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

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 its application in rice breeding

technical field

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

Background technique

Ubiquitination is a type of protein post-translational modification, which is involved in many aspects of plant stress and growth and development. The ubiquitination process involves ubiquitin ligases, ubiquitin-activating enzymes, and ubiquitin-conjugating enzymes. E3 ubiquitin ligase plays a very important role in the ubiquitination pathway, which is responsible for the specific recognition of protein substrates and the recruitment and transport of 76 amino acid ubiquitin molecules to the lysine residues of protein substrates ( Deshaies R J, Joazeiro C A. RING domain E3ubiquitin ligases [J]. Annual Review of Biochemistry, 2009, 78(1):399-434.). E3 ubiquitin ligases determine the specificity of ubiquitinated substrates, of which the RING type plays an important role in plant resistance to abiotic stresses.

At present, some RING-type E3 ubiquitin ligases related to plant drought stress have been cloned and identified (Dreher K, Callis J.Ubiquitin,hormones and biotic stress in plants[J].Annals of Botany,2007,99(5):787 -822.). CHYR1 is a RING-type E3 ubiquitin ligase cloned from Arabidopsis thaliana, and its expression was significantly up-regulated under ABA-induced and drought stress conditions (Ding S, Zhang B, Qin F. Arabidopsis RZFP34/CHYR1, a Ubiquitin E3 ligase, regulates stomatal movement and drought tolerance via SnRK2.6-mediated phosphorylation[J]. Plant Cell, 2015, 27(11):3228.). In addition, ubiquitin ligases DRIP1 and DRIP2 act as negative regulators to regulate the drought stress response process of Arabidopsis (Functional study of Arabidopsis DRIP1-interacting protein DIF1/2 [D]. University of Chinese Academy of Sciences, 2014.). The RING-type E3 ubiquitin ligase RmalH1 in pepper was significantly increased in plant drought tolerance when heterologously overexpressed in Arabidopsis thaliana, demonstrating that RmalH1 is a positive regulator of plant drought stress response (HanbaY T, Shibasaka M, Hayashi Y, et al. .Overexpression of the BarleyAquaporin HvPIP2; 1increases internal CO ₂ conductance and CO ₂ assimilation in the leaves of transgenic rice plants [J]. Plant & Cell Physiology, 2004, 45(5):521-529.). OsRD-CP1 is the homologous gene of RmalH1 in rice, which positively regulates the response of rice to drought stress, but its specific mechanism is still unclear (Functional analysis of Arabidopsis F-box gene AtPP2-B11 and apple RING-type pan- Family analysis of ligase E3 [D]. Shandong Agricultural University, 2011.).

Some RING-type E3 ubiquitin ligases in plants also affect plant resistance to abiotic stress by regulating ABA synthesis. The carboxyl terminus of AIRP4 protein contains the RING domain of C3HC4. In AIRP4-overexpressing plants, ABA content increases, while AIRP4 gene mutation affects root extension and stomatal closure. The expression levels of drought-induced marker genes in overexpressing plants are also significantly higher than those in wild-type and wild-type plants. atairp4 mutant, indicating that AIRP4 positively regulates the drought resistance response and negatively regulates the salt resistance response process by affecting the amount of endogenous ABA (Wang H, Lu Y, Jiang T, et al. The Arabidopsis U-box/ARM repeat E3 ligaseAtPUB4 influences growth and degeneration of tapetal cells, and its mutation leads to conditional male sterility [J]. Plant Journal, 2013, 74(3):511-523.). Overexpression of the RING-type E3 ligase gene PeRH2 in Populus euphratica promotes ABA biosynthesis and enhances the drought resistance of transgenic tobacco (Zhang H, Cui F, Wu Y, et al. The RING finger ubiquitin E3 ligase SDIR1 targets SDIR1-INTERACTING PROTEIN1 for degradation to modulate the salt stress response and ABA signaling in Arabidopsis [J]. Plant Cell, 2015, 27(1):214-227.).

NLA gene is a kind of E3 ubiquitin ligase, and experimental results in rice show that OsNLA1 negatively regulates Pi accumulation in rice (Granato T C, Raper C D.Proliferation ofmaize(Zea mays L.)roots inresponse to localized supply ofnitrate[J ]. J Exp Bot, 1989, 40: 263-275.). In Arabidopsis thaliana, NLA also mediates the response to low nitrogen stress (the mechanism of miR827-NLA-NRT1.7 pathway regulating nitrate nitrogen from source to sink in Arabidopsis [D]. Chinese Academy of Agricultural Sciences, 2016. ). The above results show that the E3 ubiquitin ligase NLA has a greater impact on plant stress and phosphorus balance, but there is no research on the growth and development of rice seeds and plants.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the problems existing in the prior art, and to provide a ubiquitin ligase gene OsNLA2, a protein and its application in rice breeding. The present invention finds that the OsNLA2 gene in rice has an important effect on rice seed size, 1000-grain weight and quantity, and rice plant height. Overexpression can increase rice seed size and 1000-grain weight, and can also increase rice plant height under low nitrogen. Increase the number and weight of seeds per plant in rice. Therefore, overexpression or gene knockout can be applied to different aspects of genetic improvement of rice seeds and plant height.

The first object of the present invention is to provide a ubiquitin ligase gene OsNLA2.

The second object of the present invention is to provide a protein encoded by the ubiquitin ligase gene OsNLA2.

The third object of the present invention is to provide an application of the above ubiquitin ligase gene OsNLA2 in rice breeding.

The object of the present invention is achieved through the following technical solutions:

The present invention takes the ubiquitin ligase gene OsNLA2 as the object, and clones the cDNA sequence of OsNLA2 from rice Zhonghua 11. By constructing an OsNLA2 gene overexpression vector, the overexpression vector was introduced into Zhonghua 11 to obtain OsNLA2 gene overexpression plants. Compared with Zhonghua 11, the seed size and 1000-grain weight were significantly increased. It was found that the height of overexpression plants was significantly higher than that of Zhonghua 11 plants under low nitrogen. By constructing a knockout vector of the OsNLA2 gene, the knockout vector was introduced into Zhonghua 11 to obtain a gene knockout plant of the OsNLA2 gene. Compared with Zhonghua 11, the number and weight of rice seeds per plant were significantly increased. Hydroponics under nitrogen and high nitrogen showed that the height of mutant plants was significantly lower than that of Zhonghua 11 plants under low nitrogen. These results suggest that normal rice seed size and 1000-grain weight can be increased by increasing OsNLA2 gene expression, thereby improving rice grain shape and yield, as well as increasing rice plant height under low nitrogen conditions. By knocking out OsNLA2 gene expression, the number of seeds per plant in normal rice can be increased, thereby increasing the yield per plant in rice.

Based on the function of the OsNLA2 gene found in the present invention, it can be used in rice breeding. The rice selection is to increase the seed size and 1000-grain weight of the rice, or to increase the yield per plant of the rice, or to increase the plant height of the rice under low nitrogen. Specifically, overexpression technology can be used to increase the expression of OsNLA2 gene, so as to increase rice seed size and 1000-grain weight, so as to achieve the purpose of improving rice grain shape. The purpose of increasing rice yield. Overexpressing plants can also increase rice plant height by hydroponics under low nitrogen.

The amino acid sequence of the OsNLA2 protein encoded by the OsNLA2 gene is shown in SEQ ID NO.1:

MKFGAIYEEYLREQQDKYLTKCSHVEYKRLKKVLKKCRVGRSLQEDCPNGDQQEGNNESP

DICKCNSCTLCDQMFFTELTKEASEIAGCFSSRVQRLLNLHVPSGFLRYIWRVRQCFIDD

QQIMVQEGRMLLNYVTMNAIAIRKILKKYDKIHGSVSGRDFKSKMQTDHIELLQSPWLIE

LGAFHLNCNSSDIDETVGFLKNEFFKNFSCDLTEARPLMTMAISETMKYEYSLTCPICLD

TLFNPYALSCGHLFCKGCACGAASVYIFQGVKSAPPEAKCPVCRSDGVFAHAVHMTELDL

LIKTRSKDYWRQRLREERNEMVKQSKEYWDSQAMLSMGI;

The cDNA sequence of the OsNLA2 gene is preferably as shown in SEQ ID NO.2:

atgaagttcggtgcaatatatgaagagtatcttcgggaacagcaagacaaatacctaaca

aagtgctcacatgtggagtacaaacgtctcaaaaaggtactgaagaaatgtcgagttggt

cgctcattgcaagaagactgccccaatggtgaccagcaggaggggaacaacgaatctcca

gatatttgcaaatgcaattcatgcacattgtgtgatcaaatgttctttacagaacttact

aaggaggcttcagaaatagctggctgtttcagctctagagtacaacgtctcctaaatctt

catgtcccttcaggatttctacgctatatttggcgtgtaaggcaatgtttcatagatgat

caacaaatcatggttcaagaaggcagaatgttacttaattatgtaaccatgaatgctatc

gctatccgtaaaattctgaagaagtatgacaaaatacatggttctgtcagtggtagagat

ttcaagagcaagatgcaaactgatcatattgaactgttgcagtccccttggctgatagaa

ctgggtgctttccatctaaactgcaatagttcagatattgatgaaactgtggggttcctt

aagaatgagttcttcaagaatttttcctgtgatttgaccgaagcacgaccactaatgact

atggctatttctgaaactatgaagtatgagtacagcctaacttgtccaatttgcttggat

actttgttcaacccatatgcacttagctgtggccatctcttttgcaaaggctgtgcttgt

ggagctgcttctgtgtacatctttcaaggtgttaagtctgcacctcctgaggcgaagtgt

cctgtatgccgatcggatggtgtctttgctcatgctgtgcatatgactgaacttgacttg

ctcatcaaaacaaggagcaaggattactggagacagagactgcgagaagagcggaatgag

Atggttaagcaatccaaagaatactgggactctcaggctatgctgtcaatgggaatttga.

It should be understood that under the premise of not affecting the activity of the OsNLA2 protein (that is, not in the active center of the protein), those skilled in the art can make various substitutions, additions and/or deletions to the amino acid sequence shown in SEQ ID NO. Several amino acids yield amino acid sequences with equivalent functions. Therefore, OsNLA2 protein also includes proteins with equivalent activity obtained by substituting, replacing and/or adding one or several amino acids to the amino acid sequence shown in SEQ ID NO.1.

In addition, it should be understood that, taking into account the degeneracy of codons and the codon preferences of different species, those skilled in the art can use codons suitable for expression in a particular species as needed.

The advantages and beneficial effects of the present invention are as follows:

(1) The overexpression of the cloned OsNLA2 gene in the present invention increases the rice seed size and thousand-grain weight, indicating that the OsNLA2 gene is more effective in improving the grain shape of rice. Therefore, improving the expression of the OsNLA2 gene through genetic engineering technology can genetically improve the grain shape of rice.

(2) The present invention can also reduce the expression of the OsNLA2 gene through gene knockout technology, so that the number of seeds per plant of rice can be increased. Therefore, the variety improvement of plants can also be carried out by combining gene editing technology and molecular breeding.

(3) The overexpression of the OsNLA2 gene of the present invention increases the plant height of rice under low nitrogen, indicating that the OsNLA2 gene is critical for rice to adapt to the soil under low nitrogen. Therefore, improving the expression of the OsNLA2 gene through genetic engineering technology can make rice adapt to low nitrogen surroundings.

(3) The successful cloning of the OsNLA2 gene confirmed that the ubiquitin ligase gene not only plays a role in plant adversity and stress, but also plays an important role in the growth and development of plant seeds and plants, which can enrich the knowledge of plant ubiquitin ligase, It has a great role in promoting the genetic improvement of plant seed grain shape and yield and plant height.

Description of drawings

Figure 1 is a statistical histogram of the expression levels of 3 lines (OE1, OE2 and OE3) of the control Zhonghua 11 (ZH11) and OsNLA2 gene overexpressing plants;

Figure: Data were analyzed by SPSS software for variable analysis (ANOVA), using Duncan's at the 0.05 level Significant difference analysis, lowercase letters indicate significant differences among different groups.

Figure 2 is the sequence alignment of the three lines (C1, C2 and C3) of the T1 generation of the mutant plant of the OsNLA2 gene to the sequence alignment of the control wild type Zhonghua 11 (WT);

In the figure: 3 lines (C1, C2 and C3) are all homozygous mutants, and the two lines C1 and C2 are missing 5bp and 12bp, respectively. One line of C3 increased by 1bp.

Figure 3 is a phenotype diagram composed of 30 rice grains of different materials of the OsNLA2 gene;

In the figure: the order is the control Zhonghua 11 (ZH11), the three lines of the overexpressed plants (OE1, OE2 and OE3), the mutant Three lines of somatic plants (C1, C2 and C3).

Figure 4 is a bar graph of thousand kernel weight of different materials of OsNLA2 gene.

In the figure: the order is the control Zhonghua 11 (ZH11), the three lines of the overexpressed plants (OE1, OE2 and OE3), the mutant Three lines of somatic plants (C1, C2 and C3). Data were analyzed by SPSS software for variable analysis (ANOVA) using Duncan's Significant differences were analyzed at the 0.05 level, and lowercase letters indicate significant differences among different groups.

Figure 5 is a rice phenotype diagram of a single rice plant of different materials of OsNLA2 gene.

In the figure: the order is the control Zhonghua 11 (ZH11), the three lines of the overexpressed plants (OE1, OE2 and OE3), the mutant Three lines of somatic plants (C1, C2 and C3).

Figure 6 is a bar graph of the seed weight per plant of different materials of the OsNLA2 gene.

In the figure: the order is the control Zhonghua 11 (ZH11), the three lines of the overexpressed plants (OE1, OE2 and OE3), the mutant Three lines of somatic plants (C1, C2 and C3). Data were analyzed by SPSS software for variable analysis (ANOVA) using Duncan's Significant differences were analyzed at the 0.05 level, and lowercase letters indicate significant differences among different groups.

Figure 7 is a phenotype diagram of a single rice seedling of different materials of OsNLA2 gene under low nitrogen and high nitrogen.

In the figure: the order is the control Zhonghua 11 (ZH11), the three lines of the overexpressed plants (OE1, OE2 and OE3), the mutant Three lines of somatic plants (C1, C2 and C3). LN was 0.5 mM ammonium nitrate low nitrogen treatment, HN was 2.0 mM ammonium nitrate high nitrogen treatment.

Figure 8 is a histogram of the plant height of a single rice seedling of different materials of the OsNLA2 gene under low nitrogen and high nitrogen.

In the figure: the order is the control Zhonghua 11 (ZH11), the three lines of the overexpressed plants (OE1, OE2 and OE3), the mutant Three lines of somatic plants (C1, C2 and C3). LN was 0.5 mM ammonium nitrate low nitrogen treatment, HN was 2.0 mM ammonium nitrate high nitrogen treatment. data SPSS software was used for analysis of variables (ANOVA), and Duncan's was used for significant difference analysis at the 0.05 level. Asterisks (*) in the same group indicate significant differences compared with the respective control Zhonghua 11 in different treatments.

Detailed ways

The present invention will be further described in detail below with reference to the 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 methods used are all conventional methods, and can be performed according to the described recombination techniques (see Molecular Cloning, Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Ma X et al, A robust CRISPR/Cas9 system forconvenient, high-efficiency multiplex genome editing inmonocot and dicotplants. Mol Plant. 2015, 8(8):1274- 1284.) is completed; the materials, reagents, etc. used can be obtained from commercial sources.

Example 1 Construction of OsNLA2 gene overexpression plants

The RNA of Hua 11 in rice was extracted and reverse transcribed into cDNA, using primer pairs:

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

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

After amplifying the cDNA of the OsNLA2 gene by PCR, the overexpression vector OsNLA2-p1306 of the OsNLA2 gene was constructed by (KpnI and BamHI were linked to the pCAMBIA-1306 vector (pCAMBIA-1306 vector was purchased from Cambia), and the OsNLA2-p1306 overexpression vector was constructed. The overexpression vector was introduced into the normal rice variety Zhonghua 11 using the genetic transformation method.

All the obtained transgenic seedlings were transplanted in a basket with soil, watered regularly, fertilized, and planted in the field when the seedlings were about 10 cm tall. After the seedlings grew up, the genomic DNA was extracted to detect the transgenic plants by PCR. , the detection primer pair is:

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

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

If a 517bp fragment is amplified, the transgenic plants are positive plants. A single positive plant was harvested and planted until a homozygous transgenic plant was identified in the T2 generation, that is, an OsNLA2 gene overexpressing plant was obtained.

Take the leaves of OsNLA2 gene overexpressing plants, extract RNA and reverse transcribed it into cDNA, and detect the expression level of OsNLA2 gene in overexpressing plants by real-time fluorescence quantitative PCR. The results show (Fig. 1) The expression level of OsNLA2 gene in overexpressing plants Compared with the control Zhonghua 11, if the expression level of the control is set as 1, the average expression levels of the three lines of the overexpressed plants are 141.226, 139.015, and 119.085. Primer pairs used in real-time fluorescent quantitative PCR:

F5: caaagtgctcacatgtggagt, SEQ ID NO.7;

R5: aacagccagctatttctgaagcct, SEQ ID NO.8;

30 seeds were randomly selected from the three lines of the above-mentioned overexpression plants and the control Zhonghua 11, and after removing the shells by hand, the rice grains were lined up in a circle head to tail. 1000 seeds were randomly selected from the shelled seeds to count the thousand-grain weight, and it was found that the thousand-grain weight of the overexpressed plants was significantly higher than that of Hua 11 in the control (Fig. 4). One plant was randomly selected from the three lines of the above-mentioned overexpression plants and the control Zhonghua 11, and all the rice seeds with shells were arranged in a circle. The number of seeds per plant of the expressing plants was decreased compared with that of Hua 11 in the control (Figure 5), and the corresponding weight was found to find that the weight of each seed of the overexpression plants was significantly lower than that of the control Hua 11 (Figure 6).

The seeds of the overexpressed plant and Zhonghua 11 were soaked in distilled water for 3 days on the petri dish and cultivated for 7 days, and then transferred to the rice nutrient solution for cultivation. Nitrogen) and 2mM (high nitrogen), cultured for 40 days respectively, observed phenotype, and counted rice plant height. It can be seen from Figure 7 that under low nitrogen culture, the plant height of the OsNLA2 gene overexpressing plant increased compared with the control Zhonghua 11 plant, and the difference was significant. However, the difference was not as significant under high nitrogen as under low nitrogen. The plant height statistics of overexpressing plants and controls under different nitrogen cultures are shown in FIG. 8 .

The above results show that the OsNLA2 gene can increase the rice seed size and 1000-grain weight by increasing the expression level, thereby improving the grain shape of rice. Overexpressing plants can also increase rice plant height under low nitrogen.

Example 2 Construction of OsNLA2 gene mutant plants

Using a single target sequence:

F6: tgttaagtctgcacctcctgagg, SEQ ID NO. 9;

Using the above single target sequence, a gene knockout vector OsNLA2-C of the OsNLA2 gene was constructed (for the method, refer to Max et al, A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant. 2015 , 8(8):1274-1284). The gene knockout expression vector was introduced into the normal japonica rice variety Zhonghua 11 by the method of genetic transformation mediated by Agrobacterium EHA105. The mutant plants were sequenced in the T0 generation to confirm that the gene had been knocked out in three lines (Fig. 2), and continued independent breeding to the T1 generation to obtain an independent mutant plant line of the OsNLA2 gene. The seed size of the OsNLA2 gene mutant plants was significantly smaller than that of the control Zhonghua 11 plants, and the 1000-grain weight was reduced. As shown in Figures 3 and 4, the seeds were collected from a single plant. The results showed that the mutant plants had significantly increased grain filling per plant, and each plant Yield increases, as shown in Figures 5 and 6. The seeds of mutant plants and Zhonghua 11 were soaked with distilled water for 3 days on a petri dish and cultivated for 7 days, and then transferred to rice nutrient solution for cultivation. Nitrogen) and 2mM (high nitrogen), cultured for 40 days respectively, observed phenotype, and counted rice plant height. It can be seen from Figures 7 and 8 that the plant height of the OsNLA2 gene mutant plants was reduced compared with the control Zhonghua 11 plants under low nitrogen and high nitrogen culture, and reached a significant difference.

The above results show that by knocking out the expression of the OsNLA2 gene, the seed size and 1000-grain weight of rice can be reduced, and the plant height of low-nitrogen and high-nitrogen rice plants can be reduced, but the knockout of the gene can increase the number of seeds per plant and the weight of rice per plant.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

sequence listing

<110> Wuhan Aidijing Biotechnology Co., Ltd.

<120> Ubiquitin ligase gene OsNLA2, protein and its application 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

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

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

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

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

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

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

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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

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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

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

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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

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

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

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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 ttttttcctgt 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

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<213> Primer R3 (OsNLA2)

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atggatccaa ttcccattga cagcatagcc tg 32

<210> 5

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<213> Primer F4 (OsNLA2)

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gatgttggcg acctcgtatt 20

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<211> 22

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<213> Primer R4 (OsNLA2)

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tcgttatgtt tatcggcact tt 22

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<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 (2)

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 selection is to increase rice grain size and thousand-grain weight by increasing the expression of OsNLA2 gene, and increase rice plant height under low nitrogen. 2. the application of a 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 selection is to increase the number and weight of rice per plant by knocking out the expression of the OsNLA2 gene.

Images