Rice CONSTANS-LIKE 5 gene and its interacting protein OsIDS1 in the regulation of rice heading date

Abstract

The application relates to the technical field of agricultural bioengineering, and particularly provides application of a rice CONSTANS-LIKE 5 gene and an interaction protein OsIDS1 in regulating a rice heading stage. The CONSTANS-LIKE 5 gene is cloned from rice Nipponbare NIP, is a transcription factor protein with 372 amino acids, and belongs to the CCT / B-box zinc finger protein family. Through a genetic transformation experiment, it is proved that overexpression of the CONSTANS-LIKE 5 gene promotes rice heading under short-day conditions, and knockout or silencing of the CONSTANS-LIKE 5 gene inhibits rice heading under short-day conditions; overexpression of the OsIDS1 gene inhibits rice heading under short-day conditions, and knockout or silencing of the OsIDS1 gene promotes rice heading under short-day conditions.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of agricultural bioengineering, and particularly relates to application of rice CONSTANS-LIKE 5 gene and its interacting protein OsIDS1 in regulation of rice heading stage. BACKGROUND

[0002] Rice is the largest global consumption of staple crops, feeding more than half of the world's population. Heading stage is a very important agronomic trait for seasonal and regional adaptability. In the past 20 years, many genes controlling heading stage have been cloned. The mechanism of rice flowering uses leaf to recognize day length, and then transmits the signal to the activation of the flower organ. If a cultivated variety flowers too early, it will cause insufficient use of light and temperature, resulting in reduced yield; conversely, if a variety has too late heading stage, it is difficult to complete the entire flowering and grain development cycle, also leading to reduced yield. In rice, heading stage is affected by exogenous factors such as photoperiod, temperature and nutrition, among which photoperiod is a major control factor. Rice itself is a short-day plant, which promotes heading under short-day conditions and inhibits heading under long-day conditions. The currently cloned heading stage regulation genes either have a large effect or affect the yield and plant type of rice. A slight delay of 3-7 days in heading stage can allow rice to have an additional period of vegetative growth, accumulate biomass, and improve rice yield. Cloning and mining of heading stage genes are of great significance for improving rice adaptability and improving rice varieties. SUMMARY

[0003] In view of the above problems in the prior art, the application provides application of rice CONSTANS-LIKE 5 gene and its interacting protein OsIDS1 in regulation of rice heading stage. The application also provides a complete set of genes and gene modification vectors that can be used for improvement of new varieties, relatively free regulation of rice plant height, and provides new gene resources and methods for modern agricultural molecular breeding.

[0004] To achieve the above application purposes, the application adopts the following technical solutions:

[0005] The application provides, in a first aspect, application of rice CONSTANS-LIKE 5 gene in regulation of rice heading stage. The CDS sequence of the CONSTANS-LIKE 5 gene is shown as SEQ ID No. 1, and the amino acid sequence of the protein encoded by the CONSTANS-LIKE 5 gene is shown as SEQ ID No. 2. The application is as follows: overexpression of the CONSTANS-LIKE 5 gene promotes rice heading under short-day conditions, and knockout or silencing of the CONSTANS-LIKE 5 gene inhibits rice heading under short-day conditions.

[0006] Further, the overexpression of the CONSTANS-LIKE 5 gene promotes rice heading under short-day conditions, comprising the following steps:

[0007] (1) Extract total RNA from rice leaves, and obtain cDNA by reverse transcription. Use the cDNA as a template, and perform PCR amplification using primer pair CONSTANS-LIKE 5-F1 / CONSTANS-LIKE 5-R1. The base sequence of the CONSTANS-LIKE 5-F1 is shown in SEQ ID No. 6, and the base sequence of the CONSTANS-LIKE 5-R2 is shown in SEQ ID No. 7.

[0008] (2) Connect the PCR product obtained in step (1) into an expression vector to obtain an overexpression recombinant vector;

[0009] (3) Transform the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain;

[0010] (4) Transform the overexpression recombinant strain into a rice plant, and screen and obtain a CONSTANS-LIKE 5 gene overexpression strain.

[0011] Further, the knockout or silencing of the CONSTANS-LIKE 5 gene inhibits rice heading under short-day conditions, comprising the following steps:

[0012] (1) Design a target point CONSTANS-LIKE 5-sg of the CONSTANS-LIKE 5 gene and a primer pair CONSTANS-LIKE 5-F2 / CONSTANS-LIKE 5-R2 of the target point CONSTANS-LIKE 5-sg, and construct a CRISPR / Cas9 vector;

[0013] (2) Construct an Agrobacterium genetic engineering bacterium containing the CRISPR / Cas9 vector of step (1);

[0014] (3) Transform the Agrobacterium genetic engineering bacterium of step (2) into a rice plant to obtain a homozygous mutant strain containing no T-DNA and stably inherited.

[0015] Further, the base sequence of the target point CONSTANS-LIKE 5-sg is shown in SEQ ID No. 5, the base sequence of the CONSTANS-LIKE 5-F2 is shown in SEQ ID No. 3, and the base sequence of the CONSTANS-LIKE 5-R2 is shown in SEQ ID No. 4.

[0016] The application provides the application of the rice OsIDS1 gene in regulating the heading stage of rice, the CDS sequence of the OsIDS1 gene is shown as SEQ ID No. 8, the amino acid sequence of the protein encoded by the OsIDS1 gene is shown as SEQ ID No. 9, the application is as follows: overexpression of the OsIDS1 gene inhibits the heading of rice under short-day conditions, and knockout or silencing of the OsIDS1 gene promotes the heading of rice under short-day conditions.

[0017] Further, the overexpression of the OsIDS1 gene under short-day conditions inhibits the heading of rice, and the method comprises the following steps:

[0018] (1) total RNA is extracted from rice leaves, cDNA is obtained by reverse transcription, and cDNA is used as a template to perform PCR amplification by using a primer pair OsIDS1-F1 / OsIDS1-R1, the base sequence of the OsIDS1-F1 is shown as SEQ ID No. 13, and the base sequence of the OsIDS1-R2 is shown as SEQ ID No. 14;

[0019] (2) the PCR product obtained in the step (1) is connected into an expression vector to obtain an overexpression recombinant vector;

[0020] (3) the overexpression recombinant vector is transformed into agrobacterium to obtain an overexpression recombinant strain;

[0021] (4) the overexpression recombinant strain is transformed into a rice plant, and an OsIDS1 gene overexpression strain is screened and obtained.

[0022] Further, the knockout or silencing of the OsIDS1 gene under short-day conditions promotes the heading of rice, and the method comprises the following steps:

[0023] (1) a target OsIDS1-sg of the OsIDS1 gene and a primer pair OsIDS1-F2 / OsIDS1-R2 of the target OsIDS1-sg are designed to construct a CRISPR / Cas9 vector;

[0024] (2) an agrobacterium genetic engineering bacterium containing the CRISPR / Cas9 vector in the step (1) is constructed;

[0025] (3) the agrobacterium genetic engineering bacterium in the step (2) is transformed into a rice plant to obtain a homozygous mutant strain containing no T-DNA and being stably inherited.

[0026] Further, in step (1), the base sequence of the target point OsIDS1-sg is shown as SEQ ID No. 12, the base sequence of OsIDS1-F2 is shown as SEQ ID No. 10, and the base sequence of the OsIDS1-R2 is shown as SEQ ID No. 11

[0027] The third aspect of the present application provides a protein OsIDS1 interacting with CONSTANS-LIKE 5, the amino acid sequence of the CONSTANS-LIKE 5 is shown as SEQ ID No. 2, and the amino acid sequence of the protein OsIDS1 is shown as SEQ ID No. 9.

[0028] The present application has the following beneficial effects:

[0029] The present application clones the rice heading stage regulation gene CONSTANS-LIKE 5 from the rice variety Nipponbare NIP. The method for extending and shortening the rice heading stage genes is obtained by using the CONSTANS-LIKE 5 gene and the downstream interaction gene. Under short-day conditions, the CONSTANS-LIKE 5 gene knockout or silencing vector of the present application can extend the heading stage of rice after being transformed into rice; overexpression of the CONSTANS-LIKE 5 gene can shorten the heading stage of rice, and the time difference is within 7 days on average. On the other hand, the protein OsIDS1 interacting with the CONSTANS-LIKE 5 is provided, and the OsIDS1 gene knockout or silencing vector can shorten the heading stage of rice after being transformed into rice; overexpression of the OsIDS1 gene can significantly extend the heading stage of rice. The set of genes and gene modification vectors provided by the present application can be used for improvement of new varieties, and the relative free regulation of rice height provides new gene resources and methods for modern agricultural molecular breeding. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Phenotype, expression amount, photoperiod response and homologous gene analysis of the CONSTANS-LIKE 5 gene knockout and overexpression plants in the background of the rice Nipponbare NIP: Figure 1 a in the above table is the heading stage phenotype of the phenotype of the CONSTANS-LIKE 5 knockout and overexpression plants in the background of the Nipponbare NIP; Figure 1 b in the above table is the heading stage statistics of the phenotype of the CONSTANS-LIKE 5 knockout and overexpression plants in the background of the Nipponbare NIP; Figure 1 c in the above table is the rhythmic fluctuation of the expression amount of the CONSTANS-LIKE 5 under short-day conditions in NIP; Figure 1 d in the above table is the homologous analysis of the cloned CONSTANS-LIKE 5.

[0031] Figure 2 Homology comparison of amino acid sequences between the protein for controlling heading date of rice used in the present application and the OsCOL5 of the rice genome annotation.

[0032] Figure 3 Heading date and heading phenotype of the CONSTANS-LIKE 5 interacting protein IDS1 knockout line and overexpression: Figure 3 a in the table is the interaction of CONSTANS-LIKE 5 and IDS1 in yeast; Figure 3 b in the table is the heading date of the COL5 interacting protein OsIDS1 knockout line; Figure 3 c in the table is the heading phenotype of the CONSTANS-LIKE 5 interacting protein OsIDS1 knockout line and overexpression. DETAILED DESCRIPTION

[0033] The specific embodiments of the present application are described below to facilitate the understanding of the present application for those skilled in the art, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

[0034] The CONSTANS-LIKE 5 gene provided by the present application is cloned from rice Nipponbare NIP, which is a 372-amino-acid transcription factor protein belonging to the CCT / B-box zinc finger protein family, the CDS sequence of the CONSTANS-LIKE 5 gene is shown as SEQ ID No. 1, and the amino acid sequence of the protein encoded by the CONSTANS-LIKE 5 gene is shown as SEQ ID No. 2. The interacting protein of the CONSTANS-LIKE 5 gene provided by the present application is OsIDS1, the CDS sequence is shown as SEQ ID No. 8, which encodes an AP2 transcription factor, and the amino acid sequence is shown as SEQ ID No. 9.

[0035] Example 1 Construction of knockout and overexpression vectors of CONSTANS-LIKE 5 gene and OsIDS1 gene

[0036] (1) Construction of CONSTANS-LIKE 5 gene knockout vector

[0037] The CONSTANS-LIKE 5 gene knockout vector is constructed based on the CRISPR-cas9 system. A suitable target site, i.e. CONSTANS-LIKE 5-sg, is designed, and the base sequence is shown as SEQ ID No. 5. The primer pair for designing the target site is CONSTANS-LIKE 5-F2 / CONSTANS-LIKE 5-R2, and the base sequences are shown as SEQ ID No. 3 and SEQ ID No. 4, respectively. The PCR product containing the target site is obtained by PCR amplification. The purified product is connected to the plasmid to obtain the successfully constructed gene knockout vector.

[0038] (2) Construction of CONSTANS-LIKE 5 gene overexpression vector

[0039] Total RNA is extracted from rice leaves, and cDNA is obtained by reverse transcription. The cDNA is used as a template, and the primer pair CONSTANS-LIKE 5-F1 / CONSTANS-LIKE 5-R1 is used for PCR amplification. The base sequence of CONSTANS-LIKE 5-F1 is shown as SEQ ID No. 6, and the base sequence of CONSTANS-LIKE 5-R2 is shown as SEQ ID No. 7. The PCR product obtained by amplification is constructed into the vector of pCambia1300 skeleton to obtain the overexpression recombinant vector.

[0040] (3) Construction of OsIDS1 gene knockout vector

[0041] The OsIDS1 gene knockout vector is constructed based on the CRISPR-cas9 system. A suitable target site, i.e. OsIDS1-sg, is designed, and the base sequence is shown as SEQ ID No. 12. The primer pair for designing the target site is OsIDS1-F2 / OsIDS1-R2, and the base sequences are shown as SEQ ID No. 10 and SEQ ID No. 11, respectively. The PCR product containing the target site is obtained by PCR amplification. The purified product is connected to the plasmid to obtain the successfully constructed gene knockout vector.

[0042] (4) Construction of OsIDS1 gene overexpression vector

[0043] Total RNA is extracted from rice leaves, and cDNA is obtained by reverse transcription. The cDNA is used as a template, and the primer pair OsIDS1-F1 / OsIDS1-R1 is used for PCR amplification. The base sequence of OsIDS1-F1 is shown as SEQ ID No. 13, and the base sequence of OsIDS1-R2 is shown as SEQ ID No. 14. The PCR product obtained by amplification is constructed into the vector of pCambia1300 skeleton to obtain the overexpression recombinant vector.

[0044] The vector plasmids obtained from (1), (2), (3), (4) were transformed into Agrobacterium, and then the Agrobacterium was used to infect rice callus, and positive plants were obtained after detection.

[0045] Comparison of heading date of wild type and knock-out material and overexpression material of CONSTANS-LIKE 5 gene and OsIDS gene in Example 2

[0046] The genetic materials created in Example 1 were planted in the field in Shunyi, Beijing, and the wild type Nipponbare, the knock-out material and overexpression material of CONSTANS-LIKE 5 gene and OsIDS gene were planted. After the heading began, the heading time of each material was recorded, and the photos were taken.

[0047] The following: COL5-KO represents the knock-out material of CONSTANS-LIKE 5 gene, COL5-OE represents the overexpression material of CONSTANS-LIKE 5 gene, IDS1-KO represents the knock-out material of OsIDS gene, and IDS1-OE represents the overexpression material of OsIDS gene.

[0048] The results show that: compared with the wild type, COL5-KO( Figure 1 a) and IDS1-OE( Figure 3 c) all delay the heading date, COL5-OE( Figure 1 a) and IDS1-KO( Figure 3 c) all advance the heading date. The heading time of COL5-KO is increased by about 3-4 days compared with the control, the heading time of COL5-OE is advanced by about 5 days( Figure 1 b), and the heading time of IDS1-KO is advanced by about 7 days( Figure 3 b).

[0049] In the short-day incubator, the expression level of COL5 is significantly induced by darkness, and shows rhythmic changes within 48 hours. COL5-KO has no obvious response( Figure 1 c).

[0050] Through sequence homology analysis and evolutionary analysis, the CCT / B-box zinc finger transcription factor is rice CONSTANS-LIKE 5( Figure 1 d and Figure 2 ).

[0051] Example 3: A protein OsIDS1 interacting with CONSTANS-LIKE 5 and its regulation on the heading date of rice

[0052] OsIDS1 and CONSTANS-LIKE 5 interacted with each other by yeast two-hybrid screening Figure 3 a), and we found that the OsIDS1 loss-of-function mutant was significantly earlier in flowering time, and the IDS1 OE overexpression was significantly delayed in heading date by heading date detection Figure 3 b, c).

Claims

1. Oryza sativa CONSTANS-LIKE 5 The use of the gene in regulating the heading date of rice is characterized in that, The CONSTANS-LIKE 5 CDS sequence of the gene is shown as SEQ ID No. 1, the CONSTANS-LIKE 5 amino acid sequence of the protein encoded by the gene is shown as SEQ ID No. 2; the application is applied to overexpression CONSTANS-LIKE 5 The gene promotes rice heading under short-day conditions, and the knockout or silencing CONSTANS-LIKE 5 The gene inhibits rice heading under short-day conditions.

2. The rice plant of claim 1 CONSTANS-LIKE 5 use of the gene in regulating heading date in rice, characterized in that, The overexpression CONSTANS-LIKE 5 Genes that promote rice heading under short day conditions, comprising the steps of: (1) Extract total RNA from rice leaves, and obtain cDNA by reverse transcription. Use cDNA as a template, and perform PCR amplification by using primer pair CONSTANS-LIKE 5-F1 / CONSTANS-LIKE 5-R1, wherein the base sequence of the CONSTANS-LIKE 5-F1 is shown as SEQ ID No. 6, and the base sequence of the CONSTANS-LIKE 5-R2 is shown as SEQ ID No. 7; (2) Connect the PCR product obtained in step (1) into an expression vector to obtain an overexpression recombinant vector; (3) Transform the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain; (4) The overexpression recombinant strain is transformed into rice plants, and overexpression CONSTANS-LIKE 5 gene strains are screened and obtained.

3. The rice plant of claim 1 CONSTANS-LIKE 5 The use of the gene in regulating the heading date of rice is characterized in that, the knock-out or silencing CONSTANS-LIKE 5 Genes that repress heading in rice under short-day conditions, comprising the steps of: (1) Design CONSTANS-LIKE 5 a target point CONSTANS-LIKE 5-sg of a gene and a primer pair CONSTANS-LIKE 5-F2 / CONSTANS-LIKE 5-R2 of the target point CONSTANS-LIKE 5-sg for constructing a CRISPR / Cas9 vector; a base sequence of the target point CONSTANS-LIKE 5-sg is shown as SEQ ID No. 5, a base sequence of the CONSTANS-LIKE 5-F2 is shown as SEQ ID No. 3, and a base sequence of the CONSTANS-LIKE 5-R2 is shown as SEQ ID No. 4; (2) Construct an Agrobacterium genetic engineering bacterium containing the CRISPR / Cas9 vector in step (1); (3) Transform the Agrobacterium genetic engineering bacterium in step (2) into a rice plant to obtain a homozygous mutant line which does not contain T-DNA and is stably inherited.

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