CN111172179B

CN111172179B - Ubiquitin ligase gene OsNLA2, protein and application thereof in rice breeding

Info

Publication number: CN111172179B
Application number: CN202010062965.9A
Authority: CN
Inventors: 方中明; 王高华; 段芳
Original assignee: Wuhan Aidijing Biotechnology Co ltd
Current assignee: Wuhan Aidijing Biotechnology Co ltd
Priority date: 2020-01-19
Filing date: 2020-01-19
Publication date: 2020-09-08
Anticipated expiration: 2040-01-19
Also published as: CN111172179A

Abstract

The invention discloses a ubiquitin ligase gene OsNLA2, a protein and application thereof in rice breeding. The amino acid sequence of the OsNLA2 gene coding protein is shown as SEQ ID NO.1, and the cDNA sequence is shown as SEQ ID NO. 2. According to the invention, through constructing rice OsNLA2 gene overexpression plants and OsNLA2 gene mutant plants, the fact that the grain size and thousand grain weight of normal rice can be increased by improving OsNLA2 gene expression, the height of the rice under low nitrogen can also be increased, and the number and weight of normal single rice can be increased by knocking out OsNLA2 gene expression. The OsNLA2 gene can be used for rice breeding to improve rice grain type, thousand grain weight and rice plant height in a low-nitrogen environment; and can also be used in rice breeding to improve rice yield. The OsNLA2 gene has important application value in the aspects of plant seed size, weight, yield, plant height increase under low nitrogen and the like.

Description

Ubiquitin ligase gene OsNLA2, protein and application thereof in rice breeding

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to a ubiquitin ligase gene OsNLA2, a protein and application thereof in rice breeding.

Background

Ubiquitination is a type of post-translational modification of proteins that is involved in many aspects of plant stress and growth development. The ubiquitination process involves ubiquitin ligase, ubiquitin activating enzyme and ubiquitin conjugating enzyme. E3ubiquitin ligase plays a very important role in the ubiquitination pathway, which is responsible for the specific recognition of protein substrates and the recruitment and translocation of 76 amino acid ubiquitin molecules to lysine residues of protein substrates (Desheies R J, Joazeiro C. RING domain E3ubiquitin ligands [ J ]. Annual Review of Biochemistry,2009,78(1): 399-434.). E3ubiquitin ligase determines the specificity of ubiquitination substrates, of which RING type plays an important role in plants against abiotic stress.

Some RING-type E3ubiquitin ligases related to plant drought stress have been cloned and identified (Dreher K, Callis J. ubiquitin, hormons and biological stress in plants [ J]Annals of botanic, 2007,99(5): 787-. CHYR1 is a RING-type E3Ubiquitin ligase cloned from Arabidopsis thaliana, and the gene expression is significantly up-regulated under ABA-induced and drought stress conditions (Ding S, Zhang B, Qin F. Arabidopsis RZFP34/CHYR1, a Ubiquitin E3 ligand, regulation storage expression and gravity tolerancevia SnRK2.6-mediated phosphorylation [ J]Plant Cell,2015,27(11): 3228). In addition, ubiquitinLigase DRIP1 and DRIP2 as negative regulators regulate drought stress response process of Arabidopsis (functional study of Arabidopsis DRIP1 interacting protein DIF1/2 [ D)]University of chinese academy of sciences, 2014). The drought tolerance of plants is obviously improved when RING type E3ubiquitin ligase Rmal H1 in pepper is subjected to heterologous overexpression in Arabidopsis thaliana, and Rmal H1 is proved to be a positive regulator of plant drought stress response (Hanbay T, Shibasaka M, Hayashi Y, et al. overexpression Soft-shelled Aquaporin HvPIP 2; 1increa internal CO₂conductance and CO₂assimilation in the leaves of transgenic rice plants[J].Plant&CellPhysiology,2004,45(5): 521-. OsRD-CP1 is a homologous gene of Rmal H1 in rice, positively regulates the rice drought stress response process, but the specific mechanism is still unclear (the function analysis of Arabidopsis F-box gene AtPP2-B11 and the family analysis of apple RING type ubiquitin ligase E3 [ D)]Shandong university of agriculture, 2011).

Some RING type E3ubiquitin ligases of plants affect the resistance of plants to abiotic stress, also by regulating ABA synthesis. The carboxyl end of the AIRP4 protein contains a RING structural domain of C3HC4, the ABA content is increased in AIRP4 overexpression plants, the AIRP4 gene mutation affects root extension and pore closing, the drought-induced marker gene expression quantity in the overexpression plants is also obviously higher than that of wild type and atairp4 mutants, and the AIRP4 positively regulates drought-resistant reaction and negatively regulates salt-resistant response processes by affecting the quantity of endogenous ABA (Wang H, Lu Y, Jiang T, et al. the Arabidopsis U-box/ARM repeat E3 mutant AtPUB4 inhibits growth and generation of target cells and quality polypeptides to control stability [ J ]. Plant Journal,2013,74(3): 523). Overexpression of the RING-type E3 ligase gene PeRH2 of Populus euphratica promotes ABA biosynthesis and enhances drought resistance of transgenic tobacco (Zhang H, Cui F, Wu Y, et al. the RING finger ubiquitin E3 strain SDIR1 targetsSDIR1-INTERACTING PROTEIN1 for deletion of the saline and ABA signaling in Arabidopsis [ J ]. Plant Cell 2015,27(1): 214-.

The NLA gene is one of E3ubiquitin ligases, and experimental results in rice show that OsNLA1 negatively regulates Pi accumulation in rice (Granato T C, Rapid C D. promotion of mail (Zea mays L.) roots involved to localized of rice [ J ] Exp Bot,1989,40: 263-plus 275.). NLA also mediates the response of low nitrogen stress in Arabidopsis (miR827-NLA-NRT1.7 pathway regulates the mechanism of Arabidopsis nitrate nitrogen from source to sink [ D ] Acad Sci of the national academy of agricultural sciences, 2016.). The results show that the E3ubiquitin ligase NLA has large influence on plant stress and phosphorus balance, but no research is carried out on the growth and development of rice seeds and plants at present.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides a ubiquitin ligase gene OsNLA2, a protein and application thereof in rice breeding. The invention discovers that the OsNLA2 gene in rice has important effects on the size, the thousand seed weight and the quantity of rice seeds and the plant height of the rice, the overexpression can increase the size and the thousand seed weight of the rice seeds, the plant height of the rice can be increased under low nitrogen, and the gene knockout can increase the quantity and the weight of the single-plant seeds of the rice. Therefore, the gene can be applied to different aspects of genetic improvement of rice seeds and plant heights based on overexpression or gene knockout.

The first purpose of the invention is to provide a ubiquitin ligase gene OsNLA 2.

The second purpose of the invention is to provide a protein coded by ubiquitin ligase gene OsNLA 2.

The third purpose of the invention is to provide the application of the ubiquitin ligase gene OsNLA2 in rice breeding.

The purpose of the invention is realized by the following technical scheme:

the invention takes ubiquitin ligase gene OsNLA2 as an object, and clones a cDNA sequence of OsNLA2 from rice Zhonghua 11. An OsNLA2 gene overexpression vector is constructed and is introduced into the middle flower 11, an OsNLA2 gene overexpression plant is obtained, the seed size and the thousand seed weight of the OsNLA2 gene overexpression plant are obviously improved compared with those of the middle flower 11, and the height of the overexpression plant is obviously increased compared with that of the middle flower 11 plant under low nitrogen and high nitrogen through water culture. The gene knockout vector of the OsNLA2 gene is constructed and is introduced into the middle flower 11, so that a gene knockout plant of the OsNLA2 gene is obtained, the number and the weight of seeds of a single rice plant are obviously improved compared with those of the middle flower 11, and meanwhile, the mutant plant is obviously reduced in height compared with the middle flower 11 under low nitrogen and high nitrogen through cultivation. These results indicate that by increasing the expression of OsNLA2 gene, the size and thousand seed weight of normal rice can be increased, thus improving the rice grain type and yield, and increasing the plant height of rice under low nitrogen. By knocking out OsNLA2 gene expression, the number of normal rice single plant seeds can be increased, and the rice single plant yield can be improved.

Based on the function of the OsNLA2 gene discovered by the invention, the gene can be used for rice breeding. The rice breeding is to increase the size and thousand seed weight of rice seeds, or increase the yield of a single rice plant, or increase the plant height of rice under low nitrogen. Specifically, the expression of the OsNLA2 gene can be improved through an overexpression technology, so that the size and the thousand seed weight of rice seeds are increased, the purpose of improving the rice grain type is achieved, the expression of the OsNLA2 gene is reduced through a gene knockout technology, the number of the rice single plant seeds is increased, and the purpose of improving the rice yield is achieved. The over-expression plant can also be subjected to water culture under low nitrogen to increase the plant height of rice.

The amino acid sequence of OsNLA2 protein coded by the OsNLA2 geneAs shown in SEQ ID NO. 1:

MKFGAIYEEYLREQQDKYLTKCSHVEYKRLKKVLKKCRVGRSLQEDCPNGDQQEGNNESP

DICKCNSCTLCDQMFFTELTKEASEIAGCFSSRVQRLLNLHVPSGFLRYIWRVRQCFIDD

QQIMVQEGRMLLNYVTMNAIAIRKILKKYDKIHGSVSGRDFKSKMQTDHIELLQSPWLIE

LGAFHLNCNSSDIDETVGFLKNEFFKNFSCDLTEARPLMTMAISETMKYEYSLTCPICLD

TLFNPYALSCGHLFCKGCACGAASVYIFQGVKSAPPEAKCPVCRSDGVFAHAVHMTELDL

LIKTRSKDYWRQRLREERNEMVKQSKEYWDSQAMLSMGI；

the cDNA sequence of the OsNLA2 gene is preferredAs shown in SEQ ID NO. 2:

atgaagttcggtgcaatatatgaagagtatcttcgggaacagcaagacaaatacctaaca

aagtgctcacatgtggagtacaaacgtctcaaaaaggtactgaagaaatgtcgagttggt

cgctcattgcaagaagactgccccaatggtgaccagcaggaggggaacaacgaatctcca

gatatttgcaaatgcaattcatgcacattgtgtgatcaaatgttctttacagaacttact

aaggaggcttcagaaatagctggctgtttcagctctagagtacaacgtctcctaaatctt

catgtcccttcaggatttctacgctatatttggcgtgtaaggcaatgtttcatagatgat

caacaaatcatggttcaagaaggcagaatgttacttaattatgtaaccatgaatgctatc

gctatccgtaaaattctgaagaagtatgacaaaatacatggttctgtcagtggtagagat

ttcaagagcaagatgcaaactgatcatattgaactgttgcagtccccttggctgatagaa

ctgggtgctttccatctaaactgcaatagttcagatattgatgaaactgtggggttcctt

aagaatgagttcttcaagaatttttcctgtgatttgaccgaagcacgaccactaatgact

atggctatttctgaaactatgaagtatgagtacagcctaacttgtccaatttgcttggat

actttgttcaacccatatgcacttagctgtggccatctcttttgcaaaggctgtgcttgt

ggagctgcttctgtgtacatctttcaaggtgttaagtctgcacctcctgaggcgaagtgt

cctgtatgccgatcggatggtgtctttgctcatgctgtgcatatgactgaacttgacttg

ctcatcaaaacaaggagcaaggattactggagacagagactgcgagaagagcggaatgag

Atggttaagcaatccaaagaatactgggactctcaggctatgctgtcaatgggaatttga。

it is understood that the amino acid sequence shown in SEQ ID NO.1 can be variously substituted, added and/or deleted by one or more amino acids by those skilled in the art to obtain an amino acid sequence with equivalent functions without affecting the activity of the OsNLA2 protein (i.e., without being in the active center of the protein). Therefore, the OsNLA2 protein also comprises a protein with equivalent activity obtained by substituting, replacing and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1.

Furthermore, it will be appreciated that, given the degeneracy of codons and the preference of codons for different species, one skilled in the art can use codons suitable for expression in a particular species as desired.

The invention has the following advantages and beneficial effects:

(1) after the overexpression of the OsNLA2 gene cloned by the invention, the size and thousand seed weight of rice seeds are increased, which shows that the OsNLA2 gene is obvious in improving the rice grain type, so that the rice grain type can be genetically improved by improving the expression of the OsNLA2 gene through a gene engineering technology.

(2) The invention can also reduce the expression of the OsNLA2 gene by a gene knockout technology, so that the number of seeds of a single rice plant is increased, and therefore, the variety improvement of the plant can also be carried out by combining a gene editing technology and molecular breeding.

(3) After the OsNLA2 gene is over-expressed, the plant height of rice under low nitrogen is increased, which shows that the OsNLA2 gene is very key for the rice to adapt to soil under low nitrogen, therefore, the rice can adapt to low nitrogen environment by improving the expression of the OsNLA2 gene through a gene engineering technology.

(3) Successful cloning of the OsNLA2 gene proves that the ubiquitin ligase gene not only plays a role in plant stress and stress, but also plays an important role in plant seed and plant growth and development, can enrich the cognition of plant ubiquitin ligase, and has great promotion effect on the genetic improvement of plant seed grain type, yield and plant strain height.

Drawings

FIG. 1 is a histogram of the expression levels of flower 11(ZH11), OsNLA2 gene overexpression plants of 3 lines (OE1, OE2 and OE3) in the control;

in the figure: data were analyzed for variables (ANOVA) using SPSS software, at a 0.05 level using Duncan' s Analysis of significance of difference, lower case letters indicate significant difference between different groups.

FIG. 2 is a sequence alignment of 3 lines (C1, C2 and C3) of T1 generation mutant plants of OsNLA2 gene against control wild type middle flower 11(WT) sequence;

in the figure: 3 strains (C1, C2 and C3) are all homozygous mutants, 5bp and 12bp of the two strains C1 and C2 are respectively deleted, C3A line was increased by 1 bp.

FIG. 3 is a phenotypic picture of the composition of 30 rice grains of different materials of OsNLA2 gene;

in the figure: the control flower 11(ZH11), the over-expression plant 3 strains (OE1, OE2 and OE3), and the mutation are sequentially arranged in sequence The somatic plants are 3 lines (C1, C2 and C3).

FIG. 4 is a thousand-grain weight bar graph of different materials of OsNLA2 gene.

In the figure: the control flower 11(ZH11), the over-expression plant 3 strains (OE1, OE2 and OE3), and the mutation are sequentially arranged in sequence The somatic plants are 3 lines (C1, C2 and C3). Data were analyzed for variables (ANOVA) using SPSS software using Duncan's software Differential significance analysis was performed at the 0.05 level, with lower case letters indicating significant differences between the different groups.

FIG. 5 is a rice phenotype map of individual rice plants of different materials of OsNLA2 gene.

FIG. 6 is a bar graph of the weight of each seed of different materials of OsNLA2 gene.

FIG. 7 is a table diagram of individual rice plantlet phenotype of different materials of OsNLA2 gene under low nitrogen and high nitrogen.

In the figure: the control flower 11(ZH11), the over-expression plant 3 strains (OE1, OE2 and OE3), and the mutation are sequentially arranged in sequence The somatic plants are 3 lines (C1, C2 and C3). LN was 0.5mM ammonium nitrate low nitrogen treatment and HN was 2.0mM ammonium nitrate high nitrogen treatment.

FIG. 8 is a histogram of plant heights of individual rice plantlets under low nitrogen and high nitrogen for different materials of OsNLA2 gene.

In the figure: the sequence is that the control flower 11(ZH11),3 strains of over-expression plants (OE1, OE2 and OE3), mutation The somatic plants are 3 lines (C1, C2 and C3). LN was 0.5mM ammonium nitrate low nitrogen treatment and HN was 2.0mM ammonium nitrate high nitrogen treatment. Data of Analysis of variables (ANOVA) with SPSS software, significance of differences at 0.05 level using Duncan's, not The same group asterisk indicates a significant difference compared to flowers 11 in the respective control of the different treatments.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the experimental procedures used are conventional and can be carried out according to recombinant techniques already described (see molecular cloning, A laboratory Manual, 2 nd edition, Cold spring harbor laboratory Press, Cold spring harbor, N.Y.; Ma X et al, A robust CRISPR/Cas9 system for containment, high-efficiency multiplex genome editing in monoclonal and important plants. mol plant 2015,8(8): 1274. C. 1284.); the materials, reagents and the like used are all commercially available.

Example 1 construction of OsNLA2 Gene overexpression plants

Extracting RNA of rice middle flower 11, reversely transcribing the RNA into cDNA, and performing primer pair:

F3：5'-atggtaccatgaagttcggtgcaatatatgaa-3'(KpnI)，SEQ ID NO.3；

R3：5'-atggatccaattcccattgacagcatagcctg-3'(BamHI)，SEQ ID NO.4；

after amplifying cDNA of OsNLA2 gene by PCR, a overexpression vector OsNLA2-p1306 of OsNLA2 gene was constructed by ligating KpnI and BamHI into pCAMBIA-1306 vector (pCAMBIA-1306 vector is purchased from Cambia corporation). the overexpression vector was introduced into flower 11 of normal rice variety by Agrobacterium EHA 105-mediated genetic transformation.

Transplanting all the obtained transgenic seedlings into a basket with soil, watering and fertilizing at regular intervals, planting the seedlings in a field when the seedlings grow to be about 10cm in height, extracting genome DNA (deoxyribonucleic acid) and detecting transgenic plants through PCR (polymerase chain reaction), wherein a detection primer pair is as follows:

F4：5'-gatgttggcgacctcgtatt-3'，SEQ ID NO.5；

R4：5'-tcgttatgtttatcggcacttt-3'，SEQ ID NO.6；

if 517bp fragments are amplified, the transgenic plants are positive plants. And (4) harvesting and planting a single positive plant until a homozygous transgenic plant is identified at the T2 generation, namely obtaining an OsNLA2 gene overexpression plant.

Taking OsNLA2 gene over-expression plant leaves, extracting RNA and carrying out reverse transcription on the RNA to obtain cDNA, detecting the expression quantity of the OsNLA2 gene in an over-expression plant through real-time fluorescence quantitative PCR, wherein the result shows that (shown in figure 1) the expression quantity of the OsNLA2 gene in the over-expression plant is improved compared with that of a control flower 11, and if the expression quantity of the control is determined to be 1, the average value of the expression quantities of three strains of the over-expression plant is 141.226, 139.015 and 119.085 in sequence. Primer pairs for real-time fluorescent quantitative PCR:

F5：caaagtgctcacatgtggagt，SEQ ID NO.7；

R5：aacagccagctatttctgaagcct，SEQIDNO.8；

randomly taking 30 seeds from the three lines of the over-expression plants and the control flower 11, after pulling the shell by hand, arranging the head and the tail of the rice in a circle, finding that the size of the seeds of the over-expression plants is increased compared with that of the control flower 11 (figure 3), showing that the size of the rice grains of the over-expression plants is increased compared with that of the control flower 11 (figure 4), randomly taking 1000 seeds from the seeds with the shell to count thousand seed weight, and finding that the thousand seed weight of the over-expression plants is obviously increased compared with that of the control flower 11 (figure 4). Randomly selecting one of the three lines of the over-expression plants and the flower 11 in the control, arranging all rice seeds with shells into a circle, finding that the number of the seeds of the over-expression plants is smaller than that of the flower 11 in the control (figure 5), which shows that the number of the seeds of each over-expression plant is smaller than that of the flower 11 in the control (figure 6), and correspondingly weighing the seeds of each over-expression plant to find that the weight of the seeds of each over-expression plant is obviously smaller than that of the flower 11 in the control (figure 6).

Soaking the over-expression plant and the seeds of the middle flower 11 in distilled water for 3 days on a culture dish, culturing for 7 days, transferring to a rice nutrient solution for culturing, wherein the formula of the nutrient solution refers to the formula of international rice, but ammonium nitrate is adjusted to 0.5mM (low nitrogen) and 2mM (high nitrogen), culturing for 40 days respectively, observing the phenotype, and counting the plant height of the rice. As can be seen from FIG. 7, under low nitrogen culture, the plant height of the OsNLA2 gene overexpression plant is increased compared with that of the control flower 11 plant, and the difference is obvious. Whereas the difference is not significant at high nitrogen but at low nitrogen. Statistical data for plant heights of over-expressed plants and controls under different nitrogen cultures are shown in FIG. 8.

The results show that the OsNLA2 gene can increase the size and the thousand seed weight of rice seeds by increasing the expression quantity, thereby improving the rice grain type. The over-expression plant can also increase the height of the rice plant under low nitrogen.

Example 2 construction of OsNLA2 Gene mutant plants

Using a single target sequence:

F6：tgttaagtctgcacctcctgagg，SEQ ID NO.9；

using the single target sequence, a gene knockout vector OsNLA2-C of OsNLA2 gene was constructed (see Ma X et al, A robust CRISPR/Cas9 system for restriction, high-efficiency multiplex gene editing in monocot and dicot plants. mol plant.2015,8(8): 1274. sup. 1284). The gene knockout expression vector is introduced into the flower 11 of the normal japonica rice variety by adopting an agrobacterium EHA105 mediated genetic transformation method. Sequencing the mutant plant at the T0 generation, determining that three strains (shown in figure 2) are knocked out of the gene, continuously and independently breeding to the T1 generation, and obtaining the independent mutant plant strain of the OsNLA2 gene. The seed size of the OsNLA2 gene mutant plant is obviously smaller than that of a flower 11 plant in a control, the thousand seed weight is reduced, as shown in figures 3 and 4, a single plant collects seeds, the result shows that each grouted seed of the mutant plant is obviously increased, and the yield of each plant is increased, as shown in figures 5 and 6. Soaking the mutant plant and the seeds of the middle flower 11 in distilled water for 3 days on a culture dish, culturing for 7 days, transferring to a rice nutrient solution for culturing, wherein the formula of the nutrient solution refers to that of international rice, but ammonium nitrate is adjusted to 0.5mM (low nitrogen) and 2mM (high nitrogen), culturing for 40 days respectively, observing the phenotype, and counting the plant height of the rice. From fig. 7 and 8, it can be seen that under low-nitrogen and high-nitrogen culture, the plant height of the OsNLA2 gene mutant plant is reduced and reaches a significant difference compared with that of the control flower 11 plant.

The results show that the expression of the OsNLA2 gene is knocked out, so that the size of rice seeds can be reduced, the thousand seed weight can be reduced, the plant height of low-nitrogen and high-nitrogen rice plants can be reduced, and the number of the rice seeds and the weight of each plant can be increased by knocking out the gene.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> Wuhan Aidi crystal Biotech Co., Ltd

<120> ubiquitin ligase gene OsNLA2, protein and application thereof in rice breeding

<160>9

<170>SIPOSequenceListing 1.0

<210>1

<211>339

<212>PRT

<213> amino acid of OsNLA2 gene (OsNLA2)

<400>1

Met Lys Phe Gly Ala Ile Tyr Glu Glu Tyr Leu Arg Glu Gln Gln Asp

1 5 10 15

Lys Tyr Leu Thr Lys Cys Ser His Val Glu Tyr Lys Arg Leu Lys Lys

20 25 30

Val Leu Lys Lys Cys Arg Val Gly Arg Ser Leu Gln Glu Asp Cys Pro

35 4045

Asn Gly Asp Gln Gln Glu Gly Asn Asn Glu Ser Pro Asp Ile Cys Lys

50 55 60

Cys Asn Ser Cys Thr Leu Cys Asp Gln Met Phe Phe Thr Glu Leu Thr

65 70 75 80

Lys Glu Ala Ser Glu Ile Ala Gly Cys Phe Ser Ser Arg Val Gln Arg

85 90 95

Leu Leu Asn Leu His Val Pro Ser Gly Phe Leu Arg Tyr Ile Trp Arg

100 105 110

Val Arg Gln Cys Phe Ile Asp Asp Gln Gln Ile Met Val Gln Glu Gly

115 120 125

Arg Met Leu Leu Asn Tyr Val Thr Met Asn Ala Ile Ala Ile Arg Lys

130 135 140

Ile Leu Lys Lys Tyr Asp Lys Ile His Gly Ser Val Ser Gly Arg Asp

145 150 155 160

Phe Lys Ser Lys Met Gln Thr Asp His Ile Glu Leu Leu Gln Ser Pro

165 170 175

Trp Leu Ile Glu Leu Gly Ala Phe His Leu Asn Cys Asn Ser Ser Asp

180 185 190

Ile Asp Glu Thr Val Gly Phe Leu Lys Asn Glu Phe Phe Lys Asn Phe

195 200 205

Ser Cys Asp Leu Thr Glu Ala Arg Pro Leu Met Thr Met Ala Ile Ser

210 215 220

Glu Thr Met Lys Tyr Glu Tyr Ser Leu Thr Cys Pro Ile Cys Leu Asp

225 230 235 240

Thr Leu Phe Asn Pro Tyr Ala Leu Ser Cys Gly His Leu Phe Cys Lys

245 250 255

Gly Cys Ala Cys Gly Ala Ala Ser Val Tyr Ile Phe Gln Gly Val Lys

260 265 270

Ser Ala Pro Pro Glu Ala Lys Cys Pro Val Cys Arg Ser Asp Gly Val

275 280 285

Phe Ala His Ala Val His Met Thr Glu Leu Asp Leu Leu Ile Lys Thr

290 295 300

Arg Ser Lys Asp Tyr Trp Arg Gln Arg Leu Arg Glu Glu Arg Asn Glu

305 310 315 320

Met Val Lys Gln Ser Lys Glu Tyr Trp Asp Ser Gln Ala Met Leu Ser

325 330 335

Met Gly Ile

<210>2

<211>1020

<212>DNA

<213> cDNA of OsNLA2 Gene (OsNLA2)

<400>2

atgaagttcg gtgcaatata tgaagagtat cttcgggaac agcaagacaa atacctaaca 60

aagtgctcac atgtggagta caaacgtctc aaaaaggtac tgaagaaatg tcgagttggt 120

cgctcattgc aagaagactg ccccaatggt gaccagcagg aggggaacaa cgaatctcca 180

gatatttgca aatgcaattc atgcacattg tgtgatcaaa tgttctttac agaacttact 240

aaggaggctt cagaaatagc tggctgtttc agctctagag tacaacgtct cctaaatctt 300

catgtccctt caggatttct acgctatatt tggcgtgtaa ggcaatgttt catagatgat 360

caacaaatca tggttcaaga aggcagaatg ttacttaatt atgtaaccat gaatgctatc 420

gctatccgta aaattctgaa gaagtatgac aaaatacatg gttctgtcag tggtagagat 480

ttcaagagca agatgcaaac tgatcatatt gaactgttgc agtccccttg gctgatagaa 540

ctgggtgctt tccatctaaa ctgcaatagt tcagatattg atgaaactgt ggggttcctt 600

aagaatgagt tcttcaagaa tttttcctgt gatttgaccg aagcacgacc actaatgact 660

atggctattt ctgaaactat gaagtatgag tacagcctaa cttgtccaat ttgcttggat 720

actttgttca acccatatgc acttagctgt ggccatctct tttgcaaagg ctgtgcttgt 780

ggagctgctt ctgtgtacat ctttcaaggt gttaagtctg cacctcctga ggcgaagtgt 840

cctgtatgcc gatcggatgg tgtctttgct catgctgtgc atatgactga acttgacttg 900

ctcatcaaaa caaggagcaa ggattactgg agacagagac tgcgagaaga gcggaatgag 960

atggttaagc aatccaaaga atactgggac tctcaggcta tgctgtcaat gggaatttga 1020

<210>3

<211>32

<212>DNA

<213> primer F3(OsNLA2)

<400>3

atggtaccat gaagttcggt gcaatatatg aa 32

<210>4

<211>32

<212>DNA

<213> primer R3(OsNLA2)

<400>4

atggatccaa ttcccattga cagcatagcc tg 32

<210>5

<211>20

<212>DNA

<213> primer F4(OsNLA2)

<400>5

gatgttggcg acctcgtatt 20

<210>6

<211>22

<212>DNA

<213> primer R4(OsNLA2)

<400>6

tcgttatgtt tatcggcact tt 22

<210>7

<211>21

<212>DNA

<213> primer F5(OsNLA2)

<400>7

caaagtgctc acatgtggag t 21

<210>8

<211>24

<212>DNA

<213> primer R5(OsNLA2)

<400>8

aacagccagc tatttctgaa gcct 24

<210>9

<211>23

<212>DNA

<213> Single target F6(OsNLA2)

<400>9

tgttaagtct gcacctcctg agg 23

Claims

1. An application of ubiquitin ligase gene OsNLA2 in rice breeding is characterized in that:

the amino acid sequence of the OsNLA2 protein is shown in SEQ ID NO. 1;

the rice breeding method is characterized in that the rice grain size and thousand seed weight of rice are increased by improving OsNLA2 gene expression, and the plant height of the rice is improved under low nitrogen.

2. An application of ubiquitin ligase gene OsNLA2 in rice breeding is characterized in that:

the amino acid sequence of the OsNLA2 protein is shown in SEQ ID NO. 1;

the rice breeding method is characterized in that the quantity and the weight of rice of a single rice plant are increased by knocking out OsNLA2 gene expression.