Application of rice malic acid synthase gene in improving nutritional quality of seeds

Abstract

The invention discloses application of a rice malic acid synthase gene in improving the nutritional quality of seeds. The invention reports that the OsIPMS1 and OsIPMS2 genes can regulate and control the nutritional quality in polished rice grains for the first time in rice, and tests show that the mutation of the OsIPMS1 and OsIPMS2 genes improves the content of protein and amino acid in the polished rice grains. It is proved that the OsIPMS1 and OsIPMS2 genes regulate and control the nutritional quality of rice seeds, and the genes are beneficial to cultivation of rice varieties with high protein and amino acid.

Description

Technical Field

[0001] This invention discloses two rice nutritional quality-related proteins, belonging to the field of rice genetic engineering, and involves the cloning, functional verification, and application of two α-isopropylmalate synthase genes that control rice nutritional quality. Background Technology

[0002] Rice (Oryza sativa L.) is one of the most important food crops in my country. With the improvement of people's living standards, the importance attached to rice quality is increasing. Rice quality includes milling quality, appearance quality, eating quality, and nutritional quality. Currently, a large number of genetic studies related to milling, appearance, and eating quality have been conducted. However, due to the complexity of nutritional quality genetics and the relative difficulty in detection, there are few reports on the localization and gene cloning of nutritional quality. Elucidating the genetic laws of rice nutritional quality is of great significance for rice quality improvement. Previous reports have shown that OsIPMS1 can enhance glycolysis and the tricarboxylic acid cycle biochemical reactions during seed germination, providing more energy for seed germination and seedling growth. At the same time, the seed priming effect is also closely related to the level of OsIPMS1 mRNA in priming seeds. OsIPMS1 can be used as a biomarker to determine the optimal cessation time of rice seed priming. However, the application of OsIPMS1 and OsIPMS2 in the breeding of rice varieties with high protein and amino acid content has not yet been reported. Summary of the Invention

[0003] The purpose of this invention is to provide the isolation, cloning, functional verification, and application of two rice nutritional quality-related protein genes.

[0004] The objective of this invention can be achieved through the following technical solutions:

[0005] The two genes of rice α-isopropylmalate synthase, OsIPMS1 and OsIPMS2, of this invention have nucleotide sequences as shown in SEQ ID NO.1 and SEQ ID NO.2, and amino acid sequences as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0006] The application of any one or more of the rice OsIPMS1 and OsIPMS2 genes in the breeding of rice varieties with high protein and amino acid content, characterized in that the nucleotide sequences of the OsIPMS1 and OsIPMS2 genes are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0007] As a preferred embodiment of the present invention, knocking out the OsIPMS1 gene and / or the OsIPMS2 gene in rice can increase the protein and amino acid content in rice grains.

[0008] The application of any one or both of the proteins encoded by rice OsIPMS1 and OsIPMS2 described in this invention in the cultivation of rice varieties with high protein and amino acid content.

[0009] The method for obtaining and verifying the gene function of rice OsIPMS1 and OsIPMS2 gene mutants according to the present invention includes the following steps:

[0010] (1) Obtain the nucleotide and amino acid sequences of the rice OsIPMS1 and OsIPMS2 genes;

[0011] (2) Design target sites and their primers, perform PCR amplification using pCBC-MT1T2 as a template, purify and recover the PCR product, and obtain the MT1T2-PCR intermediate vector containing OsIPMS1 and OsIPMS2 gRNA target sequences.

[0012] (3) The MT1T2-PCR gel recovery product containing the target fragments of OsIPMS1 and OsIPMS2 genes obtained in step (2) is constructed into the pHUE411 vector to obtain the pHUE411+MT1T2-PCR vector containing fragments of OsIPMS1 and OsIPMS2 genes.

[0013] (4) Transform Agrobacterium with the target fragments of the OsIPMS1 and OsIPMS2 genes obtained in step (3) into Agrobacterium; transform Agrobacterium with the transformation plasmid into rice;

[0014] (5) Screening and identification of rice mutants, and identification of germination phenotype.

[0015] Furthermore, the rice OsIPMS1 and OsIPMS2 CRISPR / Cas9 mutant gRNA target sequences (19bp target fragments with the same sequence in the OsIPMS1 and OsIPMS2 genes) constructed in step (2) are shown in SEQ ID NO.5 and SEQ ID NO.6; the primer sequences for PCR amplification using pCBC-MT1T2 as a template are shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, where SEQ ID NO.7 and SEQ ID NO.8 are primer sequences for the OsIPMS1 gene gRNA target sequence, and SEQ ID NO.9 and SEQ ID NO.10 are primer sequences for the OsIPMS2 gene gRNA target sequence.

[0016] In step (3), the pHUE411 vector was digested with BsaI enzyme, and the pHUE411+MT1T2-PCR vector was obtained by homologous recombination.

[0017] In step (5), the upstream primer sequence used for screening the OsIPMS1 homozygous mutant is shown in SEQ ID NO.11, and the downstream primer sequence is shown in SEQ ID NO.12. The upstream primer sequence used for screening the OsIPMS2 homozygous mutant is shown in SEQ ID NO.13, and the downstream primer sequence is shown in SEQ ID NO.14.

[0018] The method for detecting the expression levels of OsIPMS1 and OsIPMS2 genes during seed filling process, as described in this invention, includes the following steps:

[0019] (1) Collect rice seeds at different grain-filling times after flowering;

[0020] (2) RNA was extracted from each sample using the OMEGAHP Plant RNA Kit (R6837-02);

[0021] (3) Use II. The Reverse Transcriptase system (Vazyme Biotech Co., Ltd.) kit is used to reverse transcribe cDNA, which is then used as a template.

[0022] (4) Analysis was performed using quantitative real-time PCR. The primer sequences for quantitative real-time PCR detection were as follows: 5'-GCCGAGAGCTCTTAGGAGGTCT-3' and 5'-TTTC CCAAGAACAATACCAAACTCG-3' for OsIPMS1 and 5'-CTTCCCAACAATACCAAACTCG-3' for OsIPMS2.

[0023] As shown in further step (1), rice seeds were sampled 0, 5, 10, 15, 20, 25, 30 and 35 days after flowering; after being frozen with liquid nitrogen, they were stored at -80℃, and three samples were taken at each time point.

[0024] In step (4), the rice internal reference gene OsActin primers are used. The sequence of the upstream primer is shown as 5'-AGGAAGGCTGGAAGAGGACC-3', and the sequence of the downstream primer is shown as 5'-CGGGAAATTGTGAGGGACAT-3'.

[0025] Beneficial effects:

[0026] This invention isolates and clones the OsIPMS1 and OsIPMS2 genes from rice and identifies the functions of these two genes in regulating nutritional quality, which is of great significance for improving the nutritional quality of rice.

[0027] The present invention has the following advantages:

[0028] (1) The present invention isolates and clones the OsIPMS1 and OsIPMS2 genes from rice, and demonstrates for the first time that the OsIPMS1 and OsIPMS2 genes are involved in the regulation of the nutritional quality of rice seeds by constructing CRISPR / Cas9 mutants.

[0029] (2) This invention provides a foundation for improving the nutritional quality of rice and provides important genetic resources for improving rice quality, which is of great significance to production. Attached Figure Description

[0030] Figure 1 Expression of rice OsIPMS1 and OsIPMS2 genes during seed development

[0031] Figure 2 Protein and amino acid content of single and double mutants of rice OsIPMS1 and OsIPMS2 in the japonica rice variety Nipponbare Seiko rice.

[0032] Figure 3 The protein and amino acid content of OsIPMS1 and OsIPMS2 double mutants in the promoted varieties Wuyunjing 23 and Huanghua glutinous rice. Detailed Implementation

[0033] This invention is further described in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified, the methods used in the embodiments are all conventional methods. The primers and sequencing were performed by Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd. Various restriction endonucleases used in the experiments were purchased from Kangrun Jingxing (Suzhou) Biotechnology Co., Ltd. The genome extraction kit was purchased from Zhengzhou Suling Biotechnology Co., Ltd., and the reverse transcription kit was purchased from Novizan Biotechnology Co., Ltd. The plasmid extraction kit and gel extraction kit were purchased from Meiji Biotechnology Co., Ltd. All methods were performed in accordance with the instructions.

[0034] Example 1: Gene Cloning and Mutant Construction

[0035] The nucleotide sequences of the rice OsIPMS1 and OsIPMS2 genes shown in SEQ ID NO.1 and SEQ ID NO.2, and the amino acid sequences shown in SEQ ID NO.3 and SEQ ID NO.4 were obtained using the rice MSU7.0 database (http: / / rice.uga.edu / index.shtml).

[0036] Log in to http: / / skl.scau.edu.cn / targetdesign / to screen for targets. The target sequences of the OsIPMS1 and OsIPMS2 genes are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. Primers were designed based on the target sequences. Primers for the OsIPMS1 target sequence are shown in SEQ ID NO.7 and SEQ ID NO.8, and primers for the OsIPMS2 target sequence are shown in SEQ ID NO.9 and SEQ ID NO.10. PCR amplification was performed using pCBC-MT1T2 as a template with the four primers shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10. The amplification system was: 1 μL pCBC-MT1T2, 1 μL each primer, 15 μL Mix, and 10 μL ddH2O. The amplification program was as follows: 98℃ for 30 s, 98℃ for 10 s, 58℃ for 5 s, 72℃ for 5 s, 32 cycles, 72℃ for 1 min. The PCR product was purified and recovered to obtain the target sequence MT1T2-PCR containing the gRNA of the OsIPMS1 and OsIPMS2 genes. The pHUE411 vector was digested with BsaI, and the gel-recovered product of MT1T2-PCR was constructed into the pHUE411 vector using homologous recombination to obtain the pHUE411+MT1T2-PCR vector.

[0037] The obtained pHUE411+MT1T2-PCR vector was transformed into Agrobacterium. Agrobacterium carrying the transformation plasmid was then transformed into wild-type japonica rice varieties Nipponbare and Wuyunjing 23, and indica rice variety Huanghuazhan. The PCR amplification products were sequenced and compared with wild-type strains. Single and double mutants of OsIPMS1 and OsIPMS2 were screened against the Nipponbare background. Two homozygous double mutants were screened against the Wuyunjing 23 and Huanghuazhan backgrounds, respectively. The upstream and downstream primers for screening the homozygous CRISPR / Cas9 mutant of the OsIPMS1 gene are shown in SEQ ID NO.11 and SEQ ID NO.12, and the upstream and downstream primers for screening the homozygous CRISPR / Cas9 mutant of the OsIPMS2 gene are shown in SEQ ID NO.13 and SEQ ID NO.14.

[0038] Example 2: Gene Expression Analysis During Seed Development

[0039] Seeds of the Nipponbare rice variety were collected at 0, 5, 10, 15, 20, 25, 30, and 35 days after flowering. After liquid nitrogen freezing, the seeds were rapidly ground into powder. RNA was extracted from each sample using the OMEGAHP Plant RNA Kit (R6837-02). II. The Reverse Transcriptase system (Vazyme Biotech Co., Ltd.) kit was used to reverse transcribe cDNA, which was then used as a template for analysis. Real-time PCR was employed for analysis. Primers for OsIPMS1 were shown as 5'-GCCGAGAGCTCTTAGGAGGTCT-3' and 5'-TTTCCCAAGAACAATACCAAACTCG-3', and primers for OsIPMS2 were shown as 5'-CGTCGAGAACTCTTAGGAGGTCTGTA-3' and 5'-CTTCCCAAGAACAATACCAAACTCA-3'. Primers for the rice internal reference gene OsActin were used. The sequence of the upstream primer was shown as 5'-AGGAAGGCTGGAAGAGGACC-3', and the sequence of the downstream primer was shown as 5'-CGGGAAATTGTGAGGGACAT-3'.

[0040] The results showed that the expression levels of OsIPMS1 and OsIPMS2 were highest during the 10-15 day period after seed filling. Figure 1 ).

[0041] Example 3: Phenotypic Analysis of Gene Mutants

[0042] Using single mutants (Osipms1-1, Osipms1-2, Osipms2-1, Osipms2-2) and double mutants (Osipms1 / 2) with a Nipponbare background, as well as Osipms1 / 2 mutants with Huang Huazhan and Wu Yunjing 23 backgrounds and wild-type Nipponbare (WT) rice seeds, the protein and amino acid content in polished rice seeds were compared using a Coomassie Brilliant Blue assay kit (catalog number: KMSP-1-W) and an AAA L-8900 automatic amino acid analyzer. The results showed that compared with wild-type polished rice, the protein content and free amino acid content in polished rice from the Osipms1 and Osipms2 single mutants and the Osipms1 / 2 double mutant were significantly increased. Figure 2 The results showed that mutating this gene plays an important role in significantly increasing the protein and amino acid content in polished rice grains.

[0043] Example 4: Phenotypic Analysis of Gene Mutants in Conventional Materials

[0044] The protein and amino acid content in polished rice grains were compared using two mutants, Osipms1 / 2-a and Osipms1 / 2-b, constructed with the background of the popular japonica rice variety Wuyunjing 23, and two mutants, Osipms1 / 2-1 and Osipms1 / 2-2, constructed with the background of the popular indica rice variety Huanghuazhan. The results showed that, compared with the control seeds, the protein and amino acid content in the polished rice grains of the mutants was significantly increased. Figure 3 It is evident that mutating this gene plays a crucial role in improving the nutritional quality of grains in rice varieties that are widely cultivated.

Claims

1. The application of any one or more of the rice OsIPMS1 and OsIPMS2 genes in the breeding of rice varieties with high protein and amino acid content, characterized in that... The nucleotide sequences of the OsIPMS1 and OsIPMS2 genes are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

2. The application according to claim 1, characterized in that... Knocking out the OsIPMS1 and / or OsIPMS2 genes in rice can increase the protein and amino acid content in rice grains.

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