Application of rice CONSTANS-LIKE 5 gene and interaction protein OsIDS1 thereof in regulation and control of heading stage of rice

Abstract

The invention relates to the technical field of agricultural bioengineering, and particularly provides application of a rice CONSTANS-LIKE 5 gene and an interaction protein OsIDS1 thereof in regulating and controlling the heading stage of rice. The CONSTANS-LIKE 5 gene is cloned from rice Nipponbare NIP, is a transcription factor protein of 372 amino acids, and belongs to a CCT / B-box ginc finger protein family. Genetic transformation experiments prove that the overexpressed CONSTANS-LIKE 5 gene promotes the heading of the rice under a short-day condition, and the knockout or silencing CONSTANS-LIKE 5 gene inhibits the heading of the rice under the short-day condition; overexpression of the OsIDS1 gene inhibits rice heading under a short-day condition, and knockout or silencing of the OsIDS1 gene promotes rice heading under the short-day condition.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural bioengineering, and specifically relates to application of rice CONSTANS-LIKE 5 gene and its interacting protein OsIDS1 in regulating rice heading period. Background Art

[0002] Rice is the most consumed staple crop in the world, feeding more than half of the world's population. Heading date is an agronomic trait that is very important for seasonal and regional adaptability. In the past 20 years, many genes that control heading date have been cloned. The mechanism of rice flowering uses leaves to identify the length of daylight and then transmits the signal to the activation of floral organs. If a cultivated variety blooms too early, it will result in insufficient use of light and temperature, resulting in reduced yields; on the contrary, if a variety heads too late, it will be difficult to complete the entire flowering and grain development cycle, also resulting in reduced yields. In rice, heading date is affected by exogenous factors such as photoperiod, temperature and nutrition, among which photoperiod is a major controlling factor. Rice itself is a short-day plant. Heading will be promoted under short-day conditions, while heading will be inhibited under long-day conditions. The heading date regulatory genes that have been cloned so far either have a large effect or affect rice yield and plant type. A slight delay of 3-7 days in heading date can allow rice to get more vegetative growth for a period of time, accumulate biomass, and increase rice yield. Cloning and discovering heading period genes are of great significance for improving rice adaptability and improving rice varieties. Summary of the invention

[0003] In view of the above-mentioned existing situation in the prior art, the purpose of the present invention is to provide an application of rice CONSTANS-LIKE 5 gene and its interacting protein OsIDS1 in regulating the heading period of rice. The present invention also provides a set of genes and gene transformation vectors that can be used for the improvement of new varieties, relatively freely regulating the height of rice plants, and providing new gene resources and methods for modern agricultural molecular breeding.

[0004] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0005] The first aspect of the present invention provides an application of the rice CONSTANS-LIKE 5 gene in regulating the heading period of rice, the CDS sequence of the CONSTANS-LIKE 5 gene is shown in SEQ ID No.1, and the amino acid sequence of the protein encoded by the CONSTANS-LIKE 5 gene is shown in SEQ ID No.2; the application method is: overexpressing the CONSTANS-LIKE 5 gene to promote rice heading under short-day conditions, and knocking out or silencing the CONSTANS-LIKE 5 gene to inhibit rice heading under short-day conditions.

[0006] Furthermore, the overexpression of CONSTANS-LIKE 5 gene to promote rice heading under short-day conditions comprises the following steps:

[0007] (1) extracting total RNA from rice leaves, reverse transcribing to obtain cDNA, using the cDNA as a template, and performing PCR amplification using primers CONSTANS-LIKE 5-F1 / CONSTANS-LIKE 5-R1, wherein 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) connecting the PCR product obtained by amplification in step (1) into an expression vector to obtain an overexpression recombinant vector;

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

[0010] (4) Transforming the overexpression recombinant strain into rice plants, and screening and obtaining the CONSTANS-LIKE5 gene overexpression strain.

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

[0012] (1) Design the target site CONSTANS-LIKE 5-sg of the CONSTANS-LIKE 5 gene and the primer pair CONSTANS-LIKE 5-F2 / CONSTANS-LIKE 5-R2 of the target site CONSTANS-LIKE5-sg, and construct the CRISPR / Cas9 vector;

[0013] (2) constructing an Agrobacterium genetically engineered bacterium containing the CRISPR / Cas9 vector described in step (1);

[0014] (3) Transforming rice plants with the genetically engineered Agrobacterium described in step (2) to obtain a homozygous mutant strain that does not contain T-DNA and has stable inheritance.

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

[0016] The second aspect of the present invention provides an application of the rice OsIDS1 gene in regulating the heading period of rice, wherein the CDS sequence of the OsIDS1 gene is shown in SEQ ID No.8, and the amino acid sequence of the protein encoded by the OsIDS1 gene is shown in SEQ ID No.9; the application method is: overexpressing the OsIDS1 gene to inhibit rice heading under short-day conditions, and knocking out or silencing the OsIDS1 gene to promote rice heading under short-day conditions.

[0017] Furthermore, the overexpression of the OsIDS1 gene to inhibit rice heading under short-day conditions comprises the following steps:

[0018] (1) extracting total RNA from rice leaves, reverse transcribing to obtain cDNA, using the cDNA as a template, and performing PCR amplification using primers OsIDS1-F1 / OsIDS1-R1, wherein the base sequence of OsIDS1-F1 is shown in SEQ ID No. 13, and the base sequence of OsIDS1-R2 is shown in SEQ ID No. 14;

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

[0020] (3) transforming the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain;

[0021] (4) Transforming the overexpression recombinant strain into rice plants, and screening and obtaining the OsIDS1 gene overexpression strain.

[0022] Furthermore, the knocking out or silencing of the OsIDS1 gene to promote rice heading under short-day conditions comprises the following steps:

[0023] (1) Design the target site OsIDS1-sg of the OsIDS1 gene and the primer pair OsIDS1-F2 / OsIDS1-R2 of the target site OsIDS1-sg, and construct the CRISPR / Cas9 vector;

[0024] (2) constructing an Agrobacterium genetically engineered bacterium containing the CRISPR / Cas9 vector described in step (1);

[0025] (3) Transforming rice plants with the genetically engineered Agrobacterium described in step (2) to obtain a homozygous mutant strain that does not contain T-DNA and has stable inheritance.

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

[0027] The third aspect of the present invention provides a protein OsIDS1 that interacts with CONSTANS-LIKE 5, the amino acid sequence of 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] Beneficial effects of the present invention:

[0029] The present invention clones the rice heading period regulating gene CONSTANS-LIKE 5 from the rice variety Nipponbare NIP. The CONSTANS-LIKE 5 gene and the downstream interacting genes are used to obtain methods for respectively extending and shortening the rice heading period genes. Under short-day conditions, the CONSTANS-LIKE 5 gene knockout or silencing vector of the present invention can extend the rice heading period after transforming rice; overexpression of the CONSTANS-LIKE 5 gene can shorten the rice heading period, and the time difference is within 7 days on average. On the other hand, the present invention provides a protein OsIDS1 that interacts with CONSTANS-LIKE 5, and the OsIDS1 gene knockout or silencing vector can shorten the rice heading period after transforming rice; overexpression of the OsIDS1 gene can significantly extend the rice heading period. The complete set of genes and gene transformation vectors provided by the present invention can be used for the improvement of new varieties, relatively freely regulating rice plant height, and providing new gene resources and methods for modern agricultural molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Phenotype, expression level, photoperiod response and homologous gene analysis of CONSTANS-LIKE 5 knockout and overexpression plants in rice Nipponbare NIP background: Figure 1 a in the figure shows the heading period phenotype of the CONSTANS-LIKE 5 knockout and overexpression plants in the Nipponbare NIP background; Figure 1 b shows the heading date statistics of the CONSTANS-LIKE 5 knockout and overexpression plants in the Nipponbare NIP background; Figure 1 c in the figure is the rhythmic fluctuation of the expression of short-day CONSTANS-LIKE 5 in NIP; Figure 1 d in FIG. 5 is the CONSTANS-LIKE 5 homology analysis of the clones of the present invention.

[0031] Figure 2 The amino acid sequence homology comparison is between the protein controlling rice heading period used in the present invention and OsCOL5 annotated in the rice genome.

[0032] Figure 3 Heading date and heading phenotypes of knockout and overexpression lines of CONSTANS-LIKE 5 interacting protein IDS1: Figure 3 a in the figure indicates the interaction between CONSTANS-LIKE 5 and IDS1 in yeast; Figure 3 b in the figure is the heading period of the COL5 interacting protein OsIDS1 knockout line; Figure 3 (c) shows the heading phenotype of the CONSTANS-LIKE 5 interacting protein OsIDS1 knockout line and overexpression line. DETAILED DESCRIPTION

[0033] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

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

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

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

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

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

[0039] Total RNA was extracted from rice leaves and reverse transcribed to obtain cDNA. The cDNA was used as a template and PCR amplification was performed using primers CONSTANS-LIKE 5-F1 / CONSTANS-LIKE 5-R1. The base sequence of CONSTANS-LIKE 5-F1 was shown in SEQ ID No. 6, and the base sequence of CONSTANS-LIKE 5-R2 was shown in SEQ ID No. 7. The PCR product obtained by amplification was constructed into a vector with a pCambia1300 backbone to obtain an overexpression recombinant vector.

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

[0041] The construction of the OsIDS1 gene knockout vector was based on the CRISPR-cas9 system. A suitable target site, namely OsIDS1-sg, was designed, and its base sequence was shown in SEQ ID No.12. The primer pair OsIDS1-F2 / OsIDS1-R2 of the target site was designed, and its base sequences were shown in SEQ ID No.10 and SEQ ID No.11, respectively. A PCR product containing the target site was obtained by PCR amplification. The purified product was connected to the plasmid to obtain a successfully constructed gene knockout vector.

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

[0043] Total RNA was extracted from rice leaves and reverse transcribed to obtain cDNA. Using the cDNA as a template, PCR amplification was performed on OsIDS1-F1 / OsIDS1-R1 using primers. The base sequence of OsIDS1-F1 is shown in SEQ ID No. 13, and the base sequence of OsIDS1-R2 is shown in SEQ ID No. 14. The PCR product obtained by amplification was constructed into a vector with a pCambia1300 backbone to obtain an overexpression recombinant vector.

[0044] After the vector plasmids obtained by (1), (2), (3) and (4) are transformed into Agrobacterium, the rice callus is infected with Agrobacterium and positive plants are obtained after testing.

[0045] Example 2 Comparison of heading period between wild type and knockout and overexpression materials of CONSTANS-LIKE 5 and OsIDS genes

[0046] The knockout materials and overexpression materials of the wild-type Nipponbare NIP CONSTANS-LIKE 5 gene and OsIDS gene were planted in the field in Shunyi, Beijing, using the genetic materials created in Example 1. After heading, the heading time of each material plant was recorded and photographed.

[0047] Below: COL5-KO indicates CONSTANS-LIKE 5 gene knockout material, COL5-OE indicates CONSTANS-LIKE5 gene overexpression material, IDS1-KO indicates OsIDS gene knockout material, and IDS1-OE indicates OsIDS gene overexpression material.

[0048] The results showed that compared with the wild type, COL5-KO( Figure 1 a) and IDS1-OE( Figure 3 c) The heading period was delayed, COL5-OE ( Figure 1 a) and IDS1-KO( Figure 3 c) The heading period is advanced. The heading period of COL5-KO is about 3-4 days earlier than that of the control, and the heading period of COL5-OE is about 5 days earlier ( Figure 1 b), the heading time of IDS1-KO is advanced by about 7 days ( Figure 3 b).

[0049] In a short-day incubator, the culture conditions were 8 hours of light and 16 hours of darkness. The expression level of COL5 was significantly increased by darkness and showed rhythmic changes within 48 hours. COL5-KO did not respond significantly ( Figure 1 c).

[0050] Through sequence homology analysis and evolution 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 rice heading date

[0052] Through yeast two-hybrid screening, we found that rice OsIDS1 interacts with CONSTANS-LIKE 5 ( Figure 3 a) Through the detection of heading date, we found that the flowering time of the OsIDS1 editing loss-of-function mutant was significantly advanced, and the heading date of IDS1OE overexpression was significantly delayed ( Figure 3 b, c).

Claims

1. Application of rice CONSTANS-LIKE 5 gene in regulating rice heading period, characterized in that: The CDS sequence of the CONSTANS-LIKE 5 gene is shown in SEQ ID No. 1, and the amino acid sequence of the protein encoded by the CONSTANS-LIKE 5 gene is shown in SEQ ID No. 2; the application approach is: overexpressing the CONSTANS-LIKE 5 gene to promote rice heading under short-day conditions, and knocking out or silencing the CONSTANS-LIKE 5 gene to inhibit rice heading under short-day conditions.

2. The use of the rice CONSTANS-LIKE 5 gene in regulating the heading period of rice according to claim 1, characterized in that: The method of overexpressing the CONSTANS-LIKE 5 gene to promote rice heading under short-day conditions comprises the following steps: (1) extracting total RNA from rice leaves, reverse transcribing to obtain cDNA, using cDNA as a template, and performing PCR amplification using primers CONSTANS-LIKE 5-F1 / CONSTANS-LIKE 5-R1, wherein the base sequence of CONSTANS-LIKE 5-F1 is shown in SEQ ID No. 6, and the base sequence of CONSTANS-LIKE 5-R2 is shown in SEQ ID No. 7; (2) connecting the PCR product obtained by amplification in step (1) into an expression vector to obtain an overexpression recombinant vector; (3) transforming the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain; (4) Transforming the overexpression recombinant strain into rice plants, and screening and obtaining the CONSTANS-LIKE 5 gene overexpression strain.

3. The use of the rice CONSTANS-LIKE 5 gene in regulating the heading period of rice according to claim 1, characterized in that: The knockout or silencing of the CONSTANS-LIKE 5 gene inhibits rice heading under short-day conditions, comprising the following steps: (1) Design the target site CONSTANS-LIKE 5-sg of the CONSTANS-LIKE 5 gene and the primer pair CONSTANS-LIKE 5-F2 / CONSTANS-LIKE 5-R2 of the target site CONSTANS-LIKE 5-sg, and construct the CRISPR / Cas9 vector; (2) constructing an Agrobacterium genetically engineered bacterium containing the CRISPR / Cas9 vector described in step (1); (3) Transforming rice plants with the genetically engineered Agrobacterium described in step (2) to obtain a homozygous mutant strain that does not contain T-DNA and has stable inheritance.

4. The use of the rice CONSTANS-LIKE 5 gene in regulating the heading period of rice according to claim 3, characterized in that: In step (1), the base sequence of the target CONSTANS-LIKE 5-sg is shown as SEQ ID No.5, the base sequence of CONSTANS-LIKE 5-F2 is shown as SEQ ID No.3, and the base sequence of CONSTANS-LIKE 5-R2 is shown as SEQ ID No.

4.

5. Application of rice OsIDS1 gene in regulating rice heading period, characterized in that: The CDS sequence of the OsIDS1 gene is shown in SEQ ID No.8, and the amino acid sequence of the protein encoded by the OsIDS1 gene is shown in SEQ ID No.9; the application approach is: overexpressing the OsIDS1 gene to inhibit rice heading under short-day conditions, and knocking out or silencing the OsIDS1 gene to promote rice heading under short-day conditions.

6. The use of the rice OsIDS1 gene in regulating the heading period of rice according to claim 5, characterized in that: The method of overexpressing the OsIDS1 gene to inhibit rice heading under short-day conditions comprises the following steps: (1) extracting total RNA from rice leaves, reverse transcribing to obtain cDNA, using the cDNA as a template, and performing PCR amplification on OsIDS1-F1 / OsIDS1-R1 using primers, wherein the base sequence of OsIDS1-F1 is shown in SEQ ID No. 13, and the base sequence of OsIDS1-R2 is shown in SEQ ID No. 14; (2) connecting the PCR product obtained by amplification in step (1) into an expression vector to obtain an overexpression recombinant vector; (3) transforming the overexpression recombinant vector into Agrobacterium to obtain an overexpression recombinant strain; (4) Transforming the overexpression recombinant strain into rice plants, and screening and obtaining the OsIDS1 gene overexpression strain.

7. The use of the rice OsIDS1 gene in regulating the heading period of rice according to claim 5, characterized in that: The method of knocking out or silencing the OsIDS1 gene to promote rice heading under short-day conditions comprises the following steps: (1) Design the target site OsIDS1-sg of the OsIDS1 gene and the primer pair OsIDS1-F2 / OsIDS1-R2 of the target site OsIDS1-sg, and construct the CRISPR / Cas9 vector; (2) constructing an Agrobacterium genetically engineered bacterium containing the CRISPR / Cas9 vector described in step (1); (3) Transforming rice plants with the genetically engineered Agrobacterium described in step (2) to obtain a homozygous mutant strain that does not contain T-DNA and has stable inheritance.

8. The use of the rice OsIDS1 gene in regulating the heading period of rice according to claim 7, characterized in that: In step (1), the base sequence of the target 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 OsIDS1-R2 is shown as SEQ ID No.

11.

9. A protein OsIDS1 that interacts with CONSTANS-LIKE 5, wherein the amino acid sequence of 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.

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