WO2013010368A1 - Use of rice aerenchyma formation key gene oslsd2 - Google Patents

Abstract

Provided is the use of rice aerenchyma formation key gene OsLSD2 (Oryza sativa lysine-specific demethylase-2）. The gene, with the accession number being AK111759，can be used in improving height, nitrogen use efficiency and/or production of plants. Specifically, prepared is rice with OsLSD2 gene overexpressed in roots and leaves by genetic method, with the root aerenchyma of rice plant changed. Compared with wild-type, the transgenic plants are higher，and the nitrogen use efficiency and single plant production increase by at least 30%.

Description

 Instruction manual

 Application of key gene 0sLSD2 in rice aeral tissue formation

Technical field

 The invention belongs to the technical field of 棊 engineering and relates to the application of rice ventilating tissue to form key genes.

Mm,

Nitrogen is one of the most important nutrient elements in crops and participates in various metabolic processes in organisms. It is a component of many living substances in plants, such as: amino acids, proteins, nucleic acids, enzymes, chlorophyll and so on. Nitrogen accounts for 1.5% of the total dry weight of the plant and 16% of the total protein of the plant (Frink CR., Waggoner PE. and Ausubel JH. Nitrogen fertilizer: retrospect and prospect. Proc. Nati. Acad. Sci. USA. 1999. 96 : 1175~: 1180. ). At present, China's nitrogen fertilizer accounts for 30% of the global nitrogen fertilizer use (Peng Shaobing, Huang Jianliang, Zhong Xuhua, Yang Jianchang, Wang Guanghuo, Zou Yingbin, Zhang Fusuo, Zhu Qingsen, Roland Buresh, Christian Witt. Research Strategies for Improving Nitrogen Use Efficiency in Rice Fields in China. 2002, 3.5 (9) : 1095 ~ 1103. ), became the world's largest consumer. Among them, the application rate of nitrogen fertilizer in rice fields exceeds that of any other crops, and the loss of nitrogen fertilizer accounts for 70% of the total amount of fertilizer. A series of environmental problems caused by low nitrogen utilization rate and a large amount of nitrogen loss are common in China. Although rice is suitable for ammonium crops, studies have shown that a certain amount of nitrate nitrogen can promote the absorption of ammonium by rice, and in the late stage of rice growth and development, rice is mainly composed of nitrate nitrogen. The root development of rice is regulated by nitrate. The mechanism by which the main nitrate promotes root growth is that nitrate regulates the transport distribution of auxin. In recent years, molecular biology technology has developed rapidly, providing new tools for crop genetics and breeding. People also attach great importance to the use of biological methods to improve the utilization of nitrogen fertilizer to solve the problem of nitrogen fertilizer. Like other aquatic crops, the oxygen-secretion process of rice roots is an essential process for its growth in an anaerobic environment. This oxygen-inducing process inevitably leads to different degrees of nitrification in the rhizosphere of rice, which is produced in the root surface and rhizosphere. If nitrate is quickly absorbed by the roots, it will stimulate the crop's nitrogen nutrition and other physiological processes, which will significantly promote crop growth and development. Our 3⁄43⁄4 test results show that (Li et a].. , 2008) The nitrogen-efficient variety Yangdao 6 secretes more oxygen through its developed aerated tissue, compared with the control nitrogen inefficient variety Nongken 57. Enrichment of more nitrifying bacteria produced more nitrate, so that nitrogen-efficient rice Yangdao 6 showed more nitrate and total nitrogen in the same paddy field, and nitrogen utilization enzyme activity was significantly higher than that of the control crop 57. . The final yield and nitrogen use efficiency of Yangdao 7 were significantly higher than that of the farmer's 57. However, if the nitrates in the root table are not rapidly absorbed by the roots, there is no such stimulating effect, and these nitrates are lost by denitrification. Our study shows that the high-yield nitrogen variety Yangdao 7 not only has a well-developed aerated tissue, but its ability to absorb (Fan et al 2005) and reuse nitrate (Fan et al 2007) is significantly higher than that of farmland 57. This indicates that nitrogen-efficient rice cultivars are the best in their ability to evolve and adapt themselves from terrestrial to aquatic. They not only show evolutionary or adaptive advantages in tissue formation, but also optimize or retain more Terrestrial nitrogen absorption and utilization characteristics. Aerenchy is a collection of plant wall tissue _: some air chambers or cavities. Many aquatic and wet plants form aerated tissue in the rhizomes, and other plants differentiate or produce accelerated tissue in hypoxic environments (Watkin et al., 1998). Under flooding conditions, rice increases gas conductance by forming aerated tissue (Miyamoto et al 2001), and the root base induces a barrier that prevents oxygen from escaping (Cdjiier, 2003), thereby prolonging the transmission of oxygen to the apical direction. However, different rice varieties show great differences. Colmer (2003) compared the formation of aerated tissue and oxygen barriers in 7 dry rice, 3 rice and 2 deep-water-resistant rice varieties under flooding conditions. It was found that some dry rice roots could not form oxygen-proof spillovers. The compartments, and the oxygen exudation of the apex, have significant differences in breeds.

 Seago et al. (2005) and Evans et al. (2003) review the formation and classification of aerated tissue. Compared with fissure aerobic tissue, there are relatively many studies on lytic aerenchyma. The formation of lytic aerated tissue is controlled by programmed cell death (PCD) (Gmmwardena A, 2008 review). In the middle part of the rice roots, large-scale autolysis of the cells almost eliminates all central cells, forming hollows, and the resistance to gas transport is minimized (Delia et al., 1999). Cell death is generally thought to occur in the middle of the cortex. Transmission electron microscopy of corn cortex cells showed that the disintegration of vacuoles was the first physical process to be seen (Campbel l & Drew, 1983). The change of the mesothelium in rice cells seems to be earlier. The structure changes in hypoxia within 1 d, and the cell wall disintegrates before the whole cell disintegrates (Webb & Jackson, 1986). Kawai et al. (1998) studied the cytology of rice roots and found that acidification of cells was an early indicator of subsequent cell disintegration. This means that the acidic inclusions released by the damage of the liquid membrane can cause damage to the integrity of the plasma membrane. Unlike the formation of xylem, the aerenchytic cell wall is completely degraded (Gunawardena et al 2001). This may be related to the xyloglucan transglucosidase xei gene, the pyruvate decarboxylase pcfe gene, the glycerol phosphate dehydrogenase gpc gene, etc. (Subbaiah fe Sachs, 2003). Muhl enbock et al 2007 used Arabidopsis edsl, pad4-5 single mutant and edd/Lsd?, pad4-5/sdl double mutant to study aerenchyma formation and ethylene synthesis under hypoxic conditions, suggesting negative control of LSD1 gene EDS1 and gene expression, while EDS1 and genes are regulating the synthesis of ethylene and the formation of root aerenchyma under hypoxic conditions. From the expression site of PBZ1 gene in rice, this gene is involved in PCD of rice, including leaf senescence and aerenchyma formation in roots. By mutating this gene, the gene is involved in DM Ladder formation and is expressed in all cells that develop PCD (Kim Et al 2008). Summary of the invention

 It is an object of the present invention to provide an application of rice aeral tissue to form the key gene 0sLSD2.

The object of the present invention is achieved by the following technical scheme - the application of the key gene 0 _S L502 formed by rice control aeration tissue in increasing plant height and increasing plant nitrogen use efficiency and/or yield, the sequence accession number of which is AK111759.

Among them, the plant is preferably a monocotyledonous plant, and further preferably rice, corn or wheat, and particularly preferably rice. Rice encodes a key gene for the formation of aergic tissue, the coding product of OSSLD2, rice aerenchyma, forming key protein OSLSD2 Application in increasing plant height, increasing plant nitrogen use efficiency and/or yield.

 Advantageous effects of the present invention

 1. The present invention provides the biological function of the rice auxin transport protein gene OSLSD2 for the first time through systematic research.

2. Using specific primers to study the expression of OSLSD2 in rice, it was found that OSLSD2 was expressed in both above and below ground (Fig.

1) Increased nitrogen use efficiency after overexpression of OSLSD2 (Table 1).

 3. Overexpression of OSLSD2 increased the plant height and tiller number of rice, which was beneficial to improve rice plant type (Fig. 4) and increased yield by more than 30% (Table 2).

 The OSLSD2 overexpression material obtained by the transgenic method of the present invention causes a large increase in OSLSD2 expression in leaves (Fig.

2), thereby changing the aeration tissue of the roots of rice plants. Compared with the control wild type, the transgenic material obtained higher plants, which is beneficial to the absorption and utilization of nutrients, especially nitrogen, and nitrogen use efficiency. .

DRAWINGS

Figure 1 shows the expression characteristics of OSLSD2 in different parts of rice (leaf, root), of which h root 2 _t leaves.

Figure 2 The OSLSD2 overexpression material (0-1, 0-2 0-3, 0-4) was identified, and the expression of four overexpressing lines in leaves was significantly enhanced compared with wild type.

Figure 3 Identification of Southern copy number of 0SLSD2 overexpression material,

Among them, WT: wild type, CK: blank control; 0-1, 0-2, 0-3, 0-4: respectively represent 0sLSD2 overexpression material. Figure 4 OSLSD2 overexpression material (04) increased plant height and number of tillers compared to wild type (Wuyu 粳 7).

Figure S 0SLSD2 overexpression material (04) and wild type (Wuyu粳 7) aerated tissue micrograph.

Among them, WT; wild type, 0-4 _: indicates 0SLSD2 over-expression material, the right picture is a microscopic picture of aeration tissue at a distance of 1 cm from the apex, and the left side is a microscopic picture of a ventilated tissue at a distance of 1.5 cm from the apex.

detailed description

Example 1 Gene expression (accession number AK111759) in rice

1) The total RNA was extracted from the rice seedlings of Wuyujing No. 7 by a mass ratio of 30% NaCL0 _{2 to be} sterilized, germinated, and cultured to the second leaf and one heart, and the rice plants of the same size were selected, and the endosperm was removed and transplanted to PH 5. 5 In the IRRI nutrient solution of the International Rice Research Institute, the four-leaf one-day-in-one is replaced by the International Rice Research Institute. (Ma.o D R. The methods of plant nutri tion research . Bei jing ■: ■ Bei jing Agri cultural University 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 5毫升isopropyl醇, centrifugation and discarding, adding 1 ml of Trizol reagent, adding 0.2 mL of chloroform, centrifuging, and taking up the supernatant, adding 0.5 mL of isopropanol. The supernatant was washed with 70% ethanol, and RA was dissolved in DEPC water (volume ratio 13⁄4'). The RNA quality was detected by agarose gel electrophoresis at a mass ratio of 1.7, and the concentration and purity of total IA were measured by spectrophotometer. ;

2) Total c.DNA was synthesized into 2 μg of each RNA sample, and 50 μmol of L-Oligo dT18 was added at a volume ratio of 1%. DEPC water supplemented with 10 μL, 70 water bath 501 11, placed on ice for 5 min, then added ase inhibitor 0.5 μL and 5xRT3⁄4uffer 5uL, 10 mM dNTP 2.5 μL, M chaotic V reverse transcriptase Ι μί, volume ratio 1 %. DEPC water up to 25 μ L, after the 42 ° C water bath for 60 mi _n, 70 ° C water bath for 10 min the reaction was terminated (Oligo dT18 synthesized by Nanjing Jin Sirui Company; reverse transcription kit purchased from Fe'rmentas Corporation, Canada) ₀

3) Semi-quantitative PCR Reverse transcription synthesis of the first strand of total cDNA, followed by PCR amplification. The PCR reaction system is 20μ1, including 10 pmol L - ¹ positive and reverse primers each lu L, 10s PCR buffer 2 μ L, 2.5 mM dNTP 1.6 μ L, Taq enzyme 0, 4 L, and then supplemented with sterilized water 20 ti L (The primers were synthesized by Nanjing Jinsirui Company; the PCR reagents were purchased from Takara Company, Dalian). The amount of template added varies according to its concentration, and is corrected by the amount of cytoskeletal protein gene (O ci ) of the internal reference gene rice. The PCR program of the gene Octin is as follows: pre-denaturation at 94 °C for 4 min, denaturation at 94 °C for 30 s. The renaturation was carried out at 55 °C for 30 s, at 72 °C for 30 s, after 30 cycles, and at 72 °C for 7 min, the amplified PCR products were detected by mass ratio 1.5% agarose gel electrophoresis. After staining with ABC (EB), imaging was performed in a gel imaging system. The serial number and primer design of the gene are as follows:

 *; References, Xiaorong Fan, Li jun Jia, Yilin Li, Susan J. Smith, Anthony J Miller and Qirong Shen, 2007 Comparing nitrate storage and demobilization— in two rice ciiltivars that differ in their nitrogen use efficiency, Journal of Experimental Botany 58 (7) : 1729-40

 By expressing the gene in the non-serving part of the rice (leaf, root), it was found that £®2 was expressed in roots and leaves, and the expression in leaves was higher than that in roots (Fig. 1). Example 2 Overexpressing plants of the £3⁄4 £SS2 gene

 1) Extraction of total RNA Same as Example 1;

 2) total c. DNA synthesis as in Example 1;

3) Obtaining the full-length cDNA of the 0sLSD2 gene PCR primers were designed using the total cDNA of rice cultivar Wuyujing 7 obtained above as a template. The PCR product contained the complete 0sLSD2 reading frame (from the start codon ATG to the 3' non-coding region). The primer sequence was:

0verLSD2-F _: 5, - TTGAGGATCCGTGCCATTTACACCTC- 3 ' (SEQ ID NO. 5, containing BaniH I restriction site) 0verLSD2- _: 5, - ATATGGT 'ACCACAGACCTTGCGCCAT-3, (SEQ ID NO. 6, containing Kpn: I enzyme Cut site) PCR 20 system: 2 μL 2, 5 mM dNTP, 2 uL l Ox PGR buffer , lu L 0verLSD2-F, 1 μL 0verLSD2-R, 1 u L cDNA, 13 μL dd3⁄40.

The PCR procedure was as follows: pre-denaturation at 94 °C for 4 min, denaturation at 98 °C for 10 s, refolding at 68 °C for 2 min, after 3Q cycles, 72 V for 10 min, the amplified PCR product passed the mass ratio of 1% agarose. Gel electrophoresis detection, the size of the 822 bp fragment, the target PCR product was separated by agarose electrophoresis and then recovered by gelation. The recovered fragment was ligated to the pMD 19 vector. The total volume of the enzyme system was 10 UL, including 5 μL of the ligation solution. , 1 μL of ρ - - 19 vector, 3- 4 μL of PCR purified product, supplemented with water _: ΙΟ μ ί , then ligated overnight at 16 ° C; then transferred to E. coli DH5 a competent cells coated with After growing for 12 h to 14 h on LB solid medium of 100 μg mL 1 , the positive colonies were picked for DNA sequencing. The 0sLSD2 gene accession number was AK111759. The full-length cDNA sequence of 0sLSD2 included open reading frame (0RF) 552 bp. And the 3'-end non-coding region UTR 270 bp; the correct bacterial solution was added to an equal volume volume of 30% glycerol and stored at -70 备用, and a recombinant plasmid containing the full-length sequence of the target gene 0sLSD2 cDNA was obtained, which was named 0LSD2-T. ;

 4) Construction of overexpression vector pUbi-LSD2

Using the 0LSD2-T plasmid obtained above as a template, 0verLSD2-F (SEQ ID NO. 5) and C) verLSD2-R (SEQ ID NO. 6) as primers, PCR amplification of the siS 2 gene fragment, the PCR procedure is as follows: Pre-denaturation at 94 °C: 4 min, denaturation at 98 °C for 10 s, 68 renaturation for 2 min, after 30 cycles, 72 "C for 10 min, amplified PCR products were detected by mass ratio 1% agarose gel electrophoresis The size of the PCR product was 822 bp. The target PCR product was separated by agarose electrophoresis and then recovered by gel digestion. The recovered product was digested with restriction endonucleases Ba HI and Kpn I, and the double-excision plant overexpression vector PCAMBIA13Q0 ( The plasmid was purchased from Biovector Science Lab Co., and the digested PCR fragment and vector were separately recovered, and the vector was dephosphorylated and recovered again. The linearized vector and the enzyme-cut PCR were recovered by T4 ligase after recovery. The fragment was ligated overnight at 4 °C, transformed into DH5 _a competent cells of Bacillus megaterium, and plated on LB solid medium containing kanamycin 50 and gmL-l for 12 h, then picked positive colonies and extracted. The plasmid was digested with BamH I and Kpn I to verify the size of the fragment, and the strain was The liquid was subjected to DM sequencing, and the bacterial liquid containing the correctly cloned clone was added to an equal volume volume of 30% glycerol and stored at -70 ,, and the positive cloned plasmid was named PUM-LSD2;

Finally, the _P Ubi-LSD2 plasmid was transformed into competent cells of Agrobacterium tumefaciens EHA105 by electroporation, and plated on YEP solid medium containing kanamycin and streptomycin both at 50 μg mL- ¹ for 48 h. After that, pick positive colonies and extract the plasmid. After the B _a .nfl l, Kpn l digestion was verified, the bacterial solution was added to an equal volume volume ratio of 30 ^ΰ . Glycerol was stored at - 70 'C, and the transgene was spared.

 5) Acquisition of transgenic plants

 The Agrobacterium transformed with the pUbi LSD2 plasmid obtained above was infested with the callus of Wuyujing 7 and co-cultured for 60 days. After selection, culture, differentiation, rooting and smelting, the transgenic plants were obtained.

 5, 1) Reagents and solution abbreviations

 The abbreviations used in the medium used in the present invention are as follows: 6-BA (6-benzyl adenine); Car (penicillin); NAA (naphthaleneacetic acid); IAA (indoleacetic acid); 2, 4 -D (2,4-dichlorophenoxyacetic acid); AS (acetosyringone); CH (hydrolyzed casein); L-pro (L-valine); L-Glu (L-glutamine); MES (2-morpholineethanesulfonic acid); N6 (N6 mass element solution); B5 (BS trace element solution); AA (AA mass element); Agar (agar).

 5.2) Solution and medium formulation

 1) N& medium mother liquor per liter (2Q times):

KN0 ₃ 56.6 g

CaCl ₂ · 2H ₂ 0 3.32 g (corresponding to CaCl ₂ 2.506 g)

MgSOi · 7H ₂ 0 2.70 g

KH ₂ P0 ₄ 8.0 g

(leg ₄ ) ₂ S0 ₄ 9.26 g

2) B5 micro mother liquor per liter content (100 times

 Κτ 0750 g

H ₃ B0 ₃ 30 g

MnS0 ₄ · 3⁄40 0 g

ZnS0 ₄ · 73⁄40 2 g

Na ₂ Mo0 ₄ · 2H ₂ 0 025 g

CuS0 ₄ · 5H ₂ 0 0025 g

CoCL · 63⁄40 0025 g

Niacin 1 mg ml Pyridoxine hydrochloride (VB6) 1 mg/ml

 Thiamine hydrochloride (VBl) 10 mg/ml

 Inositol 10 mg/ml

4) Iron salt (100 times) ::

FeS0 ₄ · 7H ₂ 0 2. 78 g

Na ₂ EDTA · 2H ₂ 0 3. 73 g

5) AA large amount of mother liquor (per liter content);

 KC1 2. 95 g

MgS0 ₄ · 7H ₂ 0 0. 25 g

NaH ₂ P0 ₄ · 23⁄40 0. 15 g

6) Induced callus and subculture medium (per liter content

A large number of elements: 50 ml Bo trace 10 ml iron salt: — 10 ml niacin: 1 ml pyridoxine hydrochloride: 1 ml thiamine hydrochloride: 1 ml inositol: 10 ml L-pro; 2. 8 g sucrose: 30 g CH: 0. 3 g 2, 4-D : 8 ml Phytagel (si gma) : 4, 0 g pH; 5. 8

7) Co-cultivation medium of rice callus and Agrobacterium (per liter content):

 N6 macro element.; 50 ml B5 trace.; 10 ml iron salt: 10 ml niacin: 1 ml pyridoxine hydrochloride: I ml thiamine hydrochloride: 1 ml inositol: 2 g MES: 3. 9 g sucrose 30 g

 Phyta.gel

CH : 0:5 g 4. 0 g pH: 5. 5 Adding As to a final concentration of 200 μM at 55 °C 8) Resistant callus selection medium (per liter content):

N6 large element; 50 ml B5 trace _: 10 ml iron salt t 10 ml niacin: 1 ml pyridoxine hydrochloride: 1 ml thiamine hydrochloride: 1 ml inositol: 10 ml L-Glu _: 0. 5 g L- Pro : 0. 5 g CH : 0. 3 g 2, 4-D : 8 ml maltose./sucrose 30 g Phytagel : 4. 0 g pH: 5. 8

After sterilization (55 Γ) plus _:

 Car (Carbenicillin) 250 mg L

 First round of selection - Hgy (hygromycin) 50 mg / L

 Car (Carbenicillin) 250 mg/L

 Second round of selection - Hgy (hygromycin) 80 mg / L

 Car (Carbenicillin) 250 mg/L

 Round 3 selection - Hgy (hygromycin) 80 mg/L

9) Differentiation medium formula (per liter content) - Large amount of elements; 50 ml B5 trace.; Iron salt: 10 ml Niacin: 1 ml Pyridoxine hydrochloride: Acid thiamine: I ml Inositol: 10 ml L- Glu: 丄pro : 0. 5 g

CH : 0. 3 g 6-BA : MA: 0. 5 ml Cane Sugar: 30 g Agar: 8 g ρΗ Value: 5. 8

10) Rooting medium formula (per liter content);

 Ν6 A large number of elements: 25 ml B5 Trace: 5 ml Iron salt: 5 ml Niacin: 0. 5 ml Pyridoxine hydrochloride: 0. 5 ml Thiamine hydrochloride: 0. 5 ml Inositol: 5 ml Sucrose - 20 g Agar: 8. 0 g

 pH 5. 8

11) Medium formula (AAM) of suspension of Agrobacterium-infected callus mass (per liter content) _: AA A large number of elements: 100 ml B5 Trace: 10 ml Iron salt: 10 ml Niacin: 1 ml Pyridoxine hydrochloride: 1 ml Thiamine hydrochloride: 1 ml Inositol: 10 ml MES: 3. 9 g CH : 0. 5 g maltose - 30 g pH: 5. 5

 Add As to a final concentration of 200 μ at 55 °C

 -,,-,,-,,-,,,,One,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ,,.

12) YEP liquid medium formulation for Agrobacterium growth (per liter content);

 Yeast extract 10 g

 Peptone 10 g

 aC l 5 g

 pH 7. 0

5. 3) Agrobacterium-mediated transformation of rice

 Induction of callus: Peeled rice seeds (14 tablets in one plate) into a triangular flask, soaked for 1 min in volume with 70% ethanol (submerged seeds), poured out 70% ethanol by volume, soaked with 30% by volume of sodium hypochlorite Min, then wash 5-6 times with sterile water until clear. Use a pair of tweezers to place the seeds on the sterilized filter paper, blot the water, and finally place the Wuyujing 7 seed on the induction medium and incubate in a 30 °C light incubator for 20-30 days.

 Subculture: Select the yellow, tough, detached callus of millet size and transfer to a subculture medium for 7-14 days with sterile forceps.

Preparation of Agrobacterium: 20 μL of Agrobacterium EM105 stock solution transformed with pUbi-LSD2 plasmid was inoculated into 5 ml of YEP containing 50 mg of L- ¹ streptomycin and SO mgL- ¹ kanamycin ( Sambrook , et al . Molecular Cloning Experimental Guide. SOOl) In a liquid medium, shake at 28 °C overnight. 5。 The activated bacteria liquid 500 L, inoculated in 5 ml of the same YEP medium containing the same antibiotics, continue to culture until the absorbance of the bacterial solution at a wavelength of 600 nm (0D600) 0. 8-1.

Infecting callus and co-cultivation: The cotton callus of Wuyujing No. 7 was picked from the subculture medium and placed in a centrifuge tube. The amount of callus was less than 50 ml of the cone of the centrifuge tube. (Choose a light yellow rounded tough callus). Take the cultured Agrobacterium liquid solution. ml in a centrifuge tube at 4 V, 5000 rpm, centrifuge for 1 min, and remove the supernatant. The collected cells were suspended in a medium (AAM) containing 30 ml of suspension of Agrobacterium tumefaciens containing 200 mol of L- ¹ acetosyringone (As), and the suspension was poured into the picked callus. In, infested 5 mi n. The liquid was drained, the callus was removed, and placed on a sterile petri dish containing absorbent paper for 30-40 min. The callus was placed on a co-cultivation medium (top layer of 9 c sterile filter paper) and incubated at 25 ° C for 60 hours. Washing bacteria and antibiotic screening culture: The callus was taken out from the co-culture medium, and cleared with sterile water for 5 times, shaking for 5 min each time. Soak for 40 60 min in sterile water containing 500 mg L ^_1 carbenicillin (car). Finally, it was placed on sterile filter paper and drained for 2 h. First round of screening: Transfer the dried callus to a selection medium containing 250 mg L, ¹ carbenicillin (car) and 50 rng L' ¹ hygromycin (Hyg) for the first selection, 30 V , light culture 14 _d; a second round of screening: an initial long-resistant callus callus to 250 mgL- ¹ containing carbenicillin (CAR) and 80 mgL- ¹ hygromycin (Hyg) selection On the medium, 30. C, light culture for 10 days and then transferred to the tissue culture room for 4 days.

 Induced differentiation and rooting of resistant callus: The resistant callus of fresh yellow color is picked into the differentiation tank containing the differentiation medium, placed in a constant temperature culture chamber, and then differentiated into seedlings (about 30 days, The culture conditions of the tissue culture chamber were 24-30 V, 14 h light/8 h dark), and the seedlings were grown to a root length of about 5 cm.

 Breeding and transplanting of transgenic seedlings: Pick out the tube with the better roots and stems and leaves. (The seedling grows to the top of the tube, it should be covered in time), open the sealing membrane, add appropriate amount of sterile water (prevent the medium length Bacteria), refining the seedlings for about 3 to 7 days, then washing off the agar, transplanting it to the greenhouse for hydroponic or soil culture growth and testing.

 5. 4) Rapid detection of transgenic seedlings by hygromycin to obtain TO-generation plants

 Cut and collect fresh green leaves about 1 cm long to be detected (with incisions at both ends), placed flat on hygromycin (80 mgL-. medium, 30 V, 16 h/8 h (light) Positive/dark) positive leaves were maintained in fresh leaves for 48 h, while block necrosis appeared in leaves of negative seedlings (Zheng Yi. Establishment and application of high-efficiency transgenic system in rice. 2008). Positive by hygromycin screening TO plant 26 strains. From May to November 2010, the Nanjing University of Agriculture University archway Wenwang room planted T1 generation seeds for planting over-expressed materials. From December 2010 to May 2011, T2 generation materials were used for plot production experiments. .

 5. 5) Molecular identification of 0sLSD2 overexpressing lines

 Total RA and reverse transcription total cDNA of different lines of transgenic materials were extracted and semi-quantitatively identified (total RNA extraction, total cDNA synthesis, semi-quantitative PCR method as in Example 1), and 0-over-expressing effect was obtained. 1, 0-2, 0-3, 0-4 transgenic lines (Figure 2). Southern copy number identification was performed on several phenotypes of TO generation. The results are shown in Figure 3.

 5. 6) Expansion and physiological measurement of overexpressing lines

 As can be seen from Fig. 4, the OSLSD2 overexpression material (04) increased the plant height and the number of tillers compared with the wild type (Wuyu 粳 7). Example 3 Completion of root paraffin sections

The wild-type (Wuyu粳7) and OSLSD2 over-expressing material (0-4) seedling roots (ie, the first roots after seed germination) were selected for 5-10 days, and the root tips were 1-1. 5 cm, 1. 5-2 c two roots, paraffin sections were prepared, and the aerenchyma of the roots was observed under a microscope. The results are shown in Fig. 5. The OSLSD2 overexpression material can be seen from Fig. 5. (0-4) The root aeration tissue is developed compared with the wild type (Wuyu粳7). Example ⁴ Acquisition of Yield Data

 The experimental site is Ledong County, Hainan Province. The time is from December 2010 to May 2011. The experimental materials are Wuyujing 7 wild type and 0SLSD2 T2 generation genetically modified material. The specific experimental implementation process is as follows:

 1) germination; blisters during the day, rinse with water, air dry at night; blisters the next day, rinse with water, warm up at night.

 2) Sowing: germination After 1 day, the seeds are partially exposed and then sown. Do not flood, do not flood after germination, until 1 leaf 1 heart period (sow 10 days).

3) Fertilizer application: 1 leaf 1 heart fertilization on the trampoline 5 kg urea / mu; after 5-6 days tiller, compound fertilizer 8 kg ₊ 7 kg urea / mu; 3 days before the seedlings urea 15 kg / mu;

 4) Transplanting and fertilizing

 On January 25th, transplanting rice, transplanting '2-3 days base fertilizer 60 kg compound fertilizer/mu, February 1 application day additional herbicide and returning green fertilizer nitrogen fertilizer 15 kg urea per mu, February 7 additional tiller fertilizer nitrogen fertilizer 15 kg urea Per acre.

 Note: Planting plots and density

10 plants were multiplied by 6 rows, the plant spacing was 15 on, the row spacing was 20 cm, and harvested on April 30. The plants were harvested, and the yield per plant was calculated after drying. The results are shown in Tables 1 and 2. It can be seen from Table 2 that the 0 _S LSD2 overexpression material increased by 30-60% compared with the wild type, and the nitrogen uptake was significantly higher than that of the wild type.

 Table 1. Differences between 0sLSD2 overexpression material (0-4) and wild type phenotype

表 2. 0sLSD2超表达材料产量与野生型的差异

Table 2. Differences in yield and wild type of 0sLSD2 overexpression material

Claims

Claim  1. The key gene for the formation of rice-controlled aerated tissue OSLSD2 is used to increase plant height and increase plant nitrogen use efficiency and/or yield. The sequence accession number of this gene is AK111759.  2. Use according to claim 1, characterized in that the plant is a monocot.  3. Use according to claim 2, characterized in that the plant is rice, maize or wheat, preferably rice. 4. The rice-controlled aerenchyma formed by the key gene OSLSD2 encodes a rice aerenchyma to form a key protein 0sLSD2 in increasing plant height and increasing plant nitrogen use efficiency and/or yield.