CN104774850A - BnNRT1-3 gene overexpression for improving rape nitrogen utilization rate - Google Patents

Abstract

The invention belongs to the technical field of rapeseed transgenesis, and in particular relates to improving the nitrogen utilization rate of rapeseed by overexpressing BnNRT1-3 genes. It is characterized in that the cDNA of BnNRT1-3 of Brassica napus BnNRT1-3 is cloned from the silique peel of Brassica napus by using the designed primers SEQ ID NO:3 and SEQ ID NO:4, and its sequence is as shown in SEQ ID NO:1 178-1914 nucleotides; the constructed BnNRT1-3 plant overexpression vector pBnNRT1-3 was transformed into Brassica napus through the Agrobacterium-mediated hypocotyl transformation method, and the cabbage with high expression of exogenous genes was screened type rape transgenic line; under the growth condition of culture solution containing 3mM nitrate, BnNRT1-3 overexpression transgenic plant compared with the wild-type control, its shoot dry weight significantly increased, showing that the BnNRT1-3 overexpression of the present invention can Improve the nitrogen use efficiency of plants.

Description

Overexpression of BnNRT1-3 Gene Improves Nitrogen Utilization Efficiency of Rapeseed

technical field

The invention belongs to the technical field of rapeseed transgenesis, and in particular relates to improving the nitrogen utilization rate of rapeseed by overexpressing BnNRT1-3 genes.

Background technique

Nitrogen (N) plays a vital role in the formation of plant yield and quality. my country is a large agricultural production country in the world, and rice, corn, wheat, rape, cotton and other crops are widely planted. Although my country's arable land area is only about 8% of the global arable land area, the use of chemical fertilizers (nitrogen fertilizers) accounts for 1/3 of the global total, close to 35%. The reasons are mainly in the following two aspects: First, the nitrogen utilization efficiency of crops in my country is low, such as the average nitrogen utilization rate of rice, corn and wheat is 28.7% (Yan Xiang et al., 2008), the average apparent nitrogen utilization rate of rape 34.6% (Zou Juan et al., 2011), which is far lower than the 50% in developed countries; second, there are irrationalities in the use of nitrogen fertilizer in my country, often in excess in developed areas and insufficient in poor areas, which further leads to The effective utilization rate drops. The excessive use of nitrogen fertilizer and the decrease of nitrogen utilization rate have led to the sharp deterioration of the agricultural ecological environment, and also brought many environmental problems, such as soil acidification, water eutrophication and ozone layer destruction. Therefore, improving nitrogen use efficiency and reducing nitrogen fertilizer use are not only conducive to the development of crop yield potential, improving crop quality, but also promoting the healthy and sustainable development of agriculture.

Regarding the improvement of plant nitrogen use efficiency, current research often focuses on how to optimize nitrogen fertilizer management, rationally apply nitrogen fertilizer, and reform planting systems. There are differences both in terms of quality and time, and it is impossible to form a unified standard. Although there are some reports on the collection, identification and screening of nitrogen nutrition efficient planting resources, such as rice (Tong Hanhua et al., 2007), corn (Zhou Liandong et al., 2003), cotton (Han Lu and Zhang Wei, 2011), in order to analyze nitrogen Efficient genetic basis, but progress has been slow. Plants mainly obtain nitrogen nutrients from the soil, which involves a series of N uptake and transport systems. Nitrate nitrogen is the main form of nitrogen utilized by plants, and nitrate transporters play an important role in plant N uptake and transport.

Plant nitrate transporters contain two gene families, among which the NRT1/PTR gene family is mainly a low-affinity transporter, called the low-affinity transport system (LATS); the NRT2 gene family is a high-affinity transporter, called the high-affinity transporter And transport system (HATS), which functions when the external nitrate ion concentration is very low. Under normal circumstances, the uptake of nitrate nitrogen by plants mainly depends on LATS. At present, the LATS members that have been studied clearly in Arabidopsis mainly include AtNRT1.1, AtNRT1.2, AtNRT1.3, AtNRT1.4, AtNRT1.5, AtNRT1.6, AtNRT1.7, AtNRT1.8 and AtNRT1.9 (Wang et al., 2012). Among them, AtNRT1.1 and AtNRT1.2 are responsible for N uptake (Liu et al., 1999; Okamoto et al., 2003); AtNRT1.4 plays an important role in maintaining the homeostasis of nitrate ions in leaves (Chiu et al., 2004 ); AtNRT1.5, AtNRT1.6, AtNRT1.8 and AtNRT1.9 are associated with N transport (Lin et al., 2008; Almagro et al., 2008; Li et al., 2010; Wang et al., 2011) ; AtNRT1.7 is responsible for N reuse (Fan et al., 2009). AtNRT1.3 is an inducible N transporter, and its function is still unclear. Studies have shown that in the legume model plant Medicago truncatula (Medicago truncatula), MtNRT1.3 is a dual-affinity transporter, which up-regulates expression when plants are deficient in nitrogen, and promotes plant N uptake (Morère-Le et al., 2011 ). In addition, in wheat, the expression pattern and response to N of the gene TaNPF6.6, which is homologous to AtNRT1.3, is also different from Arabidopsis. It is expressed in roots and shows up-regulation in response to nitrogen deficiency (Buchner and Hawkesford, 2014 ). It can be seen that the function of NRT1.3 gene is different in different species, and generally speaking, it has a positive effect on plant N uptake and utilization.

Contents of the invention

The purpose of the present invention is to determine the function of BnNRT1-3 gene in N uptake and utilization in Brassica napus, and relates to the application of BnNRT1-3 in improving nitrogen utilization efficiency of plants to cultivate crops with high nitrogen efficiency.

In order to achieve the above object, the present invention is achieved through the following technical solutions:

The present invention first clones the homologous gene of NRT1.3 in Brassica napus, and the applicant named the gene BnNRT1-3 gene, and overexpressed the BnNRT1-3 gene in Brassica napus under the control of CaMV35S promoter, which can improve the quality of rapeseed. nitrogen use efficiency.

The nucleotide sequence of the nitrate transporter BnNRT1-3 gene is shown in the sequence shown in base 1-1914 in the sequence table SEQ ID NO: 1, and its corresponding amino acid sequence is shown in SEQ ID NO: 1 As shown in the sequence shown in bases 1-1914, the protein sequence encoded by the gene is shown in SEQ ID NO:1.

The gene regulates the improvement of nitrogen use efficiency in Brassica napus mainly as an increase in the amount of plant dry matter.

Utilize primer pair F46 and R46-1 (see sequence table SEQ ID NO: 3,4), clone nitrate transporter BnNRT1-3 gene from Brassica napus by RT-PCR technology, then use this gene to construct plant overexpression vector , carry out the plant transgenic operation, and obtain the transgenic plant. The results showed that the nitrogen use efficiency of BnNRT1-3 overexpression plants was significantly higher than that of wild type (WT).

The invention confirms the function of BnNRT1-3 gene in improving plant nitrogen utilization efficiency, and lays a foundation for cultivating nitrogen-efficient plants by means of transgene.

Description of drawings

1. Sequence Listing SEQ ID NO: 1 is the nucleotide sequence of the BnNRT1-3 gene cloned in the present invention, the sequence length is 1914bp, and the sequence corresponding to the 178-1914 bases is the CDS region of the gene, encoding 578 amino acids sequence.

2. Sequence listing SEQ ID NO: 2 is the protein sequence encoded by the cloned BnNRT1-3 gene of the present invention.

3. SEQ ID NO: 3 and SEQ ID NO: 4 are specific primer pairs for amplifying the BnNRT1-3 gene, which we named as primer F46 and primer R46-1; among them: BnACT2-F/BnACT2-R is the internal reference of BnActin2 Gene amplification primers.

Fig. 1: Construction of an overexpression plant expression vector pBnNRT1-3 according to one embodiment of the present invention. Figure A in Figure 1 is a vector diagram of pCAMBIA1300; Figure B in Figure 1 is a schematic diagram of a pCAMBIA1300s vector; Figure C in Figure 1 is a schematic diagram of a pBnNRT1-3 vector constructed in the present invention.

Figure 2: Expression analysis of BnNRT1-3 overexpression transgenic lines. In the figure: WT is the wild-type (non-transgenic) control, and the rest numbers correspond to different transgenic lines. Number 1 represents the transgenic line D-1, number 2 represents the transgenic line D-2, and so on. In the figure, the amplification primer pair of BnNRT1-3 is F46/R46-1 (see Table 1 for the primer sequence); the amplification primer pair of the BnActin2 internal reference gene is BnACT2-F/BnACT2-R (see Table 1 for the primer sequence).

Figure 3: Comparison of dry weight between wild-type and BnNRT1-3 overexpression lines grown under different concentrations of nitrate. The overexpression plants and wild-type plants were cultured in 1/4 Hoagland nutrient solution for two weeks, and then moved into 1/2 Hoagland nutrient solution with different nitrate concentrations to continue to grow for three weeks. The above-ground part was taken to weigh the dry weight, and the experiment was carried out twice repeat. Among them, WT is the wild type control, and D-2, D-19 and D-25 are three overexpression lines. Error bars are standard deviations, * and ** indicate significant differences between transgenic plants and wild-type plants at P<0.05 and P<0.01 levels, respectively.

Detailed ways

Example 1 Cloning of Brassica napus BnNRT1-3 Gene

1. Cloning the cDNA sequence of BnNRT1-3 gene in Brassica napus

The genome sequence information (BnaA05g19580D) of BnNRT1-3 was obtained according to Brassica napus genome reference sequence (http://www.genoscope.cns.fr/brassicanapus/). Primer pairs F46 and R46-1 were designed according to the genome sequence information (see Table 1 of the description and SEQ ID NO: 3 and 4 in the sequence table for the primer sequence). Among them, F46 is a forward primer, which contains an XbaI enzyme-cut linker; R46-1 is a reverse primer, which contains a PstI enzyme-cut linker. Since the microarray results of Brassica napus silique peel and seeds showed that BnNRT1-3 was strongly expressed in the silique peel at the later stage of silique development, cabbage was extracted using the TransZol Plant kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) The RNA of the silique peel of rapeseed silique after 42 days of development (see the kit instruction manual for the specific operation steps). Using the RT-PCR method, the high-fidelity TransStart FastPfu DNA Polymerase (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) was used to amplify the cDNA sequence of BnNRT1-3. The PCR reaction conditions were 95°C for 1min; 40 cycles of 95°C for 20s, 54°C for 20s, and 72°C for 50s; 72°C for 5min. The cloned product was ligated into the pEASY-Blunt vector (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), an intermediate sequencing vector was constructed, named pEASY-NRT1.3, and the sequence was determined to verify the correctness of the cloned sequence.

2. Sequence information and characteristics analysis of BnNRT1-3 genes in Brassica napus

Brassica napus BnNRT1-3 gene coding region cDNA is 1737bp, encoding 578 amino acids. The cloned SEQ ID NO: 1 sequence contains 177 bases of the 5'UTR segment of the BnNRT1-3 gene. The homology of BnNRT1-3 protein with AtNRT1.3 and MtNRT1.3 was 89% and 70%, respectively. The ProtComp 9.0 software predicted that the BnNRT1-3 protein was located in the cell membrane, which was consistent with its function.

Example 2 Obtaining of BnNRT1-3 transgenic plants

1. Construction of cDNA expression vector containing BnNRT1-3 coding region

After the pEASY-NRT1.3 carrier plasmid containing the BnNRT1-3 gene in Example 1 was digested with XbaI and PstI, the fragment cut by the enzyme was connected to the carrier part of the pCAMBIA1300s vector plasmid digested with the corresponding enzyme to obtain The plant expression vector driven by the CaMV 35S promoter to overexpress BnNRT1-3, we named it pBnNRT1-3 vector (as shown in Figure 1 C). The pCAMBIA1300s vector is transformed from the pCAMBIA1300 vector (purchased from YRGene Company, catalog number: VAC0323). First, the pCAMBIA1300 vector (as shown in figure A in Figure 1) was double digested with HandⅢ and EcoRI, and the part of the multiple cloning site was excised; then the fragment containing the three elements of CaMV 35S, multiple cloning site and poly A was combined with The vector part of the digested pCAMBIA1300 vector plasmid was connected to obtain the pCAMBIA1300s vector (as shown in Figure 1, B).

The constructed BnNRT1-3 overexpression plant expression vector pBnNRT1-3 was transformed into Agrobacterium GV3101 electroporation competent cells. The specific steps are as follows: take a tube of 50 μl GV3101 electroporation competent cells and put them on ice, add 0.1 μg of the constructed pBnNRT1-3 vector plasmid after melting, and gently suck and mix with a pipette; The pipette was sucked into the electric cup pre-cooled on ice, and the Genepulser electric transfer instrument (purchased from Bio-Rad) was used to shock at 1800V for 6ms; add 500 μl liquid LB to the electric cup and mix well, transfer the bacterial solution to 2 mL centrifuge In the tube, culture at 150rpm in a shaker at 28°C for 2hrs; take 100μl of the bacterial solution and spread it on a solid LB plate (containing gentamicin 25μg/ml and kanamycin 50μg/ml), dry it and culture it in a 28°C incubator for 2- 3 days; pick bacterial plaques for PCR detection and enzyme digestion verification, positive clones are shaken and aliquoted and stored at -70°C or directly used for plant transformation.

2. Agrobacterium-mediated plant transformation method

After the pBnNRT1-3 vector constructed above was transformed into Agrobacterium GV3101, Brassica napus was transformed by Agrobacterium-mediated hypocotyl transformation method. Specific steps are as follows

(1) The hypocotyls of Brassica napus (Huashuang No. 5, a new variety of rape that has been publicly promoted and applied in China and passed the variety approval) grown for 5 days in the dark were taken, and cut into 5-7 mm long segments. The Agrobacterium with an OD=0.8 was resuspended with the infection suspension M0 and diluted 20 times to infect the hypocotyls of Brassica napus for 15 minutes.

M0 formula: MS basic medium, 30.0g/L sucrose, 100.0μM/L acetosyringone (AS), add distilled water to 1L, adjust the pH of the medium to 5.85 before sterilization.

(2) Transfer the infected Brassica napus hypocotyls into the co-cultivation medium M1, culture in the dark (light culture for 16 hours, dark culture for 8 hours with a test tube rack black cloth shading (light and dark culture for short), light intensity 230 -300μEm ^-2 s ^-1 ) total 2d.

M1 formula: MS basal medium (abbreviated as MS-general term in this field), 30.0g/L sucrose, 18.0g/L mannitol, 1.0mg/L 2,4-D, 0.3mg/L cytokine (KT ), 30.0 μM/L silver thiosulfate (STS), 300.0 mg/L Timentin (Timentin), 8.0 g/L agar, supplemented with distilled water to 1 L; the pH of the medium was adjusted to 5.85 before sterilization.

(3) Transfer the hypocotyl explants co-cultured for 2 days to the callus induction medium M2 to induce callus, and culture in the culture room (16h under light, 8h in dark, light intensity 230-300μEm ^-2 s ^{- 1} ) for 20 days. M2 formula: MS, 30.0g/L sucrose, 18.0g/L mannitol, 1.0mg/L 2,4-D, 0.3mg/L cytokinin (KT), 30.0μM/L silver thiosulfate (STS) , 300.0mg/L Timentin (Timentin), 25.0mg/L hygromycin B, 8.0g/L agar, add distilled water to 1L; adjust the pH of the medium to 5.85 before sterilization.

(4) The callus cultured in step (3) for 20 days was transferred into the differentiation medium M3, and continued to be cultured in the light culture room, subcultured once every 20 days. M3 formula: MS, 10.0g/L glucose, 0.25g/L xylose, 0.6g/L morpholineethanesulfonic acid (MES), 2.0mg/L zeatin (ZT), 0.1mg/L indole acetic acid (IAA ), 300.0mg/L Timentin (Timentin), 25.0mg/L hygromycin B, 8.0g/L agar, supplemented with distilled water to 1L; adjust the pH of the medium to 5.85 before sterilization; place in a light culture room For cultivation, the light intensity is 230-300 μEm ^-2 s ^-1 ;

(5) The plants regenerated in step (4) are transplanted into the rooting medium M4 for rooting culture. M4 formula: MS, 30.0g/L sucrose, 8.0g/L agar, add distilled water to 1L, adjust the pH of the medium to 5.85 before sterilization. Placed in a light culture room for cultivation, the light intensity is 230-300μEm ^-2 s ^-1 ;

3. Molecular identification of transgenic plants

The gene expression level of the obtained transgenic lines was detected. TransZol Up reagent (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) was used to extract the stem and leaf RNA of transgenic Brassica napus plants and wild-type Brassica napus plants, and the extraction procedure was carried out according to the instructions of the reagent. Dissolve RNA in 50 μL RNA solution, take 3 μL and use TransScript One-Step gDNA Remover and cDNA Synthesis Kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) for inversion to synthesize the first-strand cDNA (for specific methods, refer to the kit’s manual). BnACT2-F/BnACT2-R was used as the internal standard gene BnActin2 primer (see Table 1 for the primer sequence), and F46/R46-1 (see Table 1 for the primer sequence) was used as the specific primer for the exogenous gene BnNRT1-3 for expression detection. The PCR reaction conditions of the BnActin2 gene were 94°C for 3min; 94°C for 30s, 58°C for 30s, 72°C for 30s, 26 cycles; 72°C for 5min; the PCR reaction conditions for the BnNRT1-3 gene were 94°C for 3min; 30s at ℃, 2min at 72℃, 30 cycles; 10min at 72℃. The results showed that the expression of BnNRT1-3 gene was not detected in the wild-type control WT (non-transgenic plant), and the expression of BnNRT1-3 gene was different in different transgenic lines. The transgenic lines D-3, D-4, D-20, D-21, D-22 and D-26 basically had no expression of exogenous gene BnNRT1-3, while the transgenic lines D-2, D-9, D -11, D-14, D-19, D-23, D-25 and D-27 had higher expression levels of exogenous gene BnNRT1-3 (see Figure 2). Therefore, three lines with higher expression levels such as D-2, D-19 and D-25 were selected for follow-up work.

Table 1 Sequence list of primers for detection of gene expression in Brassica napus in the present invention

Example 3 Comparison of nitrogen use efficiency between BnNRT1-3 overexpression plants and wild-type plants

Select overexpression transgenic Brassica napus plants and wild-type Brassica napus seedlings that have germinated and grow consistently for one week, and place them in 1/4 improved Hoagland nutrient solution (commonly used culture solution in this field, literature: Hoagland and Arnon, 1950) to cultivate two week (the formula of the improved Hoagland complete culture solution is shown in Table 2 of the instruction manual), and then transferred to 1/2 Hoagland nutrient solution containing different concentrations of nitrate (such as 0.3mM, 3mM and 7.5mM) to continue culturing for three weeks, during which 4-5d change A fresh Hoagland nutrient solution. The control of the nitrate concentration in the culture medium is realized by adjusting the content of calcium nitrate tetrahydrate and potassium nitrate in the culture medium. Considering the accompanying loss of calcium ions and potassium ions, the corresponding amount of calcium ions and potassium ions will be supplemented. , supplemented in the form of calcium sulfate and potassium sulfate, respectively. After the cultivation was completed, the aerial part of the plant was dried in an oven at 70°C, and the dry weight was weighed, and the difference in the dry weight of the aerial part of the wild-type plant and the overexpression plant was compared. The results showed that when the nitrate concentration in the nutrient solution was increased from 3mM to 7.5mM, although the dry weight of the above-ground parts of the overexpression plants and the wild-type plants had no significant change, the dry weight of the above-ground parts of the over-expression plants under the 3mM condition was generally significant. Dry weight of aerial parts of overexpressed plants under 7.5 mM condition. However, under the condition of 3mM, the dry weight of overexpressed plants was significantly higher than that of wild-type plants (see Figure 3). It can be seen that under the condition of lower concentration of nitrate, the nitrogen use efficiency of overexpressed plants is higher, and it has a growth advantage than that under the condition of high nitrogen level (7.5mM), which further illustrates that BnNRT1-3 Application value of genes in breeding nitrogen-efficient Brassica napus.

Table 2 Improved Hoagland nutrient solution formula

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Claims (5)

1. the clone's sub-BnNRT1-3 gene of swede type rape nitrate transport, the sequence of its nucleotide sequence as shown in 1-1914 bit base in sequence table SEQ ID NO:1.

2. the clone's sub-BnNRT1-3 gene of swede type rape nitrate transport, the protein sequence of its coding is as shown in sequence table SEQ ID NO:2.

3. clone primer pair F46 and R46-1 of gene described in claim 1, its nucleotide sequence is as follows:

F46：GGTCTAGAATGTCCATTGAATCGAATCTCCTCT；

R46-1：GGCTGCAGTTATGGAATGTCTTTAAGCTCCAACTCAG。

4. utilize BnNRT1-3 gene to improve an application for swede type rape nitrogen utilization efficiency, described application method comprises the steps:

1) design primer pair as claimed in claim 3, from the pericarp of swede type rape metacyclic angle, clone BnNRT1-3 gene, the sequence of described BnNRT1-3 gene nucleotide series as shown in 1-1914 bit base in SEQ ID NO:1;

2) build the plant over-express vector of BnNRT1-3 gene, this plant over-express vector includes nucleotide sequence as claimed in claim 1, with this vector Agrobacterium;

3) by agriculture bacillus mediated swede type rape Regenerated from Hypocotyl Explants method, screening obtains the transgenic line of BnNRT1.3 gene overexpression;

4) adopt molecular detecting method to step 3) process LAN transformation plant carry out expression analysis, the strain selecting high expression level amount carries out comparing of nitrogen utilization efficiency with Wild-type non-transgenic plant;

Wherein:

Step 3) in the substratum that uses in agriculture bacillus mediated swede type rape Regenerated from Hypocotyl Explants method and culture condition as described below:

(1) infect suspension: MS, 30.0g/L sucrose, 100.0 μMs/L Syringylethanone, supplement distilled water to 1L; The pH to 5.85 of substratum is adjusted before sterilizing;

(2) Dual culture substratum: MS, 30.0g/L sucrose, 18.0g/L N.F,USP MANNITOL, 1.0mg/L 2, the cytokinin of 4-D, 0.3mg/L, 30.0 μMs/L silver thiosulfate, 300.0mg/L Ticarcillin/Clavulanate Acid (Timentin), 8.0g/L agar, supplements distilled water to 1L; The pH to 5.85 of substratum is adjusted before sterilizing; In culturing room's illumination cultivation 16, light culture 8h, illumination is by force 230-300 μ Em-2s-1;

(3) calli induction media: MS, 30.0g/L sucrose, 18.0g/L N.F,USP MANNITOL, 1.0mg/L 2, the cytokinin of 4-D, 0.3mg/L, 30.0 μMs/L silver thiosulfate, 300.0mg/L Ticarcillin/Clavulanate Acid, 25.0mg/L hygromycin B, 8.0g/L agar, supplements distilled water to 1L; The pH to 5.85 of substratum is adjusted before sterilizing; Be placed in 16h illumination in culturing room, cultivate during 8h secretly trains, intensity of illumination is 230-300 μ Em ^-2s ^-1;

(4) division culture medium: MS minimum medium, 10.0g/L glucose, 0.25g/L wood sugar, 0.6g/L morpholino b acid, 2.0mg/L zeatin, 0.1mg/L indolylacetic acid, 300.0mg/L Ticarcillin/Clavulanate Acid, 25.0mg/L hygromycin B, 8.0g/L agar, supplements distilled water to 1L; The pH to 5.85 of substratum is adjusted before sterilizing; Be placed in illumination cultivation room to cultivate, intensity of illumination is 230-300 μ Em ^-2s ^-1;

(5) root media: MS, 30.0g/L sucrose, 8.0g/L agar, supplements distilled water to 1L; The pH to 5.85 of substratum is adjusted before sterilizing; Be placed in illumination cultivation room to cultivate, intensity of illumination is 230-300 μ Em ^-2s ^-1.

5. gene according to claim 1 is improving the application in swede type rape nitrogen utilization efficiency.