Rapeseed BnaA04g00910D gene and its application in regulating flowering and drought and salt stress resistance in cruciferous plants

Abstract

The present invention relates to the field of genetic engineering technology, and in particular to the rapeseed BnaA04g00910D gene and its application in regulating the flowering and drought and salt stress resistance of cruciferous plants. The present invention heterologously expresses the BnaA04g00910D gene in the model plant Arabidopsis thaliana, and the obtained homozygous transgenic plants are significantly delayed in flowering time compared with the wild type, and the total number of leaves is significantly increased, indicating that the expression of the gene can delay the flowering of Arabidopsis thaliana; the BnaA04g00910D transgenic plants can improve the drought and salt stress tolerance of Arabidopsis thaliana, indicating that the gene can positively regulate drought stress and salt stress at the same time. Therefore, the gene can be used in the molecular breeding of flowering period traits and flowering period regulation of the cruciferous family. In addition, the gene provides a new gene source for the resistance to adverse stress of rapeseed, and has important application prospects in the cultivation of early-maturing varieties of cabbage-type rapeseed and the cultivation of salt-alkali tolerant rapeseed.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to a rapeseed BnaA04g00910D gene and an application thereof in regulating flowering and drought and salt stress resistance of cruciferous plants. Background Art

[0002] Rapeseed is the oil crop with the largest planting area and the widest distribution in my country. The rapeseed oil produced accounts for 50% of domestic vegetable oil. It is also an important source of feed protein in my country. With the development of the economy and the improvement of people's living standards, the demand for oil consumption continues to increase. How to use the existing arable land resources in China to increase oil production capacity is an urgent task at present. According to surveys, there are 100 million mu of winter fallow land in southern my country and 150 million mu of saline-alkali land in northern China with potential for agricultural development and utilization.

[0003] Winter rapeseed is the only winter oil crop in my country. Rotation with other crops can make full use of light, temperature and land resources and increase oil production capacity. However, the "double-season rice + rapeseed" three-cropping system in the south has compressed the growing period of rapeseed from 190 days to 170 days, so there is an urgent need to cultivate new rapeseed varieties with short growing periods. Flowering time is closely related to the growing period. Analyzing the genetic loci of rapeseed flowering time and identifying the functional genes that regulate flowering time will provide genetic resources and theoretical basis for the breeding of rapeseed varieties with short growing periods. Arabidopsis and rapeseed belong to the Brassica family of the Cruciferae family. They are closely related and have a high degree of homology between their genomes. Therefore, the model plant Arabidopsis is often used as a receptor for functional verification.

[0004] Rapeseed has a taproot system with a well-developed root system and deep taproots. It is an important crop that tolerates salt and alkali. In addition, drought also seriously affects the normal growth and yield of rapeseed. The drought-affected area in my country is about 13.25 million hectares each year. The drought is particularly serious during the rapeseed sowing period in the Yangtze River Basin. After rapeseed is sown, it often encounters autumn droughts lasting up to 10-15 days. Drought and high-salt stress can damage plants through osmotic stress, leading to an imbalance in ion concentrations in plants, interfering with normal cell metabolism, affecting plant photosynthesis, causing damage to plants, and even causing plant death. Therefore, it is of great significance to cultivate short-growing, salt-tolerant and drought-tolerant rapeseed varieties and to increase rapeseed yields by utilizing a large number of saline lands and drought-affected areas with agricultural potential to ensure national grain and oil security. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a rapeseed BnaA04g00910D gene and its application in regulating flowering and drought and salt stress resistance of cruciferous plants. The present invention mainly targets the Brassica napus gene BnaA04g00910D, and through molecular regulation of the expression of the gene, uses the Arabidopsis thaliana transformation system to study the function of the gene BnaA04g00910D in regulating plant flowering and abiotic stress.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a BnaA04g00910D gene, and the nucleotide sequence of the BnaA04g00910D gene is shown in SEQ ID No.1.

[0008] The present invention also provides a protein encoded by the BnaA04g00910D gene in the above technical solution, and the amino acid sequence of the protein is shown in SEQ ID No.2.

[0009] The present invention also provides application of the BnaA04g00910D gene described in the above technical solution in the flowering time of cruciferous plants.

[0010] Preferably, overexpression of the BnaA04g00910D gene delays flowering of cruciferous plants.

[0011] The present invention also provides application of the BnaA04g00910D gene described in the above technical solution in drought stress resistance of cruciferous plants.

[0012] The present invention also provides application of the BnaA04g00910D gene described in the above technical solution in salt stress resistance of cruciferous plants.

[0013] Preferably, the cruciferous plant includes Arabidopsis thaliana and / or Brassica napus.

[0014] The present invention also provides a method for obtaining transgenic cruciferous plants, comprising the following steps:

[0015] 1) Connecting the BnaA04g00910D gene described in the above technical solution to the PCAMBIA2300-GFP vector to obtain a connection vector;

[0016] 2) Transforming the ligated vector obtained in step 1) into Escherichia coli, extracting the plasmid, and obtaining the 35S::BnaA04g00910D plasmid;

[0017] 3) transferring the 35S::BnaA04g00910D plasmid obtained in step 2) into Agrobacterium tumefaciens to obtain transformed bacteria;

[0018] 4) The transformed bacteria obtained in step 3) are used to infect cruciferous plants by inflorescence infection method to obtain transgenic cruciferous plants.

[0019] Preferably, the cruciferous plants include Arabidopsis thaliana and / or cabbage, rapeseed, etc. In plant taxonomy, Brassica napus and Arabidopsis thaliana belong to the same family of cruciferous plants, have a common ancestor, are closely related, and have extensive colinearity in their genomes, and have high consistency in gene sequences and conserved domains. Therefore, the model plant Arabidopsis thaliana is often used as a receptor to perform functional verification of its genes.

[0020] The invention clones the flowering time-related gene BnaA04g00910D of Brassica napus "Zhongshuang 11" and constructs an overexpression vector 35S::BnaA04g00910D based on a homologous recombination method; adopts an Agrobacterium GV3101-mediated genetic transformation method to transfer the overexpression vector into wild-type Arabidopsis Col-0 through an inflorescence dip method, obtains resistant regenerated plants through kanamycin resistance screening, and finally determines positive transgenic plants through PCR analysis and Q-PCR; the flowering phenotype identification result shows that after overexpression of BnaA04g00910D, the bolting time and the first flower flowering time of the plant are significantly later than those of the wild type, and the number of leaves is significantly more than that of the wild type; subcellular localization shows that BnaA04g00910D is located in the cell nucleus and the cytoplasm, and the expression analysis of flowering signaling pathway marker genes shows that BnaA04g00910D causes late flowering of Arabidopsis by inhibiting the expression level of a key anthocyanin FT.

[0021] Seeds of BnaA04g00910D overexpression strains OE-L5, OE-L7, and OE-L9 were sterilized with 0.5% mercuric chloride and then placed on 1 / 2MS medium, 1 / 2MS medium containing Mannitol (200mM, 300mM), and 1 / 2MS medium containing NaCl (100mM, 125mM), and root growth was observed. Root length was measured after 10 days. Drought stress was simulated with 1 / 2MS medium containing Mannitol, and salt stress was simulated with 1 / 2MS medium containing NaCl. The results showed that compared with the wild type (WT), the root length of the BnaA04g00910D overexpression strain was significantly longer by about 2 cm on the untreated 1 / 2MS medium; on the 1 / 2MS medium containing mannitol, the root length of the overexpression strain was significantly longer than that of the wild type; on the 1 / 2MS medium containing NaCl, the root length of the overexpression strain was significantly longer than that of the wild type.

[0022] The seeds of BnaA04g00910D overexpression strains OE-L5, OE-L7, and OE-L9 were directly sown in a greenhouse (vermiculite nutrient soil 3:1 soil). After 18 days, drought and salt stress treatments were carried out. The growth status of the plants was observed after 12 days of drought treatment, and the number of surviving plants was counted after 3 days of rehydration. During salt stress treatment, 250mM NaCl was used for irrigation every 3 days, and the number of surviving plants was investigated and counted after 14 days. The results showed that after 12 days of drought treatment, although the rosette leaves of all plants wilted, the proportion of wilting leaves in the BnaA04g00910D overexpression strains was lower than that of WT; after 3 days of rehydration, the survival rate of the wild type was 30%, while the survival rate of the BnaA04g00910D overexpression strains was as high as 62%. Under salt stress, although the rosette leaves of most plants turned yellow, and some even turned grayish white, the leaves of the BnaA04g00910D overexpression strain were greener than those of the wild type, and the survival rate was higher.

[0023] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0024] 1. The BnaA04g00910D gene cloned in the present invention enriches the research on the phloem protein 2 gene family in rapeseed in regulating flowering time. At present, there are few studies on members of the phloem protein 2 gene family, and most of them focus on aphid resistance. Studies have shown that AtPP2-A13 in Arabidopsis plays an important role in winter short-day adaptation, but so far, the function of the BnaA04g00910D gene in the phloem protein 2 family member has not been studied in depth in regulating the flowering time of rapeseed. The present invention enriches the research on the function of such genes in rapeseed.

[0025] 2. The BnaA04g00910D gene cloned in the present invention provides a new genetic resource for early-maturing breeding of other crops.

[0026] 3. The present invention, through a series of studies on the regulation of flowering time of BnaA04g00910D overexpression strains, proves that the BnaA04g00910D gene can negatively regulate the flowering time of plants, and proves that the BnaA04g00910D gene is involved in the plant flowering time regulation signal pathway. The BnaA04g00910D gene can regulate the expression of the key flower pigment FT in plants and delay the flowering time of plants, which is of great significance for explaining the biological function of the BnaA04g00910D gene.

[0027] 4. The BnaA04g00910D gene provided by the present invention has important theoretical guiding significance for discovering and identifying crop flowering time genes and exploring the molecular mechanism of regulating flowering time.

[0028] 5. The development and utilization of the BnaA04g00910D gene provided by the present invention will help solve the contradiction between rapeseed and subsequent crops in production. At present, the cultivation and harvesting of rapeseed in production conflict with the cultivation and harvesting of rice in time, which is one of the main reasons for the idleness of rice fields, especially double-season rice fields in winter. Therefore, to develop double-season rice field rapeseed, it is necessary to solve the problem of large seasonal contradictions in double-season rice field rapeseed cultivation, and breeding early-maturing varieties is the fundamental way to solve this problem. Early flowering is the basis of early maturity. A large number of studies have shown that rapeseed has a flowering regulatory molecular network that is mostly similar to the model plant Arabidopsis thaliana. The cloning of the BnaA04g00910D gene will help cultivate early-maturing rapeseed varieties, and provide new breeding materials for further cultivating early-maturing rapeseed varieties; it has important practical guidance value in crop breeding practice, variety improvement and variety promotion.

[0029] 6. The present invention has proved that the BnaA04g00910D overexpression strain can improve the resistance of plants to drought and salt stress through a series of studies on drought and stress resistance. Drought and salinity are the most important hazards that cause crop yield losses. Cloning drought and salt stress resistance genes through genetic engineering can increase the planting range of rapeseed, providing a new gene source for further cultivating rapeseed varieties that can adapt to saline-alkali land. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0031] Figure 1 It is an identification diagram of the strain overexpressing the gene BnaA04g00910D in Example 4 of the present invention; wherein, A is the relative expression level of BnaA04g00910D in the 35S::BnaA04g00910D overexpressing transgenic strain in Arabidopsis thaliana, and the Arabidopsis Actin gene (AT1G13180) is used as a quantitative internal reference gene; B is the flowering phenotype identification of the BnaA04g00910D overexpressing transgenic strain; C is the flowering phenotype statistics of the BnaA04g00910D overexpressing transgenic strain; D is the total number of rosette leaves when the first flower of the BnaA04g00910D overexpressing transgenic strain turns white; E is the expression level of the flowering key gene FT in the BnaA04g00910D transgenic material; F is the expression level of the flowering key gene Ful in the BnaA04g00910D transgenic material;

[0032] Figure 2 This is the subcellular localization map of the BnaA04g00910D-GFP protein in Example 6 of the present invention;

[0033] Figure 31 is a phenotypic identification diagram of drought and salt stress of each genetic material of BnaA04g00910D in Example 8 of the present invention; A is a root length phenotype identification of BnaA04g00910D overexpression transgenic strain cultured on 1 / 2MS medium containing 200mM mannitol, 300mM mannitol, 100mM sodium chloride and 125mM sodium chloride for 10 days; B is a drought and salt stress phenotype statistics of BnaA04g00910D overexpression transgenic strain;

[0034] Figure 4 Figure 8 is a phenotypic identification diagram of the BnaA04g00910D overexpressing transgenic strain in Example 8 of the present invention after rehydration and salt stress after drought stress; A is the phenotypic identification of the BnaA04g00910D overexpressing transgenic strain after 12 days of drought stress and 3 days of rehydration, and the phenotypic identification of sodium chloride stress for 14 days; B is the phenotypic statistics of the survival rate of the BnaA04g00910D overexpressing transgenic strain under drought and sodium chloride stress. DETAILED DESCRIPTION

[0035] The present invention provides a BnaA04g00910D gene, the nucleotide sequence of the BnaA04g00910D gene is shown in SEQ ID No. 1, and is specifically as follows:

[0036] ATGGGCGCGAATATCTCAGGCGTTACGCCGGAGTTTGACCGGAGCAGTGA

[0037] ACCCAGATTATCCGATTTGCCGGAGAACTGTGTAGCGCTGATCATGATGCG

[0038] GCTTGACCCGCCGGAGATTTGTCGGCTCGCTCGTCTCAGCAGGGTCTTCCG

[0039] TCGCGCGTCGTCAGCTGATTTCGTGTGGGAGTCGAAGCTGCCTCCGAGTTA

[0040] CCGAGCTATTGCGCGGAAGGTGTTCGACGAAATTACTCTGAGGAAGTTGTT

[0041] GATAAAGAAGGATCTTTACGCGAAGCTCTGCAGGCCTAATCTCTTCGAGGG

[0042] TGGCACAAAGGAGCTGTGGATTGATAAGAACACTGGTCGTCTCTGTGTATC

[0043] GGTTTCTTCAAAGGCGTTAAGGATTACTGGAATTGATGATCGGAGATACTG

[0044] GAGTCATATCCCAACAGATGAATCCAGGTTCCACTCAGTTGCGTATGTTCA

[0045] ACAGATATGGTGGTTCGAAGTAGGAGGAGAGTTTGAGATCCAGTTTCCATC

[0046] CGGAACATACAGTCTCTTCTTCCGCATCCAGCTCGGGAAAACATCAAAGA

[0047] GACTTGGACGGAGAATCTGCAACTCCGACCACATCCACGGATGGGACATT

[0048] AAACCTGTTAGGTTCCAGCTCGCAACTTCAGACAACCAACAAGCTGTTTC

[0049] GCTGCGTTATCTGAACAACAACCCTGGACACTGGAGTCACTATCACGTTGG

[0050] AGATTTCAAGGTGGTAAATCCAGATGTATCAACAGGAGTCAAGTTCTCGAT

[0051] GACTCAAATTGATTGCACTCACACGAAAGGTGGCCTGTGCATAGACTCTGTTCTTATAGTACCAAAAGAAAATGCAAGAAAGGTCGTTGAATCAGAATAG.

[0052] The present invention provides a protein encoded by the BnaA04g00910D gene in the above technical solution, the amino acid sequence of the protein is shown in SEQ ID No. 2, and is specifically as follows:

[0053] MGANISGVTPEFDRSSEPRLSDLPENCVALIMMRLDPPEICRLARLSRVFRRASSADFVWESKLPPSYRAIARKVFDEITLRKLLIKKDLYAKLCRPNLFEGGTKELWIDKNTGRLCVSVSSKALRITGIDDRRYWSHIPTDE SRFHSVAYVQQIWWFEVGGEFEIQFPSGTYSLFFRIQLGKTSKRLGRRICNSDHIHGWDIKPVRFQLATSDNQQAVSLRYLNNNPGHWSHYHVGDFKVVNPDVSTGVKFSMTQIDCTHTKGGLCIDSVLIVPKENARKVVESE.

[0054] The present invention also provides the use of the BnaA04g00910D gene in the above technical solution in regulating the flowering of cruciferous plants. In the present invention, overexpression of the BnaA04g00910D gene preferably delays the flowering of cruciferous plants.

[0055] The present invention also provides application of the BnaA04g00910D gene described in the above technical solution in drought stress resistance of cruciferous plants.

[0056] The present invention also provides application of the BnaA04g00910D gene described in the above technical solution in salt stress resistance of cruciferous plants.

[0057] In the present invention, the cruciferous plant includes Arabidopsis thaliana and / or Brassica napus.

[0058] The present invention provides a method for obtaining transgenic cruciferous plants, comprising the following steps:

[0059] 1) Connecting the BnaA04g00910D gene described in the above technical solution to the PCAMBIA2300-GFP vector to obtain a connection vector;

[0060] 2) Transforming the ligated vector obtained in step 1) into Escherichia coli, extracting the plasmid, and obtaining the 35S::BnaA04g00910D plasmid;

[0061] 3) transferring the 35S::BnaA04g00910D plasmid obtained in step 2) into Agrobacterium tumefaciens to obtain transformed bacteria;

[0062] 4) The transformed bacteria obtained in step 3) are used to infect cruciferous plants by inflorescence infection method to obtain transgenic cruciferous plants.

[0063] The BnaA04g00910D gene described in the above technical solution is connected to the PCAMBIA2300-GFP vector to obtain a connection vector. The present invention does not specifically limit the method for connecting the BnaA04g00910D gene to the PCAMBIA2300-GFP vector, and those skilled in the art can use conventional methods to connect.

[0064] The present invention transfers the obtained ligated vector into E. coli, extracts the plasmid, and obtains the 35S::BnaA04g00910D plasmid. The present invention does not specifically limit the method of transferring the ligated vector into E. coli and extracting the plasmid, and those skilled in the art can use conventional methods.

[0065] The present invention transfers the obtained 35S::BnaA04g00910D plasmid into Agrobacterium tumefaciens to obtain transformed bacteria. The present invention does not specifically limit the method for transferring the 35S::BnaA04g00910D plasmid into Agrobacterium tumefaciens, and those skilled in the art can use conventional methods.

[0066] The invention uses the inflorescence infection method to infect the obtained transformed bacteria into the cruciferous plant to obtain the transgenic cruciferous plant. The invention does not specifically limit the method of inflorescence infection of the transformed bacteria into the cruciferous plant, and those skilled in the art can use conventional methods.

[0067] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0068] Example 1

[0069] TA cloning and sequence determination of BnaA04g00910D gene

[0070] Take the tender leaf part of Brassica napus "Zhongshuang 11", use TriZol Reagent (Invitrogen, Catalog No. 15596026) to extract total RNA, use Nanodrop to detect the content and purity of total RNA, take 1.0 μg of total RNA for reverse transcription reaction, the reverse transcriptase used is M-MLV (Promega, Catalog No. M1701), and the steps of reverse transcription reaction refer to the instructions for use of the reverse transcriptase. The first-strand cDNA synthesized by the reverse transcription reaction is used as a template, and the primer sequences (5′-3′) used are shown in SEQ ID No. 3 and SEQ ID No. 4:

[0071] SEQ ID No.3: ATGGGCGCGAATATCTCAGG;

[0072] SEQ ID No.4: CTATTCTGATTCAACGACCTTTC;

[0073] Conventional PCR amplification was performed, and the PCR reaction system was (50 μL): 1 μL cDNA, 5 μL 10×Buffer, 5 μL dNTP (2.5 mM), 2.5 μL MgSO4, 1 μL each of forward / reverse primers (10 μM), 0.5 μL Taq enzyme (5 U / μL) and 34 μL ddH2O (set up a 2-tube system to amplify the target gene in large quantities). After adding the sample on ice, mix well. The PCR reaction conditions were: 94°C for 5 min; 94°C for 30 sec, 52°C for 30 sec; 72°C for 30 sec, 30 cycles; 72°C for 10 min. PCR amplification obtained a fragment with a length of 861 bp. The fragment was recovered by 1% agarose gel electrophoresis, and the DNA quality was detected by Nanodrop instrument. Simple Cloning Vector was used for TA cloning (Quanshijin Company, catalog number CT111-01), and the reaction system was: 4 μL of recovered product, 1 μL Simple Cloning Vector, incubate at 25℃ for 5min. The ligation product was transformed into E. coli DH5α competent cells by heat shock method, and 400μL of fresh liquid LB medium was added for 20min recovery, and then applied to LB plates containing ampicillin antibiotics, and inverted overnight culture at 37℃. White monoclonal colonies were selected, amplified and cultured in liquid LB medium containing ampicillin antibiotics, and sequenced. Sequencing analysis showed that the sequence contained a complete open reading frame with a total length of 861 bases, as shown in SEQ ID No.1, and the encoded protein contained 286 amino acid residues, as shown in SEQ ID No.2.

[0074] Example 2

[0075] Construction of BnaA04g00910D gene overexpression vector

[0076] The BnaA04g00910D gene TA cloning plasmid of Example 1 was used as a template and the primers BnaA04g00910D-F / R were used to clone the BnaA04g00910D gene. The primer sequences (5′-3′) are shown in SEQ ID No. 5 and SEQ ID No. 6:

[0077] SEQ ID No.5:

[0078] GACTGGTACGAGCTCGGTACCATGGGCGCGAATATCTCAGG;

[0079] SEQ ID No.6:

[0080] GCCCTTGCTCACCATGGATCCTTCTGATTCAACGACCTTTC.

[0081] The BnaA04g00910D gene was connected to PCAMBIA2300-GFP by homologous recombination, and the connection system (20 μL) was as follows: 5 μL of PCAMBIA2300-GFP linear vector, 5 μL of BnaA04g00910D PCR purified product, 2 μL of enzyme (Exnase II), 4 μL of 5×CE II Buffer, and deionized water was added to a total volume of 20 μL and incubated at 37°C for 30 minutes. In this embodiment, PCAMBIA2300-GFP vector was used, and other vectors can also be used to construct a recombinant expression vector of BnaA04g00910D. The recombinant plasmid was transferred into Escherichia coli DH5α by heat shock method, and a single clone was obtained by kanamycin resistance screening and sequencing. Escherichia coli DH5α is a strain commonly used for transformation in the laboratory. In this embodiment, DH5α is used as an example, and other strains that can be used for transformation can be used to prepare a recombinant strain containing BnaA04g00910D. Sequencing analysis showed that the BnaA04g00910D gene sequence contained a complete open reading frame with a total length of 861 bases, as shown in SEQ ID No.1, and its encoded protein contained 286 amino acid residues, as shown in SEQ ID No.2.

[0082] Example 3

[0083] Genetic transformation of BnaA04g00910D gene

[0084] The DH5α recombinant strain plasmid of the BnaA04g00910D gene was extracted by alkaline lysis and named 35S::BnaA04g00910D. After the competent cells of Agrobacterium GV3101 were frozen and thawed, 5 μL of the recombinant plasmid was added, mixed, and ice bathed for 5 minutes; frozen in liquid nitrogen for 5 minutes; water bathed in a constant temperature water bath at 37°C for 5 minutes; fresh liquid LB medium without antibiotics was added, mixed, and activated at 28°C, 220rpm / min for 2 hours. Spread on LB plates containing kanamycin and rifampicin, inverted and dark cultured at 28°C for 2 days, use the BnaA04g00910D-F / R primers in Example 2 to detect positive monoclonal clones, amplify and culture in liquid LB medium with kanamycin and rifampicin resistance, and use for genetic transformation of wild-type Arabidopsis Col-0.

[0085] Agrobacterium-mediated genetic transformation of wild-type Arabidopsis Col-0: amplify the cultured Agrobacterium, centrifuge at 4000rpm / min at room temperature for 15min, discard the supernatant, add sucrose suspension and mix well (300mL ddH2O, add 15g sucrose and 70μL SilwetL-77). By inflorescence infection method, soak the inflorescence of wild-type Col-0 in Agrobacterium suspension for 60sec, and culture in dark conditions for 24h. The BnaA04g00910D gene was amplified by PCR identification.

[0086] Example 4

[0087] Molecular identification of transgenic plants

[0088] Tender leaf tissues of the transgenic positive plants identified above were taken, and total RNA was extracted using the TriZol (Invitrogen, Catalog No. 15596026) method. The first-strand cDNA was synthesized using reverse transcriptase (Promega, Catalog No. M1701). The Arabidopsis Actin gene was used as an internal reference gene, and the primers BnaA04g00910D-qF / qR were used to detect the expression level of the positive transgenic plants of the BnaA04g00910D gene. The primer sequences (5′-3′) used are as follows:

[0089] SEQ ID No.7: TGATTTCGTGTGGGAGTCGA;

[0090] SEQ ID No.8: CCGATACACAGAGACGACCA;

[0091] SEQ ID No.9: CCCGCTATGTATGTCGCCA;

[0092] SEQ ID No. 10: AACCCTCGTAGATTGGCACA.

[0093] like Figure 1 As shown, the expression levels of the BnaA04g00910D gene in the lines L1#, L2#, L5#, L7#, and L9# of the transgenic plant 35S::BnaA04g00910D-1-9 were significantly higher than those in the wild type, indicating that the target gene BnaA04g00910D was successfully transferred into the wild type Arabidopsis thaliana.

[0094] Example 5

[0095] Identification of flowering phenotypes of various genetic materials of BnaA04g00910D

[0096] The three BnaA04g00910D transgenic positive strains OE-L5, OE-L7, OE-L9 and the wild type with relatively high expression levels in Example 4 were selected as identification materials. The experimental materials were planted in a greenhouse, the temperature was controlled at 22°C, the photoperiod conditions were 16h light / 8h dark, and 35-40 plants were planted for phenotypic investigation in each strain. The flowering date from sowing to the first flower turning white was used as the standard for recording the flowering period of a single plant, the average number of actual statistical flowering dates of a single plant was used as the flowering period of the strain, and the total number of rosette leaves when the first flower turned white was recorded. The results are as follows: Figure 1 As shown in Table 1, after overexpressing BnaA04g00910D, the bolting and first flower flowering time of the plants were significantly later than those of the wild type, and the number of leaves was significantly more than that of the wild type.

[0097] Table 1 Identification of flowering phenotypes of Arabidopsis transgenic lines overexpressing BnaA04g00910D

[0098]

[0099]

[0100] Example 6

[0101] Subcellular localization of BnaA04g00910D

[0102] In order to clarify the possible cellular location where BnaA04g00910D functions, the present invention uses the tobacco transient transformation system to express the fusion protein of BnaA04g00910D-GFP. The BnaA04g00910D gene and the subcellular localization vector P2300-GFP are connected by the homologous recombination method in Example 2, and after the sequencing is correct, the Agrobacterium GV3101 is transformed. After 2 days of the tobacco transformation experiment, it was found under laser confocal microscopy that Figure 2 As shown, BnaA04g00910D-GFP under the green fluorescence channel has fluorescence in the cell nucleus and cytoplasm. Combining white light and composite light, it can be more clearly seen that BnaA04g00910D-GFP is expressed in the cell nucleus, indicating that the BnaA04g00910D gene can be located in the cell nucleus and cytoplasm.

[0103] Example 7

[0104] Quantitative analysis of flowering-related genes in various genetic materials of BnaA04g00910D

[0105] The results of subcellular localization in Example 6 showed that the BnaA04g00910D gene was located in the cell nucleus. Therefore, the present invention further studied the possible signal pathways of this gene regulating plant flowering. Figure 1As shown: the expression levels of key anthocyanins FT and Ful in the BnaA04g00910D overexpression line were significantly lower than those in the wild type, indicating that BnaA04g00910D negatively regulates key anthocyanins FT and Ful, leading to late flowering of plants.

[0106] In summary, overexpression of BnaA04g00910D in Arabidopsis thaliana through genetic engineering technology can significantly delay the flowering time of Arabidopsis thaliana, which preliminarily reveals that BnaA04g00910D has a negative regulatory role in flowering regulation.

[0107] Example 8

[0108] Phenotypic identification of each genetic material of BnaA04g00910D under drought and salt stress

[0109] Seeds of BnaA04g00910D overexpression strains OE-L5, OE-L7, and OE-L9 were sterilized with 0.5% mercuric chloride and then placed on 1 / 2MS medium containing mannitol (200mM, 300mM) and 1 / 2MS medium containing sodium chloride (100mM, 125mM) to observe root growth and measure root length after 10 days. Drought stress was simulated with 1 / 2MS medium containing mannitol, and salt stress was simulated with 1 / 2MS medium containing sodium chloride. Figure 3 The results in Table 2 show that compared with the wild type (WT), the root length of the BnaA04g00910D overexpression strain on the untreated 1 / 2MS medium was significantly longer by about 2 cm than that of the wild type. On the 1 / 2MS medium containing mannitol, the root length of the overexpression strain was significantly longer than that of the wild type. On the 1 / 2MS medium containing sodium chloride, the root length of the overexpression strain was significantly longer than that of the wild type.

[0110] Table 2 Identification of drought and salt stress in Arabidopsis transgenic lines overexpressing BnaA04g00910D

[0111]

[0112] The seeds of BnaA04g00910D overexpression lines OE-L5, OE-L7, and OE-L9 were directly sown in a greenhouse (vermiculite-nutrient soil 3:1). After 18 days, drought and salt stress treatments were carried out: the growth status of the plants was observed after 12 days of drought treatment, and the number of surviving plants was counted after 3 days of rehydration; during salt stress treatment, 250 mM NaCl was used to irrigate every 3 days, and the number of surviving plants was investigated and counted after 14 days. Figure 4The results showed that after 12 days of drought treatment, although the rosette leaves of all plants wilted, the proportion of wilted leaves in the BnaA04g00910D overexpression strain was lower than that in the WT; after 3 days of rehydration, the survival rate of the wild type was 38%, while the survival rate of the BnaA04g00910D overexpression strain was as high as 78%. Under salt stress, although the rosette leaves of most plants turned yellow, and some even turned grayish white, the leaves of the BnaA04g00910D overexpression strain were greener than those of the WT, and the survival rate was higher.

[0113] Table 3 Number of surviving plants of Arabidopsis thaliana transgenic lines overexpressing BnaA04g00910D under drought and salt stress treatment

[0114]

[0115] In summary, overexpression of BnaA04g00910D in Arabidopsis thaliana through genetic engineering technology can significantly improve the plant's resistance to drought and salt stress, which preliminarily reveals that BnaA04g00910D has a positive regulatory role in adverse stress.

[0116] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of overexpression of BnaA04g00910D gene in Arabidopsis thaliana resistance to drought stress, wherein the nucleotide sequence of the BnaA04g00910D gene is shown in SEQ ID No.

1.

2. Application of overexpression of BnaA04g00910D gene in Arabidopsis thaliana resistance to salt stress, the nucleotide sequence of the BnaA04g00910D gene is shown in SEQ ID No.1.

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