CN114836433B - Application of rice OsNAC129 in negative regulation of grain shape and starch synthesis - Google Patents

Abstract

The invention discloses application of rice OsNAC129 in negative regulation of grain shape and starch synthesis, wherein the nucleotide sequence of the rice OsNAC129 is shown as SEQ ID NO.1 or SEQ ID NO.4, and the mutant function deletion mutation of the rice OsNAC129 leads to obvious increase of grain size and apparent amylose content, and overexpression of the rice OsNAC129 leads to reduction of grain and obvious reduction of apparent amylose content. The beneficial effects of the invention are as follows: in the invention, osNAC129 is a NAC family transcription factor, and through comprehensive phenotypic analysis of a functional deletion mutant and an over-expression transgenic plant, the OsNAC129 is found to be involved in regulation of granule shape and apparent amylose content, which indicates that OsNAC129 can be involved in regulation of rice yield and quality. In addition, through fine experimental design, osNAC129 is found to be involved in BR signal and ABA signal channels at the same time, and a great deal of researches show that BR can promote yield improvement through regulating rice plant types, spike types and grain shapes, and ABA is mainly involved in stress response and seed grouting and germination.

Description

Application of rice OsNAC129 in negative regulation of grain shape and starch synthesis

Technical Field

The invention relates to the field of rice regulation, in particular to application of rice OsNAC129 in negative regulation of grain shape and starch synthesis.

Background

Rice grain size is one of the important factors affecting rice yield, while apparent amylose content has a decisive influence on rice quality, and rice with higher apparent amylose content is generally relatively poor in quality, wherein OsGBSSI is the only enzyme responsible for amylose synthesis in rice endosperm.

Grain size and starch content in endosperm have a decisive influence on the yield and quality of rice, however their relevant regulatory mechanisms are still not clear.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides an application of rice OsNAC129 in negative regulation of grain shape and starch synthesis.

The aim of the invention is achieved by the following technical scheme. The application of the rice OsNAC129 in negative regulation of grain shape and starch synthesis is characterized in that the nucleotide sequence of the rice OsNAC129 is shown as SEQ ID NO.1 or SEQ ID NO. 4.

The application of the rice OsNAC129 in negative regulation of grain shape and starch synthesis comprises that the mutant function deletion mutation of the OsNAC129 leads to obvious increase of grain size and apparent amylose content, and overexpression of the OsNAC129 leads to reduction of grain and obvious reduction of apparent amylose content.

The nucleotide sequence of the mutant of the OsNAC129 is shown as SEQ ID NO.5, and the encoded protein sequence is shown as SEQ ID No. 9.

The beneficial effects of the invention are as follows: in the invention, osNAC129 is a NAC family transcription factor, and through comprehensive phenotypic analysis of a functional deletion mutant and an over-expression transgenic plant, the OsNAC129 is found to be involved in regulation of granule shape and apparent amylose content, which indicates that OsNAC129 can be involved in regulation of rice yield and quality. In addition, through fine experimental design, osNAC129 is found to be involved in BR signal and ABA signal channels at the same time, and a great deal of researches show that BR can promote yield improvement through regulating rice plant types, spike types and grain shapes, and ABA is mainly involved in stress response and seed grouting and germination.

Drawings

FIG. 1 OsNAC129 is highly expressed in immature seeds. (A) Expression profile of OsNAC129 in different developmental stages and tissue sites of japan. Total RNA was extracted from roots and shoots 7 days after germination, leaves, leaf sheaths, stems and young ears before flowering, and seeds at different developmental stages. ACTIN1 was used as an internal control. Data are mean ± SD of 4 biological replicates. (B) Analysis of staining for OsNAC129pro: -GUS in transgenic plants. GUS expression levels in roots (a) and shoots (b), flag leaves (c), leaf sheaths (d), stems (e), stem cross sections (f) in heading stage, young ears (g), old ears (h) and flowering small ears (i) 7 days after germination; endosperm of 3DAF (j), 5DAF (k), 7DAF (l), 10DAF (m), 15DAF (n) and 25DAF (o).

Figure 2.Osnac129 mutant grain size and apparent amylose content increased. (A) phenotypic observations of osnac129 grain maturity. (B) And (3) detecting the grain length, grain width, grain thickness and thousand grain weight of the mature seeds. Data are mean ± SD,3 replicates, P <0.01, P <0.001; t-test. (F) Brown rice grain of wild type and osnac129 mutant, cross section (upper panel) and starch grain (lower panel). Blue boxes indicate the area of the mature endosperm center scanning electron microscope for analysis of starch granules. (G) And (H) detecting Apparent Amylose Content (AAC) and Total Starch Content (TSC), respectively. Data are mean ± SD,5 replicates, P <0.001; t-test.

FIG. 3 OsNAC129-OE over-expressed plants resulted in reduced grain size and reduced apparent amylose content. (A) phenotypic observations of mature seeds of WT and OE plants. (B) And (3) detecting the grain length, grain width and thousand grain weight of the mature seeds. Data are mean ± SD,3 replicates, P <0.001; t-test. (E) And (F) detecting Apparent Amylose Content (AAC) and Total Starch Content (TSC), respectively. Data are mean ± SD,5 replicates, P <0.01, P <0.001; t-test.

Figure 4.OsNAC129 regulates cell expansion and endosperm starch synthesis simultaneously. (A) Scanning electron microscope observation of WT and osnac129 mutant palea exodermis cells. (B) Measurement of the length, width and number of the exosome cells. (B) And (C) 3 replicates (each replicate comprising at least 30 cells), (D) 4 replicates, data mean ± SD, P <0.01, P <0.001; t-test. (E) And (F) qRT-PCR detection of the size-related genes reported in WT, osnac129 mutants and OE plant 7DAF seeds to control cell elongation. UBQ10 is used as an internal control. Data are mean ± SD,4 replicates, P <0.05, P <0.01, P <0.001; t-test. (G) And (H) qRT-PCR detection of starch synthase encoding genes in WT, osnac129 and OE plant 7DAF seeds. UBQ10 is used as an internal control. Data are mean ± SD,4 replicates, P <0.01, P <0.001; t-test.

Figure 5 osnac129 is ABA-induced and is also a BR signaling pathway-related gene. (A) OsNAC129 was induced only by ABA. Seedlings of NIP at 2 weeks of age were treated with IAA,6BA, GA3, ABA and BL (50. Mu.M each hormone concentration), respectively, with sterilized water without hormone as a blank. The treated samples were collected and tested for OsNAC129 expression using qRT-PCR. Data are mean ± standard deviation, with 4 replicates. ND, not detected. (B) Two week old seedlings of WT and OE plants were treated with BL at a series of concentrations for 24 hours, respectively, and then leaf angle imaging was performed. (C) image statistics of WT and OE seedling leaf angle after BL treatment. Data were taken as mean ± standard deviation for 10 replicates. * P <0.001; t-test.

FIG. 6 preparation and identification of OsNAC 129-related transgenic material. Schematic representation of (A) osnac129 and T-DNA insertion site. The black boxes represent exons, and P1, P2, P3 and P4 are primers for insertion site identification. (B) PCR screening of homozygous mutants. (C) Southern blot detection of T-DNA insertion copy number. The PCR-derived hygromycin B phosphotransferase (HPT) fragment was used as a positive control. HindIII, salI, sphI and Xhol, restriction endonucleases digest genomic DNA of the osnac129 mutant. Detection was then performed with biotin-labeled HPT probes. (D-E) qRT-PCR detection of OsNAC129 mutants and overexpressing plants. UBQ10 was used as an internal reference. Data are mean ± standard deviation, with 4 replicates. * P <0.001; t-test. FIG. 7.Osnac129 mutant seedlings grew faster and this accelerated growth continued to heading stage. (A) two week old seedlings of WT and osnac129 mutants. (B) And (C) shoot length and root length measurements of WT and osnac129 mutants after two weeks of germination. Data are mean ± SD,10 replicates, P <0.01, P <0.001; t-test. (D) Plant types of Wild Type (WT) and osnac129 mutants during the stucco phase. (E) And (F) the plant height and tillering number of the WT and osnac129 mutants at the grouting stage. Data are mean ± SD,12 replicates, P <0.01; t-test.

Overexpression of OsNAC129 inhibited plant growth. (A) two week old seedlings of WT and OE plants. (B) And (C) determination of shoot length and root length of WT and OE plants after two weeks of germination. Data are mean ± SD,10 replicates, P <0.001; t-test. (D) the plant type of WT and OE plants in the stucco stage. The plant heights and tillering numbers of WT and OE plants in the grouting period (E) and (F). Data are mean ± SD,12 replicates, P <0.01, P <0.001; t-test.

Detailed Description

The invention will be described in detail below with reference to the attached drawings:

the application of the rice OsNAC129 in negative regulation of grain shape and starch synthesis is characterized in that the nucleotide sequence of the rice OsNAC129 is shown as SEQ ID NO.1 or SEQ ID NO. 4.

The application of the rice OsNAC129 in negative regulation of grain shape and starch synthesis comprises that the mutant function deletion mutation of the OsNAC129 leads to obvious increase of grain size and apparent amylose content, and overexpression of the OsNAC129 leads to reduction of grain and obvious reduction of apparent amylose content.

The nucleotide sequence of the mutant of the OsNAC129 is shown as SEQ ID NO.5, and the encoded protein sequence is shown as SEQ ID No. 9.

Sequence description:

1. the parent of the overexpressing strain is Japanese sunny and the DNA transferred is genomic DNA of Japanese sunny OsNAC 129.

2. The osnac129 mutant is a T-DNA insertion mutant of the Dongjin background, the inserted T-DNA sequence is shown in the genome sequence (SEQ ID NO.5, 1587-9861) and the CDS sequence, and SEQ ID NO.8 is the CDS sequence of the Dongjin mutant.

3. The genomic sequence of OsNAC129 against Dongjin was one base different from the genomic sequence of japan against sunny (SEQ ID No.1 or SEQ ID No. 4) (japan was T, dongjin was G) and the difference base position was 357. Because this base is located in an intron, the CDS sequence is not affected, so that the two rice variety coding region sequences (SEQ ID NO.2 or SEQ ID NO. 6) are identical to the protein sequences (SEQ ID NO.3 or SEQ ID NO. 8).

4.OsNAC129 genomic DNA was 1900bp long, CDS was 723bp long, and protein was 240 amino acids long. The mutant had been inserted by T-DNA and protein translation was terminated prematurely, resulting in a 143 amino acid protein (SEQ ID NO. 9) with 137 amino acids at the N-terminus identical to the wild type and the last 6 amino acids encoded by T-DNA.

High expression of OsNAC129 in Rice immature endosperm

Previous transcriptomics studies have reported that 9 seed-specific NAC transcription factors, one of which is OsNAC129, are present in rice. To fully analyze the biological function of OsNAC129, we first studied the spatial and temporal expression profile of OsNAC129 in wild type sun-dried rice in more detail by qRT-PCR techniques. The results of the test showed that the expression level of OsNAC129 was very low and barely detectable in vegetative tissues such as roots and shoots 7 days after germination, in the stems, leaves, leaf sheaths, and young ears at the filling stage (FIG. 1A). The expression level in early-in-filling seeds was also very low, however, as the seeds develop and filling process, the expression level of OsNAC129 increased rapidly and reached a peak in the seeds 7 days after fertilization, followed by a gradual decrease (fig. 1A), which was highly consistent with the rice endosperm filling process. Furthermore, it was found by more detailed analysis that OsNAC129 was mainly concentrated in endosperm for expression (fig. 1A). Furthermore, we also detected expression of OsNAC129 in glumes, seed coats and embryos, although not very high, suggesting that OsNAC129 plays an important role in seed development.

To reflect more precisely the expression pattern of oscac 129, we also prepared oscac 129Pro: : transgenic plants of GUS, and GUS staining observations were made on the transgenic plants at different developmental stages and tissue sites. As a result, it was found that there was little signal in the young roots and shoots and leaf sheaths of the leaves at the early stage of development for 7 days, but a strong GUS staining signal was present at some specific sites such as the leaf pillow and stem node sites (a-f of FIG. 1B). Young ears at early stages of development were also barely stained, however, as young ears developed, GUS staining signals appeared and gradually deepened, and glumes and stamens were stained (g-i of FIG. 1B). After flowering and filling, embryo and endosperm gradually formed, but seed seeds did not yet develop GUS signal 5 days ago, GUS signal rapidly increased after 5 days, and staining signal appeared first at the embryo-endosperm interface, followed by filling the whole endosperm (k-o of FIG. 1B). In summary, the above results fully demonstrate that OsNAC129 is not a transcription factor that is expressed fully seed-specifically, but is expressed in other tissue parts, and the expression level is highest in immature endosperm, indicating that OsNAC129 may be involved not only in seed development regulation, but also in plant growth and development regulation.

Loss of function mutations in OsNAC129 resulted in significant increases in grain size, apparent amylose content, and plant height

Subsequently, we obtained from Korean mutant pool a T-DNA insertion mutant of OsNAC129 (PFG-3A 60140) inserted at the front end of the third exon of OsNAC129 gene (FIG. 6A), which resulted in frameshift and premature termination of OsNAC 129. We verified that the mutant was correct by PCR with primers on the T-DNA and at both ends of the insertion site, respectively, and that homozygous mutants were obtained from the isolated offspring (FIGS. 6A and 6B). Furthermore, we confirmed that there was only one T-DNA insert in this mutant by southern blot hybridization experiments (FIG. 6C), and therefore, that this mutant was a specific mutant of OsNAC 129. In addition, fluorescent quantitative PCR assay showed that the expression level of OsNAC129 was very significantly reduced in mutant OsNAC129 (fig. 6D), which in combination showed that OsNAC129 was a loss-of-function mutant. We then analyzed the mature seed phenotype of OsNAC129 and the results indicated that the OsNAC129 mutants had significantly increased grain length and thousand kernel weight without significant changes in grain width and grain thickness (FIGS. 2A-2D), indicating that OsNAC129 negatively regulated grain shape. Furthermore, the osnac129 mutant mature endosperm was translucent in appearance, had no chalkiness, and had no significant differences from the wild-type endosperm, nor was scanning electron microscopy observed to indicate that the starch granule shapes in the mutant and wild-type endosperm were regularly arranged with no significant differences (fig. 2F). Subsequently we examined the starch of mutant and wild-type mature seeds and found that the apparent amylose content in the mutant was significantly increased and the total starch content was significantly reduced (fig. 2G and 2H). These results indicate that OsNAC129 is involved in both granular and endosperm starch synthesis regulation.

Description of T-DNA mutant identification primers:

first, a pair of primers P1 (TCATTTTCCGACATCAGGAAG) and P2 (ACGCACACACACACACACAC) are designed on both sides of the insertion site of the gene (the positions of the primers are shown in Table 1) for PCR amplification, and the wild type and heterozygous mutants can obtain normal amplified bands, while the homozygous mutants cannot obtain amplified bands due to the insertion of large T-DNA fragments.

Subsequently, primers P3 (CTAGAGTCGAGAATTCAGTACA) and P4 (TTGGGGTTTCTACAGGACGTAAC) were designed at the left and right ends of the T-DNA, and PCR detection was performed in combination with primers at both ends of the insertion site on the OsNAC129 gene, respectively: p1+p3 combinations, or p4+p2 combinations. Since the wild plants do not have T-DNA sequences, the amplified bands cannot be obtained by both primer sets, and the amplified bands can be obtained by homozygous mutants and heterozygous mutants.

The three groups of primers can be used together to conveniently distinguish the wild type, the homozygous mutant and the heterozygous mutant.

Considering that OsNAC129 is expressed in stem and leaf pillow, we want to know if it also plays a role in the vegetative growth and development of rice. As a result, we found that the osnac129 mutant seedlings grew faster than the wild type and this rapid growth state continued to maturity, resulting in mutant strain heights significantly higher than the wild type (FIGS. 7A-7D). However, the tillering number was not affected. These results indicate that OsNAC129 has negative regulatory function on plant height.

3. Overexpression of OsNAC129 resulted in reduced grain size, significantly reduced apparent amylose content and plant height

To further verify the biological function of oscac 129 during seed development and plant growth, we then prepared an OsNAC129 whole gene overexpressing transgenic plant (comprising a promoter region of about 2kb on the start codon ATG, an ORF region, and a terminator region of about 1kb downstream of the stop codon) in a japan background. The fluorescent quantitative PCR detection shows that the expression level of OsNAC129 is obviously improved (figure 6E), which shows that the preparation of the over-expression transgenic plant is successful. We then performed a comprehensive analysis of the phenotype of the over-expressed transgenic plants. As a result, it was found that the grain width and thousand kernel weight of the overexpressing transgenic plants were significantly reduced, and the grain length was not significantly changed, compared to the wild-type plants (FIGS. 3A-3D), and these results further confirmed that OsNAC129 negatively regulated rice grain shape. In addition, apparent amylose content was significantly reduced, total starch content was not significantly different (fig. 3E and 3F), and these results indicate that OsNAC129 was indeed involved in both rice grain and endosperm starch synthesis regulation.

Furthermore, we also examined the vegetative growth of OsNAC129 overexpressing transgenic plants. As a result, it was found that, in contrast to the mutant, the over-expressed transgenic plants grew at a significantly slower rate than the wild type, resulting in significantly lower seedling length and plant height than the wild type plants (fig. 8A-8D). In conclusion, osNAC129 negatively regulates rice grain shape, apparent amylose content and plant height.

The expression level of the grain-length-related gene and the starch synthase-encoding gene in both the OsNAC129 mutant and the OsNAC129-OE overexpressing transgenic plants was altered

The above results indicate that OsNAC129 negatively regulates granule shape, apparent amylose content and plant height. To further investigate the regulatory mechanisms, we first examined the glume cells of the osnac129 mutant. The results found that mutant glume cell lengths were significantly greater than wild type, cell widths were unchanged, and longitudinal cell numbers were slightly lower than wild type (fig. 4A-4D), suggesting that OsNAC129 was involved in granulocyte regulation primarily by affecting cell expansion. Several genes affecting grain size by regulating cell expansion have been cloned and reported, and in order to verify whether OsNAC129 is involved in grain regulation by these known genes, we performed fluorescent quantitative detection of OsNAC129 mutants and related grain genes (such as OsSRS1, osSRS3, osSRS5, osPGL1, osPGL 2) in overexpressed transgenic plants. As a result, it was found that OsPGL1 and OsPGL2 were up-regulated in mutants and down-regulated in overexpressing transgenic plants, respectively, among these genes examined (FIGS. 4E and 4F). These results indicate that ospac 129 is likely involved in the regulation of granulocyte shape by negatively regulating OsPGL1 and OsPGL 2. OsPGL1 and OsPGL2 are two atypical bHLH family transcription factors, which have been demonstrated to be positive regulators of grain length, which affect rice grain shape by positively regulating cell expansion, and which are both BR signal transduction pathway-related genes.

Furthermore, we examined the starch synthase encoding genes in mutants and over-expressed transgenic plants. As a result, it was found that many starch synthase-encoding genes such as OsAGPS2b, osAGPL2, osAGPL3, osSIIa, osSSIVb, osGBSSI, and OsSBEIIb were significantly up-regulated in the mutants (FIG. 4G). In the over-expressed transgenic plants, some starch synthase encoding genes such as OsAGPL3, osSIIa, osGBSSI, osBEIIb, and OsPHOI were significantly down-regulated (FIG. 4H). The above results further demonstrate that OsNAC129 does negatively regulate rice starch synthesis. However, the total starch content in the endosperm of the mutant and the over-expressed transgenic plants is not changed correspondingly, which implies that the regulation network of starch synthesis in the endosperm is very complex.

Expression of OsNAC129 was induced by ABA alone and involved in BR signaling

The above results all indicate that OsNAC129 is involved in both seed development and plant growth regulation, and a number of prior studies have shown that phytohormones play an important regulatory role in the overall growth and development of plants. Therefore, to further investigate what upstream signals OsNAC129 was induced to participate in the above-described regulation, we first conducted various exogenous hormone induction experiments on the expression level of OsNAC 129. As a result, it was found that only ABA was able to significantly induce expression of OsNAC129 in wild rice seedlings treated with 5 hormones (IAA, 6BA,GA3,ABA,BL) induction, while none of the other hormones induced OsNAC129 (fig. 5A). This suggests that ABA is an upstream signal of OsNAC 129.

Furthermore, our previous results demonstrate that OsNAC129 is locally expressed at the leaf pillow, which closely matches the expression pattern of BR-related genes, whereas regulation of leaf inclination in crop plants is a physiological response typical of BR. In addition, osPGL1 and OsPGL2 were negatively regulated by ospcl 129, and both genes were BR signal transduction-related genes, although OsPGL1 was not induced by BR, the sensitivity of the overexpressed transgenic plants to BR was significantly increased. Therefore we suspected that OsNAC129, although not induced by BL (the strongest active form of BR), was also and possibly involved in BR signaling pathways. To test this question, we tested seedlings of transgenic plants overexpressing OsNAC129 for exogenous BR sensitivity. As a result, it was found that the leaf angle of the seedlings of the overexpressing transgenic plants was significantly reduced in sensitivity to exogenous BL compared to the wild-type seedlings (fig. 5B and 5C), which fully confirmed that OsNAC129 was involved in BR signaling pathway and negatively regulated BR signaling.

Conclusion:

1. the present invention demonstrates that OsNAC129 is not an endosperm-specific expressed gene, but a ubiquitously expressed gene, but is expressed in the immature endosperm at its highest levels.

OsNAC129 simultaneously negatively regulates rice grain shape and apparent amylose content phenotype. And regulate grain shape mainly by regulating cell expansion. Has obvious negative regulation and control effects on the grain-shape related genes OsPGL1, osPGL2 and the amylose synthase encoding gene OsGBSSI.

OsNAC129 is involved in rice grain development and plant growth regulation through a variety of pathways such as BR signaling pathways and ABA pathways.

Materials and methods:

1. plant material and growth conditions

For over-expression of OsNAC129, a whole gene fragment (comprising a 2kb promoter sequence in front of the start codon, an Open Reading Frame (ORF) 1kb after the start codon to the stop codon) was PCR amplified from the genomic DNA of japan, and the PCR fragment was then recombinantly ligated into the pCAMBIA 1300 vector using a recombinase. For promoter-driven GUS reporter transgenic plants, a 2kb sequence before the start codon was amplified from genomic DAN using PCR, and this fragment was then introduced into pCAMBIA 1300-GN using recombinase. The correctly constructed transgenic plasmid is introduced into an agrobacterium strain EHA105 and infects callus of japonica rice variety Nipponbare to obtain a transgenic plant. The japonica rice (Oryza sativa l.) varieties Dongjin and osnac129 (pfg_3a 60140) mutants were obtained from korea university of technology (Jeong et al, 2002). The paddy field condition is that the paddy field is planted in the Shanghai test base in summer and planted in the Hainan cemetery test base in winter (Hainan island in south China), and the paddy field condition is planted in natural condition, and is mainly used for phenotype analysis, gene expression detection and seed reservation. The greenhouse growth condition is 28 ℃,11 hours day/13 hours night, and is mainly used for culturing seedlings and detecting gene expression.

DNA and RNA extraction and fluorescent quantitative PCR (qRT-PCR) detection

The rice genome DNA is separated by using a plant DNA separation kit (Tian Gen Biotechnology), and the plasmid DNA is extracted by using a plasmid DNA extraction kit (Tian Gen Biotechnology), and the plant DNA and the plasmid DNA are separated according to the operation procedure of a manufacturer. In extracting RNA, total RNA is extracted by using a plant total RNA isolation kit (Tiangen biotechnology), all tissues must be freshly collected or frozen in liquid nitrogen, and the operation instructions of the manufacturer are followed. Reverse transcription was performed using a reverse transcription kit (TAKARA) according to the manufacturer's instructions. qRT-PCR assay qPCR assay was performed using 2. Mu.l cDAN as template and 20. Mu.l reaction system using LightCycler 480 (BIORAD). Rice ubiquitin 10 (OsUBQ 10) or ACTIN1 was used as an internal reference. The primers used in the present invention are listed in supplementary Table 1.

GUS staining analysis

Histochemical analysis of GUS activity was performed as described in the previous invention (Edwards et al, 1990). Briefly, transgenic plant tissue expressing OsNAC129pro:: GUS was collected, soaked in 90% aqueous acetone, and fixed on ice for 15min. The aqueous acetone solution was then discarded and rinsed twice with deionized water. The tissues were soaked in GUS staining solution and stained overnight at 37 ℃. The next day the GUS staining solution was discarded, ethanol solution was added, the tissue was decolorized to no green, and the staining signal was observed using a split microscope and imaged.

4. Endosperm starch content detection

The detection of apparent amylose content and total starch content was performed according to the methods in the previously published literature of the laboratory (Wang et al, 2013).

5. Seed phenotype observation and glume epidermic cell statistical analysis

The grain shape analysis adopts a ten thousand-depth grain analysis system, and the grain size of mature seeds of transgenic plants is measured according to the operation rules of manufacturers. Each sample was provided with 3 biological replicates, each replicate containing no less than 300 seeds. Exotic epidermal cell measurements were performed as described in the previously published literature (Heang and Sassa,2012 a). The morphology of the starch grains was observed according to the literature method published previously (Fu and Xue, 2010).

6. Phytohormone induction assay

Wild type Japanese seedlings of 2 weeks old were collected, immersed in distilled water containing different plant hormones, and distilled water containing no plant hormone was used as a negative control. The treated samples were then collected at various time points and immediately frozen in liquid nitrogen. All samples collected were then tested for expression of OsNAC129 using qRT-PCR. RNA isolation, reverse transcription, qRT-PCR were all performed as described above.

BR sensitivity analysis

2-week-old wild type Nippon Temminck and OsNAC129-OE over-expressed seedlings were treated with BL at concentrations of 1. Mu.M, 5. Mu.M, 50. Mu.M, respectively, for 24h with sterile water as a simulated control. Seedlings were then collected, photographed, and leaf angle counted and calculated using instructions of ImageJ software.

Table 1. Primers used in the present invention.

It should be understood that equivalents and modifications to the technical scheme and the inventive concept of the present invention should fall within the scope of the claims appended hereto.

Sequence listing

<110> university of Yangzhou

<120> application of Rice OsNAC129 in negative regulation of grain shape and starch synthesis

<160> 9

<170> PatentIn Version 3.1

<210> 1

<211> 1900

<212> DNA

<213> Rice (Japanese sunny)

<400> 1

atgcacctcc ccgctgtggg catgtcacac cccaccgagg gtgagcttgt cttccactac 60

ctctatcgtc gtgccgttaa catgccgctg ccctctgaat tcatttgtga tgtcaacgtt 120

cttcctcata acccatggga catcgtccca ggtaagtata ttttgactta tgctgccata 180

tcatcttgct cccataaaaa ttgttagcac atccagatac atctcgaatt gattaatcta 240

tgtctaccag gcttgattga ttgcatggcc taatgtccga tttgttgttg gttttgtttt 300

tgtctttttc tctctcttgt tccatgatgg tgatcatgga cataggagca ctgacataga 360

gggagaaagg gaagtacttc tttatgcaga aggagataaa gtgccctagt agtcgccgta 420

gcaaccgtat cacaagtaaa gggttctgga ggtcggcagg ctcagaaaag ccagtctact 480

acaaccaggg aggtggcagc gactgcatgc tggtgggaat gaggcgcacc ctaacatttt 540

actttgggaa ttcacggacc gcagagcgca caaaatgggg tatgcaagaa tttcgccttg 600

ctggcaatgg tctttcgcct taccctgcga tgaagcatgc caccggggat ggctcaaaac 660

caccctgcaa ctgtgccgaa acaaccatcg ccaaggtaat taagatgttg acttcaaaca 720

ttttcctgtc atcatttttt aatcgtcttg ttcgtttcat ctgcaaattg aatctaataa 780

gtttgttatt ttcgagcaag tgtactattg tgaggtttag ctttgggaaa tagggagggg 840

gatttaataa atatgatgag ccgtcttcta gatataactc tcatgtcgat tttattcatg 900

gaaaggaaaa ggtagagatt gtctttgatg ccgcttaggg tacatgctat tagatctgta 960

gttatggact gctgcgatgc aaggaacgtg ggatctatac atgattcaaa ttttgtcgag 1020

acgtagggat ttatagttgg taaaatctag atatgtatat gtgtcagtgg ggagttccat 1080

gcatatgtgt cagtgggatc gagtcgatgt attataaatt ttgtcataag gttagatctg 1140

gttacgagat ctataatcga cttgaatttg acaagaagac aaaagtggtt tgttgtcagc 1200

ttgcgagcaa tgaacctatt taatgaacac catgtgttgt taactcgttt ctcccatgat 1260

ttttggagtt ttatatcaga taatgtatca accatttatt ggtcaaattt tctgtgatgt 1320

tcaaactgaa caatggagaa tactcatttt ccgacatcag gaagaaaact gataacaaat 1380

aagttaccat gtctttcaaa ttcagtagct atgttattag catccgtata taacatatat 1440

acaaaatatt cctttccaac taatagtact tcatcaattt tcccttaggt atacatctta 1500

catataccat gctttgtgcg ttcagcagca tccattaaca ggcaattcgg tgactgtttt 1560

gcagagaaat gatggtctct ctgcagttct tcgtaatgtg ctcgccgtca ccccccttgt 1620

ggaaaccgtt gtcgaacctg atggatcatg gctgatctgc cgcatctaca ggacaaggca 1680

gcgtgcattg cctgttatca ctcctcctgc catagaaaat gcaagggaaa tcattatccc 1740

tcctgccaat ggcaatgcaa gggaggctca agtccgcttt attgacttcc tgcagcaggg 1800

atcgcacatt gagtcatcct ccccctgtag ttgtatagtt ggcccctctt tggcagaggg 1860

aagtgatgaa tctgctggta gtgttgacca aaaggattga 1900

<210> 2

<211> 723

<212> DNA

<213> Rice (Japanese sunny)

<220>

<400> 2

atgcacctcc ccgctgtggg catgtcacac cccaccgagg gtgagcttgt cttccactac 60

ctctatcgtc gtgccgttaa catgccgctg ccctctgaat tcatttgtga tgtcaacgtt 120

cttcctcata acccatggga catcgtccca ggctcagaaa agccagtcta ctacaaccag 180

ggaggtggca gcgactgcat gctggtggga atgaggcgca ccctaacatt ttactttggg 240

aattcacgga ccgcagagcg cacaaaatgg ggtatgcaag aatttcgcct tgctggcaat 300

ggtctttcgc cttaccctgc gatgaagcat gccaccgggg atggctcaaa accaccctgc 360

aactgtgccg aaacaaccat cgccaagaga aatgatggtc tctctgcagt tcttcgtaat 420

gtgctcgccg tcacccccct tgtggaaacc gttgtcgaac ctgatggatc atggctgatc 480

tgccgcatct acaggacaag gcagcgtgca ttgcctgtta tcactcctcc tgccatagaa 540

aatgcaaggg aaatcattat ccctcctgcc aatggcaatg caagggaggc tcaagtccgc 600

tttattgact tcctgcagca gggatcgcac attgagtcat cctccccctg tagttgtata 660

gttggcccct ctttggcaga gggaagtgat gaatctgctg gtagtgttga ccaaaaggat 720

tga 723

<210> 3

<211> 240

<212> PRT

<213> Rice (Japanese sunny)

<220>

<400> 3

Met His Leu Pro Ala Val Gly Met Ser His Pro Thr Glu Gly Glu Leu Val Phe His Tyr

1 5 10 15 20

Leu Tyr Arg Arg Ala Val Asn Met Pro Leu Pro Ser Glu Phe Ile Cys Asp Val Asn Val

25 30 35 40

Leu Pro His Asn Pro Trp Asp Ile Val Pro Gly Ser Glu Lys Pro Val Tyr Tyr Asn Gln

45 50 55 60

Gly Gly Gly Ser Asp Cys Met Leu Val Gly Met Arg Arg Thr Leu Thr Phe Tyr Phe Gly

65 70 75 80

Asn Ser Arg Thr Ala Glu Arg Thr Lys Trp Gly Met Gln Glu Phe Arg Leu Ala Gly Asn

85 90 95 100

Gly Leu Ser Pro Tyr Pro Ala Met Lys His Ala Thr Gly Asp Gly Ser Lys Pro Pro Cys

105 110 115 120

Asn Cys Ala Glu Thr Thr Ile Ala Lys Arg Asn Asp Gly Leu Ser Ala Val Leu Arg Asn

125 130 135 140

Val Leu Ala Val Thr Pro Leu Val Glu Thr Val Val Glu Pro Asp Gly Ser Trp Leu Ile

145 150 155 160

Cys Arg Ile Tyr Arg Thr Arg Gln Arg Ala Leu Pro Val Ile Thr Pro Pro Ala Ile Glu

165 170 175 180

Asn Ala Arg Glu Ile Ile Ile Pro Pro Ala Asn Gly Asn Ala Arg Glu Ala Gln Val Arg

185 190 195 200

Phe Ile Asp Phe Leu Gln Gln Gly Ser His Ile Glu Ser Ser Ser Pro Cys Ser Cys Ile

205 210 215 220

Val Gly Pro Ser Leu Ala Glu Gly Ser Asp Glu Ser Ala Gly Ser Val Asp Gln Lys Asp

225 230 235 240

<210> 4

<211> 1900

<212> DNA

<213> Rice (Dongjin)

<220>

<400> 4

atgcacctcc ccgctgtggg catgtcacac cccaccgagg gtgagcttgt cttccactac 60

ctctatcgtc gtgccgttaa catgccgctg ccctctgaat tcatttgtga tgtcaacgtt 120

cttcctcata acccatggga catcgtccca ggtaagtata ttttgactta tgctgccata 180

tcatcttgct cccataaaaa ttgttagcac atccagatac atctcgaatt gattaatcta 240

tgtctaccag gcttgattga ttgcatggcc taatgtccga tttgttgttg gttttgtttt 300

tgtctttttc tctctcttgt tccatgatgg tgatcatgga cataggagca ctgacagaga 360

gggagaaagg gaagtacttc tttatgcaga aggagataaa gtgccctagt agtcgccgta 420

gcaaccgtat cacaagtaaa gggttctgga ggtcggcagg ctcagaaaag ccagtctact 480

acaaccaggg aggtggcagc gactgcatgc tggtgggaat gaggcgcacc ctaacatttt 540

actttgggaa ttcacggacc gcagagcgca caaaatgggg tatgcaagaa tttcgccttg 600

ctggcaatgg tctttcgcct taccctgcga tgaagcatgc caccggggat ggctcaaaac 660

caccctgcaa ctgtgccgaa acaaccatcg ccaaggtaat taagatgttg acttcaaaca 720

ttttcctgtc atcatttttt aatcgtcttg ttcgtttcat ctgcaaattg aatctaataa 780

gtttgttatt ttcgagcaag tgtactattg tgaggtttag ctttgggaaa tagggagggg 840

gatttaataa atatgatgag ccgtcttcta gatataactc tcatgtcgat tttattcatg 900

gaaaggaaaa ggtagagatt gtctttgatg ccgcttaggg tacatgctat tagatctgta 960

gttatggact gctgcgatgc aaggaacgtg ggatctatac atgattcaaa ttttgtcgag 1020

acgtagggat ttatagttgg taaaatctag atatgtatat gtgtcagtgg ggagttccat 1080

gcatatgtgt cagtgggatc gagtcgatgt attataaatt ttgtcataag gttagatctg 1140

gttacgagat ctataatcga cttgaatttg acaagaagac aaaagtggtt tgttgtcagc 1200

ttgcgagcaa tgaacctatt taatgaacac catgtgttgt taactcgttt ctcccatgat 1260

ttttggagtt ttatatcaga taatgtatca accatttatt ggtcaaattt tctgtgatgt 1320

tcaaactgaa caatggagaa tactcatttt ccgacatcag gaagaaaact gataacaaat 1380

aagttaccat gtctttcaaa ttcagtagct atgttattag catccgtata taacatatat 1440

acaaaatatt cctttccaac taatagtact tcatcaattt tcccttaggt atacatctta 1500

catataccat gctttgtgcg ttcagcagca tccattaaca ggcaattcgg tgactgtttt 1560

gcagagaaat gatggtctct ctgcagttct tcgtaatgtg ctcgccgtca ccccccttgt 1620

ggaaaccgtt gtcgaacctg atggatcatg gctgatctgc cgcatctaca ggacaaggca 1680

gcgtgcattg cctgttatca ctcctcctgc catagaaaat gcaagggaaa tcattatccc 1740

tcctgccaat ggcaatgcaa gggaggctca agtccgcttt attgacttcc tgcagcaggg 1800

atcgcacatt gagtcatcct ccccctgtag ttgtatagtt ggcccctctt tggcagaggg 1860

aagtgatgaa tctgctggta gtgttgacca aaaggattga 1900

<210> 5

<211> 10175

<212> DNA

<213> Rice (Dongjin mutant)

<220>

<400> 5

atgcacctcc ccgctgtggg catgtcacac cccaccgagg gtgagcttgt cttccactac 60

ctctatcgtc gtgccgttaa catgccgctg ccctctgaat tcatttgtga tgtcaacgtt 120

cttcctcata acccatggga catcgtccca ggtaagtata ttttgactta tgctgccata 180

tcatcttgct cccataaaaa ttgttagcac atccagatac atctcgaatt gattaatcta 240

tgtctaccag gcttgattga ttgcatggcc taatgtccga tttgttgttg gttttgtttt 300

tgtctttttc tctctcttgt tccatgatgg tgatcatgga cataggagca ctgacagaga 360

gggagaaagg gaagtacttc tttatgcaga aggagataaa gtgccctagt agtcgccgta 420

gcaaccgtat cacaagtaaa gggttctgga ggtcggcagg ctcagaaaag ccagtctact 480

acaaccaggg aggtggcagc gactgcatgc tggtgggaat gaggcgcacc ctaacatttt 540

actttgggaa ttcacggacc gcagagcgca caaaatgggg tatgcaagaa tttcgccttg 600

ctggcaatgg tctttcgcct taccctgcga tgaagcatgc caccggggat ggctcaaaac 660

caccctgcaa ctgtgccgaa acaaccatcg ccaaggtaat taagatgttg acttcaaaca 720

ttttcctgtc atcatttttt aatcgtcttg ttcgtttcat ctgcaaattg aatctaataa 780

gtttgttatt ttcgagcaag tgtactattg tgaggtttag ctttgggaaa tagggagggg 840

gatttaataa atatgatgag ccgtcttcta gatataactc tcatgtcgat tttattcatg 900

gaaaggaaaa ggtagagatt gtctttgatg ccgcttaggg tacatgctat tagatctgta 960

gttatggact gctgcgatgc aaggaacgtg ggatctatac atgattcaaa ttttgtcgag 1020

acgtagggat ttatagttgg taaaatctag atatgtatat gtgtcagtgg ggagttccat 1080

gcatatgtgt cagtgggatc gagtcgatgt attataaatt ttgtcataag gttagatctg 1140

gttacgagat ctataatcga cttgaatttg acaagaagac aaaagtggtt tgttgtcagc 1200

ttgcgagcaa tgaacctatt taatgaacac catgtgttgt taactcgttt ctcccatgat 1260

ttttggagtt ttatatcaga taatgtatca accatttatt ggtcaaattt tctgtgatgt 1320

tcaaactgaa caatggagaa tactcatttt ccgacatcag gaagaaaact gataacaaat 1380

aagttaccat gtctttcaaa ttcagtagct atgttattag catccgtata taacatatat 1440

acaaaatatt cctttccaac taatagtact tcatcaattt tcccttaggt atacatctta 1500

catataccat gctttgtgcg ttcagcagca tccattaaca ggcaattcgg tgactgtttt 1560

gcagagaaat gatggtctct ctgcagtggc aggatatatt gtggtgtaaa caaattgacg 1620

cttagacaac ttaataacac attgcggacg tttttaatgt actgaattct cgactctaga 1680

ggatccccaa catggtggag cacgacactc tcgtctactc caagaatatc aaagatacag 1740

tctcagaaga ccagagggct attgagactt ttcaacaaag ggtaatatcg ggaaacctcc 1800

tcggattcca ttgcccagct atctgtcact tcatcgaaag gacagtagaa aaggaagatg 1860

gcttctacaa atgccatcat tgcgataaag gaaaggctat cgttcaagat gcctctaccg 1920

acagtggtcc caaagatgga cccccaccca cgaggaacat cgtggaaaaa gaagacgttc 1980

caaccacgtc ttcaaagcaa gtggattgat gtgatatcta gatccccaac atggtggagc 2040

acgacactct cgtctactcc aagaatatca aagatacagt ctcagaagac cagagggcta 2100

ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct cggattccat tgcccagcta 2160

tctgtcactt catcgaaagg acagtagaaa aggaagatgg cttctacaaa tgccatcatt 2220

gcgataaagg aaaggctatc gttcaagatg cctctaccga cagtggtccc aaagatggac 2280

ccccacccac gaggaacatc gtggaaaaag aagacgttcc aaccacgtct tcaaagcaag 2340

tggattgatg tgatatctag atccccaaca tggtggagca cgacactctc gtctactcca 2400

agaatatcaa agatacagtc tcagaagacc agagggctat tgagactttt caacaaaggg 2460

taatatcggg aaacctcctc ggattccatt gcccagctat ctgtcacttc atcgaaagga 2520

cagtagaaaa ggaagatggc ttctacaaat gccatcattg cgataaagga aaggctatcg 2580

ttcaagatgc ctctaccgac agtggtccca aagatggacc cccacccacg aggaacatcg 2640

tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt gatatctaga 2700

tccccaacat ggtggagcac gacactctcg tctactccaa gaatatcaaa gatacagtct 2760

cagaagacca gagggctatt gagacttttc aacaaagggt aatatcggga aacctcctcg 2820

gattccattg cccagctatc tgtcacttca tcgaaaggac agtagaaaag gaagatggct 2880

tctacaaatg ccatcattgc gataaaggaa aggctatcgt tcaagatgcc tctaccgaca 2940

gtggtcccaa agatggaccc ccacccacga ggaacatcgt ggaaaaagaa gacgttccaa 3000

ccacgtcttc aaagcaagtg gattgatgtg atatctagat ccgaaactat cagtgtctag 3060

agcaatcact agtgaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 3120

gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 3180

aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 3240

tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 3300

tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 3360

ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 3420

tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 3480

agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 3540

cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 3600

tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatagg 3660

cctcgagacg cgtagtactg aagcttctag agctcgttaa cggtacgaat tcagacatat 3720

atatctatct aaatttattg atatcaatat gaatgtggaa aatgctaaaa taacttacat 3780

tgtgaaatga aggaagtaat tttcttcaca aattttggca cttgcactta cggcttgtga 3840

agcccgctcg atctgaagat ttttcaaacg atgccattca ccacgtgcca ctttatatca 3900

aaccttattt gtatccaaga tttctggtac tgggcattag cgatgtcaaa caaactcgac 3960

gtaaagttga gggacagagt gtgaacttat tttattccca gcagagaacg cctccaaggt 4020

ttatgggctt tctggtccat cgcaacgaca cggactttct acagggctga gattacgggc 4080

ctaaatgggc tcccccgtct tgtgctccgc gctgtttcaa gttgggccaa aatggaactg 4140

ggccgaatag tccggtgcgg ctaacggcat ccacagccaa gccaacggtg ccgttctccc 4200

gctctcgcat ttcaaaaatc gaaaataaaa tcggcagggc cgccgacgac gcaaccaccc 4260

gttgggatcc aatctactcc tacgcaccga acaactcatc gcacccaccg atatgtggga 4320

cccagctgta acgtgggccc accggcaagc gggacgtctg catcggtcgc acctacaacg 4380

aggtggccag gtcacctact gctttttctt ggagcgacgc gtgcactcgc taaccgcaca 4440

cgaaggaagc ctctggaccc acctgtcagt gaaactaacg agaggcgggt atggacttca 4500

accgagtgca aacgaaatgg cctccttgcg gtggatatta tcagaaagct ccctaactta 4560

ttaaatattc tcgatatgat ccaaccaccc gacctttgca ggcacgatgt gagatgcgag 4620

tgaattttga agacgtcaaa gggggtaaaa cgaaaaactc ccccatccct tcgcttccgc 4680

tcctgagagg tccaggctgt ctggccctat aaaacgggcg gagtgcgggg atcgcaaacc 4740

ctaagccgag acaacgcaga gaaaggcgtc ttcgtactcg cctctctccg cgcctccgcg 4800

cttttcctcc tctcccctct ctcccttctc cgccgccgtc gcagcatcaa cccaatccgc 4860

cgccaagctt ggatccgcgt cccatctccc tcaccccccg tgttcttcgt gcctgcttct 4920

gggtcagatc tgggtggatt cgcggttgtt ggatgtgggg ggctgtgttt atttgtcggt 4980

ggatctggtt gtctggatct gcgttttctc tgtcgtagtt agcggatctg atgaaatgtt 5040

tagtgttcgt gtatactggt atggtggatc tggtcctagg atgcgtggaa tggatatatg 5100

taggcgaatt ggaggattta ttttgtgaat tttgctgaaa tgatagttct aaacactgga 5160

tctgacctcg ggatgctgtt aaatgtggaa atcatggtcg atgctgtcat gaacatggtg 5220

ttcttatggt agatctgagc aatgtatgtt tcaaaattgt ttgtcacatg gaaatgctat 5280

ggttctagat gcaatagaat gatacatgcc gagatcccct ctagttgata tgatagatca 5340

tgatgtttta cagctatgtc atatgaatat gttcatttgt taccgatgta tttggatcta 5400

cttaacattt ccaaagcacg ccgcgttcta attctagatc tggtagtcat gtttgtacac 5460

gtcatccacc taatacaaat acatatgtct agtgtttggt gacactgccc gtcagatctg 5520

ttttttccag atctgtggaa caaatactcc atgcatgtat ggtagttttg aaacgatctt 5580

gtatcttcca ttgttgtagt aacaactaaa taaagtacaa ttgttcaatt attgggaatc 5640

gtattttctg tagtgccgat gtacagcata ttcatagatg tctatttagg aactcaaatt 5700

ttaaattgag gactagttat ttattatggg tcagtctttt gaattgtgtt atcttgctgt 5760

actgaaataa taatgtacca ctaaggcgct aacatgtatt ttgtcctttc aggctgactc 5820

gacggatccc ggggggcaat gagatatgaa aaagcctgaa ctcaccgcga cgtctgtcga 5880

gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga 5940

agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag 6000

ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct 6060

cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc 6120

ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct 6180

gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg 6240

gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg 6300

cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc 6360

gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg 6420

gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac 6480

agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat 6540

cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag 6600

gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga 6660

ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg 6720

atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag 6780

aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg 6840

ccccagcact cgtccgaggg caaaggaata gagtagatgc cgaccgggat ctagaggagt 6900

cgtcgtcgtc tgggggcttg atgttctgtg tgtcaaggcc tgattgataa ctgctgctat 6960

cccatgatct gccagtgtgg agttatcctg ttgccgtgtg cgtgtgtctt cgagacattt 7020

gcttgtggtg atctgatgtt tggggcttaa tgggcttaca accctcgttg ttgtaacctg 7080

tgtgccctgt tttatgtgag accgcttcgt cacttataat atgcgcgttt gttcaatttt 7140

atgtttgttt cttgtgttga ttacggttta ctctcgactg tggaacttag tatgtttcaa 7200

ggtttacgtg cttctcctgc tgacaaccca ttactggctc cgatcttaag aaaagaacag 7260

ttcgttatta gtgcattagt atagcacttc gctaaatgta gtttattgga ttggctttta 7320

tactgttttg cctaatcaac atctctgcta tctgctgtgt ttacgtttac gtgattagct 7380

ctctctctct ctctctctct ttgaaatcca cactacggcc gcacctagtg gccattgtgc 7440

gttccaggtg agtggcatgt taacgtgtac agtttgcatc gcgtcggcag tttgctgcac 7500

ttacaacgta cagtcaccat gactagccta tcgatgaatt aattcccgat ctagtaacat 7560

agatgacacc gcgcgcgata atttatccta gtttgcgcgc tatattttgt tttctatcgc 7620

gtattaaatg tataattgcg ggactctaat cataaaaacc catctcataa ataacgtcat 7680

gcattacatg ttaattatta catgcttaac gtaattcaac agaaattata tgataatcat 7740

cgcaagaccg gcaacaggat tcaatcttaa gaaactttat tgccaaatgt ttgaacgatc 7800

ggggaaattc gagctcggta gcaattcccg aggctgtagc cgacgatggt gcgccaggag 7860

agttgttgat tcattgtttg cctccctgct gcggtttttc accgaagttc atgccagtcc 7920

agcgtttttg cagcagaaaa gccgccgact tcggtttgcg gtcgcgagtg aagatccctt 7980

tcttgttacc gccaacgcgc aatatgcctt gcgaggtcgc aaaatcggcg aaattccata 8040

cctgttcacc gacgacggcg ctgacgcgat caaagacgcg gtgatacata tccagccatg 8100

cacactgata ctcttcactc cacatgtcgg tgtacattga gtgcagcccg gctaacgtat 8160

ccacgccgta ttcggtgatg ataatcggct gatgcagttt ctcctgccag gccagaagtt 8220

ctttttccag taccttctct gccgtttcca aatcgccgct ttggacatac catccgtaat 8280

aacggttcag gcacagcaca tcaaagagat cgctgatggt atcggtgtga gcgtcgcaga 8340

acattacatt gacgcaggtg atcggacgcg tcgggtcgag tttacgcgtt gcttccgcca 8400

gtggcgaaat attcccgtgc acttgcggac gggtatccgg ttcgttggca atactccaca 8460

tcaccacgct tgggtggttt ttgtcacgcg ctatcagctc tttaatcgcc tgtaagtgcg 8520

cttgctgagt ttccccgttg actgcctctt cgctgtacag ttctttcggc ttgttgcccg 8580

cttcgaaacc aatgcctaaa gagaggttaa agccgacagc agcagtttca tcaatcacca 8640

cgatgccatg ttcatctgcc cagtcgagca tctcttcagc gtaagggtaa tgcgaggtac 8700

ggtaggagtt ggccccaatc cagtccatta atgcgtggtc gtgcaccatc agcacgttat 8760

cgaatccttt gccacgtaag tccgcatctt catgacgacc aaagccagta aagtagaacg 8820

gtttgtggtt aatcaggaac tgttggccct tcactgccac tgaccggatg ccgacgcgaa 8880

gcgggtagat atcacactct gtctggcttt tggctgtgac gcacagttca tagagataac 8940

cttcacccgg ttgccagagg tgcggattca ccacttgcaa agtcccgcta gtgccttgtc 9000

cagttgcaac cacctgttga tccgcatcac gcagttcaac gctgacatca ccattggcca 9060

ccacctgcca gtcaacagac gcgtggttac agtcttgcgc gacatgcgtc accacggtga 9120

tatcgtccac ccaggtgttc ggcgtggtgt agagcattac gctgcgatgg attccggcat 9180

agttaaagaa atcatggaag taagactgct ttttcttgcc gttttcgtcg gtaatcacca 9240

ttcccggcgg gatagtctgc cagttcagtt cgttgttcac acaaacggtg atacgtacac 9300

ttttcccggc aataacatac ggcgtgacat cggcttcaaa tggcgtatag ccgccctgat 9360

gctccatcac ttcctgatta ttgacccaca ctttgccgta atgagtgacc gcatcgaaac 9420

gcagcacgat acgctggcct gcccaacctt tcggtataaa gacttcgcgc tgataccaga 9480

cgttgcccgc ataattacga atatctgcat cggcgaactg atcgttaaaa ctgcctggca 9540

cagcaattgc ccggctttct tgtaacgcgc tttcccacca acgctgatca attccacagt 9600

tttcgcgatc cagactgaat gcccacaggc cgtcgagttt tttgatttca cgggttgggg 9660

tttctacagg acgtaacata agggactgac ctacccggga cctgcatata acctgcatat 9720

aacctgtaag atttagcacc ccaagttagt catgtaatta gccacatagc aaaaaaaata 9780

gcaccgtggt agtaagaatg gaactcacct ggtacctggt acctcggatc cgtgtttgac 9840

aggatatatt ggcgggtaaa cttcttcgta atgtgctcgc cgtcaccccc cttgtggaaa 9900

ccgttgtcga acctgatgga tcatggctga tctgccgcat ctacaggaca aggcagcgtg 9960

cattgcctgt tatcactcct cctgccatag aaaatgcaag ggaaatcatt atccctcctg 10020

ccaatggcaa tgcaagggag gctcaagtcc gctttattga cttcctgcag cagggatcgc 10080

acattgagtc atcctccccc tgtagttgta tagttggccc ctctttggca gagggaagtg 10140

atgaatctgc tggtagtgtt gaccaaaagg attga 10175

<210> 6

<211> 723

<212> DNA

<213> Rice (Dongjin)

<220>

<400> 6

atgcacctcc ccgctgtggg catgtcacac cccaccgagg gtgagcttgt cttccactac 60

ctctatcgtc gtgccgttaa catgccgctg ccctctgaat tcatttgtga tgtcaacgtt 120

cttcctcata acccatggga catcgtccca ggctcagaaa agccagtcta ctacaaccag 180

ggaggtggca gcgactgcat gctggtggga atgaggcgca ccctaacatt ttactttggg 240

aattcacgga ccgcagagcg cacaaaatgg ggtatgcaag aatttcgcct tgctggcaat 300

ggtctttcgc cttaccctgc gatgaagcat gccaccgggg atggctcaaa accaccctgc 360

aactgtgccg aaacaaccat cgccaagaga aatgatggtc tctctgcagt tcttcgtaat 420

gtgctcgccg tcacccccct tgtggaaacc gttgtcgaac ctgatggatc atggctgatc 480

tgccgcatct acaggacaag gcagcgtgca ttgcctgtta tcactcctcc tgccatagaa 540

aatgcaaggg aaatcattat ccctcctgcc aatggcaatg caagggaggc tcaagtccgc 600

tttattgact tcctgcagca gggatcgcac attgagtcat cctccccctg tagttgtata 660

gttggcccct ctttggcaga gggaagtgat gaatctgctg gtagtgttga ccaaaaggat 720

tga

<210> 7

<211> 432

<212> DNA

<213> Rice (Dongjin mutant)

<220>

<400> 5

atgcacctcc ccgctgtggg catgtcacac cccaccgagg gtgagcttgt cttccactac 60

ctctatcgtc gtgccgttaa catgccgctg ccctctgaat tcatttgtga tgtcaacgtt 120

cttcctcata acccatggga catcgtccca ggctcagaaa agccagtcta ctacaaccag 180

ggaggtggca gcgactgcat gctggtggga atgaggcgca ccctaacatt ttactttggg 240

aattcacgga ccgcagagcg cacaaaatgg ggtatgcaag aatttcgcct tgctggcaat 300

ggtctttcgc cttaccctgc gatgaagcat gccaccgggg atggctcaaa accaccctgc 360

aactgtgccg aaacaaccat cgccaagaga aatgatggtc tctctgcagt ggcaggatat 420

attgtggtgt aa

<210> 8

<211> 240

<212> PRT

<213> Rice (Dongjin)

<220>

<400> 8

Met His Leu Pro Ala Val Gly Met Ser His Pro Thr Glu Gly Glu Leu Val Phe His Tyr

1 5 10 15 20

Leu Tyr Arg Arg Ala Val Asn Met Pro Leu Pro Ser Glu Phe Ile Cys Asp Val Asn Val

25 30 35 40

Leu Pro His Asn Pro Trp Asp Ile Val Pro Gly Ser Glu Lys Pro Val Tyr Tyr Asn Gln

45 50 55 60

Gly Gly Gly Ser Asp Cys Met Leu Val Gly Met Arg Arg Thr Leu Thr Phe Tyr Phe Gly

65 70 75 80

Asn Ser Arg Thr Ala Glu Arg Thr Lys Trp Gly Met Gln Glu Phe Arg Leu Ala Gly Asn

85 90 95 100

Gly Leu Ser Pro Tyr Pro Ala Met Lys His Ala Thr Gly Asp Gly Ser Lys Pro Pro Cys

105 110 115 120

Asn Cys Ala Glu Thr Thr Ile Ala Lys Arg Asn Asp Gly Leu Ser Ala Val Leu Arg Asn

125 130 135 140

Val Leu Ala Val Thr Pro Leu Val Glu Thr Val Val Glu Pro Asp Gly Ser Trp Leu Ile

145 150 155 160

Cys Arg Ile Tyr Arg Thr Arg Gln Arg Ala Leu Pro Val Ile Thr Pro Pro Ala Ile Glu

165 170 175 180

Asn Ala Arg Glu Ile Ile Ile Pro Pro Ala Asn Gly Asn Ala Arg Glu Ala Gln Val Arg

185 190 195 200

Phe Ile Asp Phe Leu Gln Gln Gly Ser His Ile Glu Ser Ser Ser Pro Cys Ser Cys Ile

205 210 215 220

Val Gly Pro Ser Leu Ala Glu Gly Ser Asp Glu Ser Ala Gly Ser Val Asp Gln Lys Asp

225 230 235 240

<210> 9

<211> 143

<212> PRT

<213> Rice (Dongjin mutant)

<220>

<400> 9

Met His Leu Pro Ala Val Gly Met Ser His Pro Thr Glu Gly Glu Leu Val Phe His Tyr

1 5 10 15 20

Leu Tyr Arg Arg Ala Val Asn Met Pro Leu Pro Ser Glu Phe Ile Cys Asp Val Asn Val

25 30 35 40

Leu Pro His Asn Pro Trp Asp Ile Val Pro Gly Ser Glu Lys Pro Val Tyr Tyr Asn Gln

45 50 55 60

Gly Gly Gly Ser Asp Cys Met Leu Val Gly Met Arg Arg Thr Leu Thr Phe Tyr Phe Gly

65 70 75 80

Asn Ser Arg Thr Ala Glu Arg Thr Lys Trp Gly Met Gln Glu Phe Arg Leu Ala Gly Asn

85 90 95 100

Gly Leu Ser Pro Tyr Pro Ala Met Lys His Ala Thr Gly Asp Gly Ser Lys Pro Pro Cys

105 110 115 120

Asn Cys Ala Glu Thr Thr Ile Ala Lys Arg Asn Asp Gly Leu Ser Ala Val Ala Gly Tyr

125 130 135 140

Ile Val Val

Claims (3)

1. The application of the rice OsNAC129 in negative regulation of grain length and apparent amylose content is provided, and the nucleotide sequence of the rice OsNAC129 is shown as SEQ ID NO.1 or SEQ ID NO. 4.

2. The use of rice OsNAC129 in negative regulation of grain length and apparent amylose content according to claim 1, wherein the mutant loss-of-function mutation comprising OsNAC129 results in a significant increase in both grain length and apparent amylose content.

3. The use of rice OsNAC129 in negative regulation of grain length and apparent amylose content according to claim 2, wherein the nucleotide sequence of the mutant of OsNAC129 is shown as SEQ ID No.5, and the encoded protein sequence is shown as SEQ ID No. 9.

Images