CN115820695B - Gene PGI1 and PGI2 for regulating rice chalkiness, and encoding protein and application thereof - Google Patents

Abstract

The invention discloses genes PGI1 and PGI2 for regulating rice chalkiness, and encoding proteins and application thereof. The gene is PGI1 or PGI2, and the nucleotide sequences of the gene are respectively shown in SEQ ID NO.1 and SEQ ID NO. 2. The amino acid sequence of the protein coded by the gene is shown as SEQ ID NO.3 and SEQ ID NO. 4. According to the invention, two genes for controlling rice chalkiness are cloned, and after PGI1 and PGI2 in wild type are knocked out respectively through a crispr/cas9 gene editing technology, the knocked-out lines show that the rice chalkiness rate is increased, so that the two genes have the function of regulating rice chalkiness, can be used for cultivating high-yield and high-quality varieties of cereal crops such as rice and the like, and provide references for cloning and researching homologous genes in other species.

Description

Gene PGI1 and PGI2 for regulating rice chalkiness, and encoding protein and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to genes PGI1 and PGI2 for regulating rice chalkiness, and encoding proteins and application thereof.

Background

Rice is one of three major food crops in the world, more than half of the population worldwide takes rice as main food, and more than 60% of the population in China takes rice as main food. With the increasing population, the farmland area is drastically reduced, and the grain production is still under tremendous pressure, so that the improvement of the grain yield is a necessary requirement for the rice production. However, with the development of the national economic level, the requirements of people on rice change from the initial full meal to the good meal due to the improvement of the living standard of people. The rice with attractive appearance, good transparency and good palatability can be more favored by consumers. Therefore, the breeding objective of breeders is also changed from the first cultivation of high-yield rice varieties to the cultivation of high-yield high-quality rice varieties. The rice quality mainly comprises four aspects of appearance quality, grinding processing quality, cooking taste quality and nutrition quality, wherein the appearance quality is taken as an important constituent factor of the rice quality and has direct influence on the rice quality. Chalkiness are important indexes for evaluating rice appearance quality, and are also the most important indexes for variety approval.

Positioning, cloning and functional analysis of rice chalky Bai Xiangguan trait genes are beneficial to molecular genetic improvement of rice quality traits and improve rice quality. In recent years, there has been an increasing research on rice quality, especially on the molecular mechanism of chalking. The localization and cloning of rice chalkiness-related genes have made tremendous progress. Most of these cloned genes are involved in nutrient synthesis, such as Wx (Yang Yong et al 2019), ssimia (Fujita et al 2007), osBEIIb (Nishi et al 2001), osGluA2 (Yang et al 2019), GPA3 (Ren et al 2014), osAAP6 (pen et al 2014), osLTP36 (Wang et al 2015), osaot (Zhao et al 2019), RISBZ1 and RPBF (Kawakatsu et al 2009), ssima and ssimia (Zhang et al 2011), FLO6 (Xiong et al 2019), etc., and mutations of these genes affect the synthesis of nutrients such as starch, protein, lipid, etc. in grains, resulting in insufficient filling of endosperm storage substances, leading to the production of chalky rice. In addition, genes related to glycolytic pathway affect the chalk generation of rice, such as OsPK2 (Cai et al 2018), osPK3 (Hu et al 2020), osPPDKB (Kang et al 2005), PFP beta (PFP 1) (Chen et al 2020; duan et al 2016), etc., and these gene mutations affect starch biosynthesis during rice filling, and thus affect rice yield and rice quality. Cloning and functional analysis of these chalky related genes provide an increasing number of basis materials for molecular design breeding for improving rice yield and quality.

Glucose phosphate isomerase (Phosphate glucose isomerase, PGI) catalyzes the second step of the glycolytic process, mainly responsible for the interconversion between glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P), which is a reversible reaction. Only fructose-6-phosphate can enter the subsequent reaction of glycolysis, and thus phosphoglucose isomerase is an indispensable enzyme for the glycolysis pathway. The glycolytic pathway can provide a basic reaction for the gluconeogenic pathway. However, no report on the influence of the gene PGI1 on the quality of rice, especially on chalkiness of rice, is currently seen.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides genes PGI1 and PGI2 for regulating rice chalkiness, and encoding proteins and application thereof.

In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

a gene for regulating rice chalkiness is PGI1 or PGI2, and the nucleotide sequences of the gene are shown in SEQ ID NO.1 and SEQ ID NO. 2, and the specific sequences are as follows:

PGI1：ATGGCGTCGTCGGCGCTCATCTGCGACACCGAGCAGTGGAAGG GCCTCCAGGCGCATGTTGGGGAGATTCAGAAAACGCACCTGCGCCATCTGATGCATGATGTTGAGCGCTGCAAGGCAATGACAGCTGAGTATGAAGGCATATATCTGGATTACTCGAGGCAGCGTGCGACTGGCGAAACCATGGAGAAGCT

GTTTAAATTGGCCGAGGCTGCAAAGCTCAAGGAGAAGATTGAGAAGATGT

TTAGAGGTGACAAGATAAATAGCACAGAGAACAGATCAGTGCTTCATGTA

GCTCTAAGGGCTCCAAGAGACGAAGTAATAAATAGCAATGGGGTCAATGT

GGTTCCCGAAGTTTGGGGTGTAAAAGATAAAATCAAGCAATTTTCAGAAA

CTTTTAGGAGTGGATCATGGGTTGGGGCAACTGGTAAAGCATTGACAAATG

TTGTGTCAGTAGGAATAGGTGGTAGCTTTCTTGGTCCTCTGTTTGTGCATGC

TGCCCTCCAGACAGATCCAGAAGCTGCAGAATCTGCCAAAGGGCGGCAAT

TAAGATTTCTTGCAAATGTCGACCCTGTTGATGTTGCACGAAGCATCAAAG

ATTTAGATCCTGAAACAACACTTGTTGTGGTAGTCTCGAAGACCTTCACAA

CAGCTGAAACAATGTTAAATGCTCGAACTCTTAAGGAGTGGATTGTCTCTT

CTCTTGGACCTGATGCTGTTGCAAAACATATGATTGCTGTCAGTACCAATCT

TGAGCTTGTGGAGAAGTTTGGAATTGACCCGAAAAATGCTTTTGCATTTTG

GGACTGGGTTGGTGGCCGCTATAGTGTTTGCAGTGCTGTTGGTGTCCTGCC

CTTATCTCTTCAGTATGGCTTTCCGATTGTTCAGAAATTTTTGGAGGGTGCA

GCCAGCATCGACAAACACTTTCGTTCATCTTCATTTGAGAAAAATATTCCTG

TACTCCTTGGTTTGCTGAGTGTGTGGAATGTTTCATTTCTCGGATATCCAGC

TAGAGCAATACTGCCCTATTCCCAAGCACTTGAGAAATTTGCACCGCATATT

CAGCAGCTTAGCATGGAGAGTAATGGAAAGGGTGTCTCCATTGATGGTGTT

CAACTGCCCTTTGAGAGTGGTGAAATTGATTTTGGTGAACCTGGAACCAAT

GGGCAACACAGCTTCTATCAATTAATCCATCAGGGAAGAGTTATTCCTTGT

GATTTTATCGGTGTCGTAAAAAGCCAGCAACCTGTTTACTTGAAAGGGGAA

ATTGTGAGCAATCATGACGAATTGATGTCCAATTTCTTTGCTCAGCCTGATG

CGCTTGCTTATGGAAAGACTCCTGAACAACTGCATAGCGAGAAAGTACCT

GAACATCTTATCCCTCATAAGACTTTTCAGGGCAACCGACCATCGCTTAGTT

TATTGCTGCCCTCATTATCTGCTTATGAGATTGGACAGCTTTTAGCCATCTAC

GAGCACCGGATTGCAGTCCAGGGTTTCCTATGGGGAATAAACTCATTTGAC

CAGTGGGGAGTGGAACTGGGCAAGTCTCTTGCCTCTCAAGTGAGAAAATC

TCTACATGCATCCCGCGTTGAAGGAAAGCCTGTCCTGGGGTTTAACAGCAG

TACTACAAGTTTGCTGACACGATATCTTGCTGTTGAGCCATCCACTCCTTAC

AACACTACCACACTGCCGAAAGTTTGA

PGI2：ATGGCGTCGTCGGCGCTAATCTGCGACACCGAGCAGTGGAAGGGCCTCCAGGCGCACGTCGGGGCGATTCAGAAGACGCACCTGCGCGATCTGATGGATGATGCCGAGCGCTGCAAGGCAATGACAGCTGAGTATGAAGGCATATTTCTGGATTACTCGAGGCAGCGTGCAACTGGCGAGACCATGGAGAAGCTGTTTAAATTGGCAGAGGCGGCAAAGCTCAAGGAGAAGATTGAGAAGATGTTTAGTGGTGACAAGATAAATAGCACAGAGAACAGATCTGTGCTTCATGTAGCTCTAAGGGCTCCAAGAGACGAAGTAATAAAAAGTGATGGGGTCAATGTGGTTCCCGAAGTTTGGGGTGTAAAAGATAAAATCAAGCAGTTTTCAGAAACTTTTAGGAGTGGATCATGGGTTGGGGCAACTGGTAAAGCATTGACAAATGTTGTGTCAGTTGGAATAGGTGGTAGCTTTCTTGGTCCTCTGTTCGTGCATGCTGCCCTCCAGACAGATCCAGAAGCTGCAGAATCTGCCAAAGGGCGACAGTTAAGATTCCTTGCAAATGTTGACCCTGTTGATGTTGCACGAAGCATCAAAGATTTAGATCCTGAAACAACACTTGTTGTGGTTGTCTCAAAGACCTTCACAACAGCTGAAACAATGTTAAATGCTCGAACTCTCAAGGAGTGGATTGTCTCTTCTCTTGGACCTGATGCTGTTGCGAAACATATGATTGCTGTCAGTACCAATCTTGAGCTTGTGGAGAAGTTTGGAATTGACCCTAAAAATGCTTTTGCATTTTGGGACTGGGTTGGTGGTCGCTATAGTGTTTGCAGTGCTGTTGGTGTCCTGCCCTTATCTCTTCAATATGGTTTTCCGATTGTTCAGAAATTTTTGGAGGGTGCAGCCAGCATCGACAAACACTTCCGTTCATCTTCATTTGAGAAAAACATACCTGTACTCCTTGGTTTGCTGAGTGTGTGGAATGTTTCCTTTCTTGGATATCCAGCTAGAGCAATATTGCCCTATTCCCAGGCACTTGAGAAATTTGCACCACATATTCAGCAGCTTAGCATGGAGAGTAATGGAAAGGGTGTCTCTATTGATGGCGTTCAATTGTCCTTTGAGACTGGTGAAATTGATTTTGGTGAACCTGGAACCAATGGGCAACACAGCTTCTATCAATTAATCCATCAGGGAAGAGTTATTCCTTGTGATTTTATCGGCGTCGTGAAAAGCCAGCAACCCGTTTACTTGAAAGGGGAAATTGTGAGCAATCATGACGAATTGATGTCCAATTTCTTTGCTCAGCCTGATGCACTTGCTTATGGAAAGACCCCTGAACAACTGCACAGCGAGAAAGTACCTGAACATCTTATCTCTCATAAGACTTTTCAGGGCAACCGACCATCACTTAGTTTATTGCTGCCCTCATTATCTGCTTATGAGATTGGACAGCTTTTATCCATCTACGAGCACCGGATTGCAGTTCAGGGTTTCCTATGGGGAATAAACTCATTTGACCAGTGGGGAGTGGAACTGGGAAAGTCTCTGGCTTCTCAAGTGAGAAAATCTCTGCATGCATCCCGCATGGAAGGAAAGCCTGTCCAGGGGTTCAACAGCAGCACTGCAAGTTTGCTGACACGATATCTCGCTGTTGAGCCATCCACTCCTTACAACACTACAACAATGCCGAAAGTTTAA。

further, the sequence of the gene PGI1 or PGI2 is a sequence which has more than 80% homology with the nucleotide sequence shown in SEQ ID NO.1 or 2 and codes the same functional protein.

The amino acid sequence of the protein coded by the gene is shown as SEQ ID NO.3 and SEQ ID NO. 4, and the specific sequence is as follows:

MASSALICDTEQWKGLQAHVGEIQKTHLRHLMHDVERCKAMTAEYEGI

YLDYSRQRATGETMEKLFKLAEAAKLKEKIEKMFRGDKINSTENRSVLHVAL

RAPRDEVINSNGVNVVPEVWGVKDKIKQFSETFRSGSWVGATGKALTNVVS

VGIGGSFLGPLFVHAALQTDPEAAESAKGRQLRFLANVDPVDVARSIKDLDP

ETTLVVVVSKTFTTAETMLNARTLKEWIVSSLGPDAVAKHMIAVSTNLELVEK

FGIDPKNAFAFWDWVGGRYSVCSAVGVLPLSLQYGFPIVQKFLEGAASIDKH

FRSSSFEKNIPVLLGLLSVWNVSFLGYPARAILPYSQALEKFAPHIQQLSMESN

GKGVSIDGVQLPFESGEIDFGEPGTNGQHSFYQLIHQGRVIPCDFIGVVKSQQP

VYLKGEIVSNHDELMSNFFAQPDALAYGKTPEQLHSEKVPEHLIPHKTFQGN

RPSLSLLLPSLSAYEIGQLLAIYEHRIAVQGFLWGINSFDQWGVELGKSLASQV

RKSLHASRVEGKPVLGFNSSTTSLLTRYLAVEPSTPYNTTTLPKV(SEQ ID NO.3)

MASSALICDTEQWKGLQAHVGAIQKTHLRDLMDDAERCKAMTAEYEGIFLDYSRQRATGETMEKLFKLAEAAKLKEKIEKMFSGDKINSTENRSVLHVALRAPRDEVIKSDGVNVVPEVWGVKDKIKQFSETFRSGSWVGATGKALTNVVSVGIGGSFLGPLFVHAALQTDPEAAESAKGRQLRFLANVDPVDVARSIKDLDPETTLVVVVSKTFTTAETMLNARTLKEWIVSSLGPDAVAKHMIAVSTNLELVEKFGIDPKNAFAFWDWVGGRYSVCSAVGVLPLSLQYGFPIVQKFLEGAASIDKHFRSSSFEKNIPVLLGLLSVWNVSFLGYPARAILPYSQALEKFAPHIQQLSMESNGKGVSIDGVQLSFETGEIDFGEPGTNGQHSFYQLIHQGRVIPCDFIGVVKSQQPVYLKGEIVSNHDELMSNFFAQPDALAYGKTPEQLHSEKVPEHLISHKTFQGNRPSLSLLLPSLSAYEIGQLLSIYEHRIAVQGFLWGINSFDQWGVELGKSLASQVRKSLHASRMEGKPVQGFNSSTASLLTRYLAVEPSTPYNTTTMPKV(SEQ ID NO.4)

an expression cassette, expression vector or cloning vector comprising the nucleotide sequence described above.

Engineering bacteria containing the genes PGI1 or PGI2, or expression cassettes, expression vectors or cloning vectors.

The application of the gene PGI1 or PGI2 in rice variety improvement.

The application of the gene PGI1 or PGI2 in preparing transgenic plants.

An agent for regulating rice chalkiness, which comprises an active ingredient for inhibiting the expression of the above genes or for knocking out the above genes.

Further, the active ingredient is a small molecule compound, shRNA, gRNA or short peptide.

The invention has the beneficial effects that:

according to the invention, two genes for controlling rice chalkiness are cloned, and after PGI1 and PGI2 in wild type are knocked out respectively through a crispr/cas9 gene editing technology, the knocked-out lines show that the rice chalkiness rate is increased, so that the two genes have the function of regulating rice chalkiness, can be used for cultivating high-yield and high-quality varieties of cereal crops such as rice and the like, and provide references for cloning and researching homologous genes in other species.

Drawings

FIG. 1 is a map of a reporter GUS vector;

FIG. 2 shows the space-time expression profile of PGI1 gene in rice whole growth period and GUS staining analysis; wherein a is the expression mode of rice in PGI1 gene: r1 (young root), R2 (old root), LB1 (seedling stage leaf), LB2 (tillering stage young leaf), LB3 (tillering stage heart leaf), LB4 (grouting stage sword leaf), S (booting stage festival), C (booting stage internode), YP3 (booting stage 3cm long young ear), YP12 (booting stage 12cm long young ear), E5-E25 (post-fertilization 5-25 days caryopsis); b is the GUS transgenic material dyeing result;

FIG. 3 shows the space-time expression profile of PGI2 gene in rice whole growth period and GUS staining analysis; wherein a is the expression mode of rice in PGI2 gene: r1 (young root), R2 (old root), LB1 (seedling stage leaf), LB2 (tillering stage young leaf), LB3 (tillering stage heart leaf), LB4 (grouting stage sword leaf), S (booting stage festival), C (booting stage internode), YP3 (booting stage 3cm long young ear), YP12 (booting stage 12cm long young ear), E5-E25 (post-fertilization 5-25 days caryopsis); b is the GUS transgenic material dyeing result;

FIG. 4 is a map of the crispr/cas9 system vector;

FIG. 5 shows the phenotype of PGI1 knockout transgenic lines and test data; wherein a is a schematic diagram of PGI1 gene editing result: "." indicates the base deletion at this position and the red letter indicates the base insertion at this position; b is the plant leaf shape comparison of the knocked-out material and the wild material; c and g are grain length comparisons of knockout material to wild type material; d and h are grain width comparisons of the knocked-out material and the wild-type material; e is the polished rice appearance of the knocked-out material compared with the wild-type material; f is the plant height comparison of the knocked-out material and the wild material; i is the thousand grain weight comparison of the knocked-out material and the wild material; j is the rice chalkiness ratio of the knockout material compared to the wild-type material; "x" indicates that the difference is significant at the 0.01 level;

FIG. 6 shows the phenotype of PGI2 knockout transgenic lines and test data; wherein a is a schematic diagram of PGI2 gene editing result: the red letter indicates the base insertion at that position; b is the plant leaf shape comparison of the knocked-out material and the wild material; c and g are grain length comparisons of knockout material to wild type material; d and h are grain width comparisons of the knocked-out material and the wild-type material; e is the polished rice appearance of the knocked-out material compared with the wild-type material; f is the plant height comparison of the knocked-out material and the wild material; i is the thousand grain weight comparison of the knocked-out material and the wild material; j is the rice chalkiness ratio of the knockout material compared to the wild-type material; "×" indicates significant differences at the 0.05 level and "×" indicates significant differences at the 0.01 level.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.

Example 1 Gene sequence acquisition

1. Sequence acquisition

The PGI1 protein consists of 567 amino acids, the corresponding gene contains 6969 nucleotides, the PGI2 protein also consists of 567 amino acids, and the corresponding gene contains 6969 nucleotides. The information of the protein and nucleotide sequence which code the same function is derived from Rice Genome Annotation Project%http://rice.uga.edu/cgi-bin/ORF_ infopage.cgi)

2. qPCR primer design

A pair of quantitative primers, AK1307-F-PGI1 (TGGACGGTGGACCAAGCAACA), AK1308-R-PGI1 (CGTGGGCTGAAAATAGTAGGG), AK1309-F-PGI2 (TTCCTCTTCCCGAATTGCTT) and AK1310-R-PGI2 (GAACCGAACAGACAGCCAAT), were designed in the 5' -UTR regions of the PGI1 and PGI2 genes, respectively, using software Primer 5.

3. Space-time expression profile analysis of PGI1 and PGI2 genes in whole growth period of rice

Extracting RNA in roots, stems, leaves, leaf sheaths, young ears, glumes and caryopsis of paddy rice and 3d and 6d caryopsis after flowering, carrying out expression quantity analysis by QPCR after reverse transcription, and the result shows that: PGI1 and PGI2 were constitutively expressed, and were expressed in all tissues of rice, and PGI1 and PGI2 were expressed in the highest amounts in the leaves (see FIG. 2 a).

4. GUS vector construction and transformation

The 2kb (PGI 1 and PGI 2) genomic fragments upstream of the ATG start codon were amplified by PCR using ZH11 DNA as template and the PCR products were gel recovered with single fragment recombinase to ligate to the linearized (HindIII/BamHI) DX2181 vector. And delivering the hundred-cell gene transgenic japonica rice ZH11 material. (FIG. 1)

5. GUS material staining

And (5) taking each tissue and organ of the positive GUS transgenic plant for GUS staining analysis.

(1) The X-Gluc Solution (50X) and GUSBuffer were mixed well in a ratio of 1:50 to prepare an X-Gluc staining Solution.

(2) Each tissue was placed in a 5ml centrifuge tube, and an appropriate amount of staining solution was added to completely submerge the tissue in the staining solution.

(3) Incubate at 37℃in the dark for 1-24h until blue color appears on the tissue.

(4) The chlorophyll in the tissues is removed repeatedly by 70% ethanol until the chlorophyll is completely removed.

(5) Samples were stored in 70% ethanol and observed and photographed using a split microscope (LEICA S60, germany). (FIGS. 2 and 3)

GUS staining in FIGS. 2 and 3 shows that PGI1 and PGI2 genes have obvious GUS signals in pollen in leaves, nodes and internodes, anthers, and show that the genes are expressed in various periods of rice growth and development.

Example 2 construction of PGI1 and PGI2 Gene knockout strains

1. Construction of the Crispr/cas9 knockout vector for PGI1 and PGI2 and genetic transformation

(1) Design and selection of guide RNA (gRNA) target site sequences

1 guide RNA target sequence of PGI1 and PGI2 was designed and synthesized based on the genomic sequences of PGI1 and PGI2, respectively.

(2) Target site CRISPR/Cas9-gRNA vector construction

The oligonucleotide strand of the target sequence of the guide RNA synthesized in the step (1) is denatured at 95 degrees, then mixed with intermediate vectors of pYL-U6a-gRNA and pYL-U6b-gRNA respectively, bsa1 endonuclease, T4 ligase and buffer are added, enzyme digestion connection is carried out in 5min at 37 ℃ and 5min at 20 ℃ on a PCR instrument for 5 cycles, then a gRNA expression cassette containing target sites is obtained through two rounds of PCR, and then the gRNA expression cassettes are sequentially loaded on the CRISPR/Cas9 vectors through a Golden gate cloning method, so that two CRISPR/Cas9-gRNA vectors containing 1 target site are obtained. (see FIG. 4)

(3) Genetic transformation

The Kittake seeds are sterilized by sodium hypochlorite solution and placed on an induction culture medium, induced calli are picked for subculture after about 2 weeks, and calli with vigorous growth are picked for agrobacteria infection after 1 week.

Two CRISPR/Cas9-gRNA vectors with 1 target spot are transferred into agrobacterium strain EH105, monoclonal shaking bacteria are selected, the callus is impregnated, the co-culture is carried out for 3 days at 25 ℃ under the dark condition, and then the transfer is carried out on a screening culture medium containing hygromycin for screening about 10 days. Transferring the selected calli to a differentiation medium for culture, and removing the calli from a test tube for transplanting after the calli grow into normal seedlings.

2. Knock-out transgenic seedling detection

Finally, extracting DNA from plant leaves, firstly screening by using hygromycin detection primers to obtain positive plants, and then respectively using amplification primers crossing target sites:

AK1279-F-PGI1：5’-TCTCCTGAGGGGGACGTTTAT-3’；

AK1280-R-PGI1：5’-ATGCTTTACCAGTTGCCCCA-3’；

AK1281-F-PGI2：5’-CTCCAGGTGCGTGAGTTGAT-3’；

AK1282-R-PGI2：5’-TGAGCTTTGCCGCCTACAAT-3’；

the sequences in the positive individuals are amplified and then sequenced by the engine sequencing company. As a result, as shown in FIGS. 5 and 6, transgenic plants whose positive Miao Cexu obtained sequences showed that PGI1 gene gave 3 premature protein translation termination in total were KO-1, KO-2 and KO-3 in FIG. 5, respectively, and transgenic plants whose PGI2 gene gave 2 premature protein translation termination in total were KO-1 and KO-2 in FIG. 6, respectively.

Example 3 effects of PGI1 and PGI2 Gene knockout on Rice phenotypes

In the T0 and T1 generation planting, agronomic traits were observed for different lines and no significant differences were observed for the knockout mutants, except chalkiness, compared to the wild type kitake. After obtaining T2 generation knockout plants with stable inheritance, field planting is repeated three times, each repeated planting is performed for 2 rows, and first sequencing detection is performed to confirm target site mutation. And then, when the agronomic characters are inspected, each side row and the mixed plant are repeatedly removed, 5-10 stable plant lines are selected, and the characters of the single plant are inspected.

As shown in fig. 5 and 6, the PGI1 and PGI2 gene knockout strains have no significant differences in rice grain length, rice grain width, thousand grain weight and plant height compared to the wild type, but the grain chalkiness rates of the PGI1 and PGI2 knockout mutant seeds are significantly increased. Thus, PGI1 and PGI2 are both rice grain chalkiness regulatory genes (fig. 5j and 6 j).

The foregoing is merely a few embodiments of the present invention and it should be noted that all modifications directly derived or suggested to a person skilled in the art from the disclosure of the present invention should be considered as being within the scope of the present invention.

Claims (2)

1. The application of the gene PGI1 or PGI2 in improving the chalkiness rate of rice seeds is provided, and the nucleotide sequences of the gene PGI1 or PGI2 are shown as SEQ ID NO.1 and SEQ ID NO. 2 respectively.

2. The application of knockout gene PGI1 or PGI2 in preparing transgenic rice with increased grain chalkiness rate is disclosed, wherein the nucleotide sequences of the gene PGI1 or PGI2 are shown in SEQ ID NO.1 and SEQ ID NO. 2 respectively.