receptor material J9712 by utilizing an agrobacterium-mediated genetic transformation method and a plant tissue culture technology, obtaining a brassica napus plant with the gene expression quantity of BnaDR1 remarkably increased relative to that of a control, and comparing with the control, through drought phenotype identification, the brassica napus with the over-expression BnaDR1 has better plant growth condition and obviously increased embryo axis length, root length and fresh weight under the condition of 15% PEG and 150 mM mannitol simulated osmotic stress; under drought stress conditions in seedling stage, compared with a control, the rape over-expressed BnaDR1 has obviously better growth vigor and obviously improved survival rate, which shows that BnaDR1 can regulate and control drought resistance of plants.

Advantageous effects

The invention takes cabbage type rape as a research material, and discovers BnaDR that the molecular regulation network of the cabbage type rape under drought stress condition and screening core genes are a key core gene of the rape in drought stress response by constructing the molecular regulation network of the cabbage type rape, which possibly plays an important role in improving drought resistance of plants.

Cabbage type rape is one of the main sources of edible vegetable oil in China, drought stress is an important environmental factor threatening the production of rape in China, and improvement of drought resistance of cabbage type rape can provide guarantee for stable rape production. In the invention, the over-expression BnaDR gene can improve the tolerance of the cabbage type rape to osmotic stress in the late germination period and the drought resistance in the seedling period. Therefore, bnaDR genes are separated from the cabbage type rape, and the biological functions of the genes in the aspect of enhancing the drought resistance of the cabbage type rape are identified, so that the method has important practical significance for cultivating new varieties of drought-resistant cabbage type rape.

The invention utilizes a PCR method to separate a related gene BnaDR for controlling the drought response of the brassica napus, obtains a brassica napus plant with BnaDR1 expression quantity increased through a genetic engineering technology, discovers that the gene can improve the drought resistance of the brassica napus through a drought resistance identification method, and confirms the function and application way of BnaDR 1.

Compared with a wild type control plant, the invention constructs BnaDR over-expressed transgenic brassica napus, and discovers that under drought stress conditions, the axis length, root length and fresh weight of the embryo of the over-expressed plant are remarkably increased, the survival rate is remarkably improved, the content of accumulated ROS is less, the ROS scavenging capability is stronger, and the damage caused by oxidative stress is lighter. These results demonstrate that over-expression BnaDR a1 in brassica napus can improve the drought resistance of the napus in the seedling stage.

According to the invention, through screening and identifying the candidate gene related to rape drought resistance, the over-expression BnaDR gene is verified to be capable of effectively improving the drought resistance of transgenic rape, which shows that BnaDR1 gene participates in the response process of rape to drought stress. Therefore, bnaDR gene is separated from rape and the functions of the gene in improving the drought resistance of rape are identified, so that the gene has very important significance for cultivating new varieties of drought-resistant rape.

Drawings

FIG. 1 is a schematic diagram of BnaDR-pMDC 83 over-expression vector construction;

FIG. 2 shows the expression level of BnaDR1 in BnaDR1 over-expressed plants, CK represents a transgenic negative control, and the remaining numbered plants are BnaDR.about.1-pMDC 83 transgenic positive brassica napus plants;

FIG. 3 shows the phenotypic identification of BnaDR1 over-expressed transgenic canola plants under conditions of simulated osmotic stress with PEG and mannitol at the late stage of germination, where CK represents the control, bnaDR-OE-3, bnaDR-OE-6, bnaDR-OE-9 are three transgenic brassica napus lines over-expressing the BnaDR1 gene.

FIG. 4 shows the results of measurements of the axis length, root length and fresh weight of the embryo of BnaDR over-expressed transgenic rape and control plants under conditions of simulated osmotic stress with PEG and mannitol at the late stage of germination.

FIG. 5 is a phenotypic characterization of BnaDR1 over-expressed transgenic canola plants under seedling drought stress conditions, where CK represents control, bnaDR-OE-3, bnaDR-OE-6, bnaDR1-OE-9 are three transgenic brassica napus lines over-expressing BnaDR1 genes.

FIG. 6A shows the results of blue nitrotetrazolium chloride (nitrotetrazolium blue chloride, NBT) staining of cotyledons under conditions of simulated osmotic stress with PEG and mannitol at the late stage of germination, where CK represents a control, bnaDR-OE-3, bnaDR-OE-6, bnaDR1-OE-9 are three transgenic brassica napus lines over-expressing the BnaDR1 gene; FIG. 6B shows the survival rates of three brassica napus lines BnaDR-OE-3, bnaDR1-OE-6, bnaDR1-OE-9 over-expressing the BnaDR1 gene under drought stress conditions.

FIG. 7 shows the results of Diaminobenzidine (DAB) staining of leaf blades of BnaDR1 over-expressed transgenic canola plants under seedling stage drought stress conditions, where CK represents control, bnaDR-OE-3, bnaDR-OE-6, bnaDR-OE-9 are three transgenic brassica napus lines over-expressing the BnaDR1 gene.

Detailed Description

The following examples define the invention and describe the method of the invention in cloning a DNA fragment comprising the full coding segment of the BnaDR gene and verifying the function of the BnaDR1 gene. From the following description and examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Example 1: cloning and isolation of cabbage type rape BnaDR gene

Extraction of total DNA of plants: taking the leaves of three leaves of Brassica napus Darmor-bzh' three-leaf one-heart stage seedlings, rapidly placing the leaves in liquid nitrogen, grinding the leaves into powder by using a precooled mortar, and extracting total RNA of the rape leaves by using RNA isolater Total RNA Extraction Reagent reagent of Nanjinopran company. Reverse transcription was performed using RNAHISCRIPT Q RT SuperMix for qPCR (+ GDNA WIPER) kit from Nanjinopran, as follows:

the pipette is sucked and beaten evenly and reacts at 42 ℃ for 2 min.

Pipetting and beating uniformly, reacting at 50 ℃ for 15 min ℃, treating at 85 ℃ for 5 s, and detecting reverse transcription by utilizing specific primers BnaActin-RTF (5'-TCTTCCTCACGCTATCCTCCG-3') (SEQ ID NO: 3) and BnaActin-RTR (5'-AACAATGGATGGACCTGACT-3') (SEQ ID NO: 4) of brassica napus reference gene BnaActin to obtain the quality of cDNA.

BnaDR1 design of specific primers: specific primers were designed within 200 bp of each of the CDS sequence upstream and downstream of BnaDR1 using PRIMER PREMIER software, the internal cleavage site of the BnaDR fragment was analyzed using SNAPGENE VIEWER software, appropriate cleavage sites SpeI and AscI and homology arms were selected in combination with the multiple cloning site on the pMDC83 backbone, and the primers were designed as BnaDR1-GF (5'-GACCTCGACTCTAGAACTAGTATGAAGGTTCATGAG-3') (SEQ ID NO: 5) and BnaDR-GR (5'-CGGGCCCCCCCTCGAGGCGCGCCACCTACCTTCAGGGT-3') (SEQ ID NO: 6) added to the 5' ends of the two primers, respectively.

BnaDR1 Gene fragment cloning System:

The PCR product was detected by agarose gel electrophoresis and specific target fragments were recovered and purified according to the method described in the recovery kit for the glue FastPure Gel DNA Extraction Mini Kit of Nanjinouzan Biotechnology Co., ltd.

Example 2: construction of BnaDR1 over-expression vector BnaDR1-pMDC83

The BnaDR amplified fragment was ligated to pCE2-TA-Blunt-Zero vector using 5 XTA/Blunt-Zero Cloning Mix from Nanjinouzan, the reaction system was as follows:

transferring the product into DH5 alpha escherichia coli competent cells by using a heat shock method, picking single colonies after overnight culture, carrying out colony PCR positive identification by using M13F (5'-TGTAAAACGACGGCCAGT-3') (SEQ ID NO: 7) and M13R (5'-CAGGAAACAGCTATGAC-3') (SEQ ID NO: 8) primer collocation, and sequencing positive clones by the Qingzhou qing department biotechnology Co-Ltd, wherein the carrier with the correct sequencing verification is named as BnaDR1OE-T.

The BnaDR OE-T plasmid and pMDC skeleton vector are digested by SpeI+AscI double enzyme digestion respectively, bnaDR exogenous fragment and pMDC83 vector fragment are recovered by using FastPure Gel DNA Extraction Mini Kit glue recovery kit of Nanjinozan biotechnology Co., ltd, T4 ligase of Beijing full-length gold biotechnology Co., ltd is used for connection, DH5 alpha Escherichia coli competent cells are transformed from the connection products, single colony after overnight culture is selected, PCR positive identification is carried out by using primer 35S-F (5'-CAAGACCCTTCCTCTAT-3') (SEQ ID NO: 9) and primer BnaDR1-GR (5'-CGGGCCCCCCCTCGAGGCGCGCCACCTACCTTCAGGGT-3') (SEQ ID NO: 6) collocation, primer BnaDR1-GF (5'-GACCTCGACTCTAGAACTAGTATGAAGGTTCATGAG-3') (SEQ ID NO: 5) and primer GFP-R (5'-CATCACCTTCACCCTC-3') (SEQ ID NO: 10), positive clone extraction is carried out, namely, the BnaDR over-expression vector is named BnaDR1-pMDC (FIG. 1).

Example 3: genetic transformation of BnaDR A1-pMDC 83 Brassica napus

The invention adopts an electrotransformation method to transfer BnaDR to pMDC83 over-expression vectors into agrobacterium: placing the competent cells of the agrobacterium GV3101 into an ice box for melting, sucking 2 mu LBnaDR of 1-pMDC of plasmid into the competent cells, and adding the plasmid into the ice box for gentle mixing; rapidly adding the mixture to the bottom of an electric rotating cup, placing the electric rotating cup into an electric rotating instrument, performing 2000V electric shock, rapidly adding 500 mu L of antibiotic-free LB liquid culture medium to the bottom of the electric rotating cup after the electric shock is finished, sucking, beating, uniformly mixing, completely sucking out, adding into a new 1.5 mL centrifuge tube, and culturing at 28 ℃ at 200 rpm for 30 min; mu.L of resuscitated Agrobacterium solution was pipetted onto LA medium plates containing 50 mg/L Kan and 50 mg/L RFP resistance and incubated at 28℃for 2 d in an inverted position.

And selecting single bacterial colony for PCR positive identification, performing amplification culture on positive bacterial liquid, mixing the positive bacterial liquid with 50% sterile glycerol in equal volume for seed preservation, and placing the mixture in a refrigerator at the temperature of minus 80 ℃ for standby.

Taking the hypocotyl of the aseptic seedling of the brassica napus as an explant, carrying out genetic transformation by using an agrobacterium infection method, and relating to various culture medium formulas as follows:

Inoculation medium (M ₀): adding 30.0 g MURASHIGE & SKOOG MEDIUM (Duchefa Biochemie), 8g Sucrose, agar, and distilled water of 1L, adjusting pH to 5.8-pH 6.0, sterilizing at 121deg.C under high temperature and high pressure for 20 min, and pouring into tissue culture tank.

Co-culture medium (M ₁): adding 30.0 g MS culture medium, 8g Sucrose, agar and 18.0 g Mannitol Mannitol into 1L distilled water, adjusting pH to 5.8-pH 6.0, sterilizing at 121deg.C under high temperature and high pressure for 20 min, adding 1.0 mg/L2, 4-D2, 4-dichlorophenoxyacetic acid, 0.3 mg/L kinetin Kinetin and 100 μm acetosyringone AS under aseptic condition, mixing, and pouring into plate.

After the callus differentiation medium (M ₂）：M₁ was pH-adjusted and sterilized, 300.0 mg/L Timentin and 25 mg/L Hygromycin (Hygromycin B, hyg) were added and mixed evenly, and the plates were poured.

Seedling medium (M ₃): 30.0 g MS medium, 10.0 g Glucose, 0.25 g xylose Xylose, 0.6 g morpholinoethanesulfonic acid MES, and after pH adjustment to kill bacteria, 2.0 mg/L Zeatin, 0.1 mg/L indoleacetic acid IAA, 300.0 mg/L Timentin, and 25 mg/L Hyg were added.

Rooting medium of strong seedling (after finishing pH sterilization by M ₄）：M₀, adding 300.0 mg/L Timentin of Timentin, pouring into a tissue culture tank.

The genetic transformation of brassica napus was carried out as follows:

(1) Sterilizing and inoculating rape seeds: selecting full and mature brassica napus J9712 seeds, placing the seeds into a sterile beaker with a sterilizing screen on an ultra-clean workbench, pouring 75% alcohol to immerse the seeds, sterilizing 1 min, discarding the alcohol, then pouring 40% sodium hypochlorite (NaClO) to sterilize the seeds by about 20 min, pouring NaClO, and then washing 3-5 times with sterile distilled water; and (3) fully burning and sterilizing tweezers on an alcohol lamp outer flame, cooling to room temperature, clamping sterile seeds, uniformly placing the seeds on an M ₀ culture medium, and culturing 30-40 grains per pot at 25 ℃ in a dark place for 5-6 d.

(2) Agrobacterium Strain activation: the BnaDR-pMDC agrobacterium strain stored in a refrigerator at-80 ℃ is activated on a medium containing 50 mg/L calicheamicin and 50 mg/L rifampicin, single colonies which are positively identified are picked up one day before infection and inoculated into LB liquid medium containing 50 mg/L calicheamicin and 50 mg/L rifampicin, the culture is carried out at 28 ℃ for 250: 250 rpm overnight until the bacterial concentration OD ₆₀₀ =0.8, the supernatant is discarded in a sterile environment after centrifugation at 5000: 5000 rpm at room temperature for 5: 5 min, the bacterial body is resuspended in DM liquid and centrifuged again, the supernatant is discarded, the process is repeated twice, and DM liquid is added for resuspension.

(3) Agrobacterium infection and co-cultivation (M ₁ stage): 3 pairs of sterile large dishes (a pair of filter papers with dried filter papers, a pair of dishes with 40 mL of DM solution with 1/1000 volume AS, and a pair of empty dishes) were prepared in an ultra clean bench. The forceps and the dissecting knife are fully burnt and sterilized on an alcohol lamp outer flame and then cooled to room temperature, rape seedlings are carefully taken out of a tissue culture pot by using forceps and put into an empty dish, roots and cotyledons are cut by using a blade, only hypocotyls are reserved, then the hypocotyls are cut into small sections of 1.0-1.5 cm by using the blade, the cut hypocotyls are placed into DM liquid containing 1 per mill of AS for soaking, after all the hypocotyls are cut and soaked in the DM liquid, the resuspended agrobacterium liquid is added into the DM liquid containing AS in 1 percent of the volume, the hypocotyls are infected by 10 min, and then the hypocotyls are transferred onto M ₁ culture medium for co-culture at 25 ℃ under the dark condition for 36-48 h.

(4) Screening differentiation (stage M ₂ and M ₃): and (3) transferring the explant from the M ₁ culture medium to the M ₂ culture medium under the aseptic condition, culturing for 15-20 days at 25 ℃ under illumination, and then picking and transferring the explant with obvious callus in the M ₂ culture medium to the M ₃ culture medium containing 250 mg/L Hyg, wherein the M ₃ culture medium is changed every 15-20 d until green seedlings are differentiated.

(5) Rooting culture of strong seedlings (stage M ₄): and (3) transferring the seedlings to an M ₄ culture medium, and culturing for 2-4 weeks at 25 ℃ under illumination until the seedlings root, wherein the culture medium can be replaced every 15: 15 d.

(6) Hardening seedlings and transplanting: opening the cover of the culture tank, adding some sterile water, placing in an illumination incubator for culturing 1 d to enable seedlings in the culture tank to adapt to external bacteria environment, then placing the culture tank in water, carefully taking out the seedlings by using tweezers, slightly washing off the culture medium on roots, then transplanting the seedlings into soil, covering a fresh-keeping bag, adding a little tap water, and moving the seedlings to a big basin or a field for growth after the seedlings grow in the soil for about 1 week.

Example 4: identification of BnaDR1-pMDC83 transgenic Brassica napus positive plants

Preparing buffer (Tris-HCl (pH=7.5) with a formula of 500 mM, naCl with 300 mM and Sucrose with 300 mM) for rapidly extracting the genomic DNA of the brassica napus, and preserving at normal temperature after sterilizing; placing 0.2 g young leaves of each transgenic rape plant into a centrifuge tube of 2mL, adding DNA buffer 500 [ mu ] L and 1 steel ball, and treating the leaves of 3 min by a proofing machine 50 Hz; placing the crushed sample in a boiling water bath for 10 min, cooling to room temperature, and centrifuging for 5 min by 12000 rpm; and (3) sucking 100 mu L of supernatant, transferring to a new centrifuge tube, and diluting for 5 times for later use.

The genomic DNA of transgenic rape plant leaves extracted according to the method is used as a template, and primers 35S-F (5'-TCAGGGTAACGGGAGAAGC-3') (SEQ ID NO: 9) are matched with primers BnaDR1-GR (5'-CGGGCCCCCCCTCGAGGCGCGCCACCTACCTTCAGGGT-3') (SEQ ID NO: 6), primers BnaDR1-GF (5'-GACCTCGACTCTAGAACTAGTATGAAGGTTCATGAG-3') (SEQ ID NO: 5) and primers GFP-R (5'-TCAGGGTAACGGGAGAAGC-3') (SEQ ID NO: 10) respectively, so that PCR positive identification is carried out according to the following system:

The amplification conditions were: 95 ℃ 5 min;95℃for 30s, 58℃for 30s, 72℃for 45s,33 cycles; 72 ℃ 5 min.

And detecting PCR amplification products by agarose gel electrophoresis, and if a band which accords with the expected size is detected, proving that the corresponding plant is BnaDR-pMDC vector transgenic positive plant.

Example 5: bnaDR1 expression quantity identification of transgenic brassica napus with over-expression

The invention utilizes a real-time fluorescent quantitative PCR (qPCR) technology to detect the expression level of BnaDR genes in transgenic brassica napus plants, and comprises the following steps:

Taking positive transgenic plants with consistent growth states and leaves at the same position of a control J9712, placing the positive transgenic plants and the leaves in a centrifuge tube of 2.0 mL, quickly placing the positive transgenic plants and the leaves in liquid nitrogen, and breaking the leaves under a low-temperature condition by a precooled freezing proofing machine; total RNA from canola leaves was extracted with RNA isolater Total RNA Extraction Reagent reagent from nandina, reverse transcribed and the quality of cDNA was examined according to the method and system described in example 1.

The cDNA of BnaDR.sup.1-pMDC.sup.83 transgenic brassica napus plants was used as template and qPCR was performed with the use of the ABI StepOneplusT quantitative PCR system and the BnaDR.sup.1 specific quantitative primers BnaDR.sup.1-QF (5'-GGGAGTTAGTGGAAGAAG-3') (SEQ ID NO: 11) and BnaDR.sup.1-QR (5'-AGCTCGAGCGTTACATTT-3') (SEQ ID NO: 12) in combination, the internal reference primers being BnaActin-QF (5'-TCTTCCTCACGCTATCCTCCG-3') (SEQ ID NO: 13) and BnaActin-QR (5'-AGCCGTCTCCAGCTCTTGC-3') (SEQ ID NO: 14). The reaction procedure was as follows:

qPCR results were analyzed by ABI StepOne ™ Software (v 2.3) and expression levels of BnaDR1 in transgenic plants were calculated by the 2 ^-△△Ct method. As shown in FIG. 2, the amount of BnaDR1 expressed was significantly increased in all transgenic positive plants compared to control plants, wherein BnaDR-OE-1, bnaDR-OE-2, bnaDR-OE-3, bnaDR-OE-6, bnaDR1-OE-9 lines had about 11-fold, 5-fold, 10-fold, 20-fold and 9-fold increased BnaDR1 expression levels, respectively, indicating that the present invention successfully created transgenic canola over-expressing BnaDR 1.3 lines (BnaDR-OE-3, bnaDR-OE-6, bnaDR-OE-9) with higher expression level and more seeds were selected for subsequent experiments.

Example 6: bnaDR1 transgenic brassica napus with increased resistance to osmotic stress in later stages of germination

Seeds of mature and full control J9712 and over-expressed strains (BnaDR 1-OE-3, bnaDR1-OE-6 and BnaDR 1-OE-9) are selected, sterilized and placed on a 1/2 MS solid medium for culturing 1 d, and the over-expressed materials and the control with consistent growth vigor after germination are respectively moved to a normal medium (1/2 MS solid medium) and an osmotic stress treatment medium (1/2 MS solid medium containing 15% PEG and 1/2 MS solid medium containing 150 mM mannitol) for continuous growth under simulated drought conditions. After culturing 7 d under light conditions, the axis length, fresh weight and root length of the underembryo of the over-expressed material and the control were measured, respectively. The results show that under normal treatment conditions, bnaDR-OE and control plants showed no significant difference in vigor; whereas under 15% PEG treatment, the embryo axis length and fresh weight were significantly increased under BnaDR-OE material compared to control (fig. 3), the average value of the hypocotyl increased from 2.80 cm to 3.84 cm, 3.46 cm and 3.86 cm, respectively, 37.14%, 23.57% and 37.86%, respectively; fresh weight average increased from 0.048 g to 0.062 g, 0.059 g, 0.061 g, 29.37%, 21.82% and 26.50%, respectively (fig. 4); root length increased significantly (fig. 3), with average values increasing from 4.52 cm to 4.90 cm, 5.16 cm, and 5.98 cm, respectively, by 28.41%, 14.16%, and 32.30%, respectively (fig. 4); under 150 mM mannitol stress conditions, the embryogenic axis length, root length and fresh weight of the BnaDR-OE material are extremely obviously increased compared with a control (figure 3), and the average value of the hypocotyl is respectively increased from 3.42 cm to 5.68 cm, 4.84 cm and 4.58 cm, and 66.08%, 41.52% and 33.92% are respectively increased; root length average increases from 5.58 cm to 6.70 cm, 5.96 cm, and 6.32 cm, respectively, by 20.07%, 6.81%, and 13.26%, respectively; the fresh weight average increased from 0.056 g to 0.094 g, 0.074 g, 0.083 g, respectively, by 66.87%, 31.89% and 48.45%, respectively (fig. 4).

The O ^2•- content in cotyledons of control and over-expressed BnaDR1 material under PEG and mannitol stress treatment was detected using NBT staining. The results show that under normal conditions, there is no significant difference in cotyledon staining levels for BnaDR-OE material and control; under drought treatment conditions simulated by PEG and mannitol, compared with J9712, the cotyledon overexpression material of the BnaDR-OE material is obviously lighter in color, which shows that the content of O ^2•- accumulated by the BnaDR-OE material is smaller and the capability of removing O ^2•- is stronger. In sum, bnaDR1 may enhance the tolerance of brassica napus to PEG and mannitol-simulated osmotic stress by faster clearance of excessively accumulated ROS (fig. 5).

Example 7: bnaDR1 transgenic brassica napus seedling drought resistance enhancement

The control J9712 and the over-expression material are planted in the same black basin, the growth environment is consistent, when the plants grow to three leaves and one heart healthily, the growth conditions of the control and the over-expression plants are observed, the growth conditions of the control and the over-expression plants are basically consistent, then water is cut off for drought stress-rehydration-drought stress treatment, compared with the control, the three plants of the over-expression BnaDR1 are less influenced by the drought stress, the growth conditions are obviously superior to those of the control, the leaf wilting degree is light, only individual plants lodge, and most plants grow normally; whereas J9712 was severely affected by drought, leaf curl, severe water loss, and death of part of the plants dried out (fig. 6A); the survival of BnaDR a1 over-expressed material after drought stress was significantly improved compared to the control (figure 6B). These results demonstrate that over-expression BnaDR a1 in brassica napus can improve the drought resistance of the napus in the seedling stage.

Accumulation of H ₂O₂ in leaves of control and BnaDR-OE plants at seedling stage under normal and drought conditions was examined using DAB staining. The staining results showed that under normal conditions, the control and BnaDR-OE material leaves were not stained, while under drought conditions and after rehydration, bnaDR-OE material was lighter in staining, and dark brown spots appeared at multiple sites on the control leaves (FIG. 7), indicating less accumulation of H ₂O₂ in the BnaDR-OE plant leaves under drought stress, less damage from oxidative stress, and therefore more drought resistance.

The cabbage type rape gene BnaDR1 related to plant drought resistance is obtained through cloning, and has certain theoretical guiding significance for researching the gene related to plant drought response. The drought response related gene obtained by cloning is derived from the brassica napus and has less adverse effect on the environment. The separated gene is used for carrying out rape drought resistance genetic improvement molecular breeding, and has very important significance for cultivating new varieties of cabbage type rape with high drought resistance.

The foregoing has shown and described the basic principles, main features and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

SEQ ID NO:1

ATGAAGGTTCATGAGTTTTCCAATGGGTTTTCGTCCTGGGAACAACATGATTCGCCATCATCCCTTAGCCTAAGCTGCAAACGCCTCCGTCCTCTCGGCCCTAAGCTCTCCGGCAGCCCTTGCTCTCCTTCTTCTTCCTCCGGCGTCACTTCCGCTACTTTCGACCTCAAGAGCTTCATTAAACCCGATCAAACCGGTCCAAGAAAATTTGAATACTCTATTGAACACCAACGAGACCTTCCTCATGTGGGGACGCACCAGGGAGGGACAAGGTGGAACCCAACTCAAGAACAGATAGGGATACTTGAGATGTTATACAAAGGTGGAATGCGCACTCCTAACGCGCAACAGATCGAGAACATCACTTCTCAACTCGGTAAATACGGAAAAATTGAAGGAAAGAATGTATTTTACTGGTTTCAGAACCACAAAGCCCGCGAGAGGCAGAAGCAGAAGAGGAACAACTTCATCAGCTTAAGTTGCCTAAGCAGCTTCAAGACCACTAATATCAATAACGCAAGTGTAACAACGAAGACAACAACATCGTCAAATGACGTGATCAGGAGAGACTCAATGGTTGAGAAGGGGGAGTTAGTGGAAGAAGCTGAGTACAAGAGGACATGTAGGAGCTGGGGATTTGAGAACTTGGAGATAGATAGCAGACGAAACATAAATAGTAGTAAAAATGCTACAATGGCAACTACTTTCAACAAAATCATTGAAAATGTAACGCTCGAGCTTTTCCCATTGCACCCTGAAGGTAGGTGA.

SEQ ID NO:2

MKVHEFSNGFSSWEQHDSPSSLSLSCKRLRPLGPKLSGSPCSPSSSSGVTSATFDLKSFIKPDQTGPRKFEYSIEHQRDLPHVGTHQGGTRWNPTQEQIGILEMLYKGGMRTPNAQQIENITSQLGKYGKIEGKNVFYWFQNHKARERQKQKRNNFISLSCLSSFKTTNINNASVTTKTTTSSNDVIRRDSMVEKGELVEEAEYKRTCRSWGFENLEIDSRRNINSSKNATMATTFNKIIENVTLELFPLHPEGR.

SEQ ID NO:3

TCTTCCTCACGCTATCCTCCG。

SEQ ID NO:4

AACAATGGATGGACCTGACT。

SEQ ID NO:5

GACCTCGACTCTAGAACTAGTATGAAGGTTCATGAG。

SEQ ID NO:6

CGGGCCCCCCCTCGAGGCGCGCCACCTACCTTCAGGGT。

SEQ ID NO:7

TGTAAAACGACGGCCAGT。

SEQ ID NO:8

CAGGAAACAGCTATGAC。

SEQ ID NO:9

CAAGACCCTTCCTCTAT。

SEQ ID NO:10

CATCACCTTCACCCTC。

SEQ ID NO:11

GGGAGTTAGTGGAAGAAG。

SEQ ID NO:12

AGCTCGAGCGTTACATTT。

SEQ ID NO:13

TCTTCCTCACGCTATCCTCCG。

SEQ ID NO:14

AGCCGTCTCCAGCTCTTGC。

Claims

1. A BnaDR protein for regulating drought resistance of brassica napus, wherein the BnaDR protein is any one of the following (a) or (b):

(a) A protein consisting of the amino acid sequence shown in SEQ ID NO. 2;

2. A BnaDR gene for regulating drought resistance of brassica napus, wherein the BnaDR gene encodes the protein of claim 1.

3. The gene according to claim 2, characterized in that: the gene is a DNA molecule of any one of the following (a 1) to (a 3);

(a1) A DNA molecule shown in SEQ ID NO. 1;

(a2) A DNA molecule which hybridizes under stringent conditions to the DNA sequence defined in (a 1) and which codes for a protein which is involved in the regulation of drought resistance of brassica napus;

(a3) A DNA molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the DNA sequence defined in (a 1) and encoding a protein involved in the regulation of drought resistance in brassica napus.

4. An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the gene of claim 2 or 3.

5. Use of a protein according to claim 1 or a gene according to claim 2 or 3 or an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising a gene according to claim 2 or 3 for regulating drought resistance and/or for breeding drought-resistant varieties of brassica napus.

6. The use according to claim 5, wherein the use comprises increasing the axis length, root length and fresh weight of the embryo under drought stress conditions in the late stage of germination of brassica napus, and increasing the survival rate of brassica napus seedlings under drought stress conditions.

7. The use according to claim 5, wherein the use comprises increasing the ability of a plant to scavenge active oxygen under drought stress conditions.

8. A method for cultivating drought-resistant transgenic plants, which is characterized in that the content or activity of the protein of claim 1 in target plants is improved to obtain transgenic plants; the transgenic plant has higher drought resistance than the target plant.

9. A method for identifying drought-resistant varieties of brassica napus, which is characterized by extracting genomic RNA of brassica napus and detecting the expression level of BnaDR genes in claim 2.

10. The method for identifying drought-resistant varieties of brassica napus according to claim 9, wherein the primer sequences are shown in SEQ ID NO. 11 and SEQ ID NO. 12.