阅读说明：本技术 OsLAC20基因在提高水稻产量中的应用 (Application of OsLAC20 gene in improving rice yield ) 是由 何瑞瑞 张玉婵 杨宇薇 杨露 陈月琴 于 2020-04-09 设计创作，主要内容包括：本发明公开了OsLAC20基因在增加水稻结实率、提高水稻产量中的应用。本发明研究发现,水稻中OsLAC20基因的敲除会显著性增加水稻的结实率,增大水稻的籽粒和穗型,表明OsLAC20基因是水稻结实率和产量的负调控基因。因此,对OsLAC20的表达调控可用于培育高产水稻,为培育高结实率或高产水稻提供了一种全新的并且简单有效的方法。(The invention discloses application of OsLAC20 gene in increasing rice setting rate and rice yield. The research of the invention finds that the knockout of the OsLAC20 gene in the rice can obviously increase the maturing rate of the rice and increase the kernel and spike type of the rice, and the OsLAC20 gene is a negative regulation gene of the maturing rate and the yield of the rice. Therefore, the expression regulation of OsLAC20 can be used for cultivating high-yield rice, and a novel, simple and effective method is provided for cultivating high-maturing-rate or high-yield rice.)

1, SEQ ID NO: 1, the OsLAC20 gene is applied to the improvement of rice yield.

SEQ ID NO: 1, the OsLAC20 gene is applied to increasing the maturing rate of rice.

3, SEQ ID NO: 1, the OsLAC20 gene is applied to increasing rice grain and spike types.

SEQ ID NO: 1, the OsLAC20 gene is applied to breeding high-maturing-rate and/or high-yield rice.

Application of the OsLAC20 gene inhibition and/or silencing preparation in breeding high-maturing-rate and/or high-yield rice.

6. A method for breeding high-maturing and/or high-yielding rice, characterized in that OsLAC20 gene is knocked out in rice or expression of OsLAC20 gene is suppressed in rice, thereby obtaining high-maturing and/or high-yielding rice.

7. The method of claim 5, wherein the OsLAC20 gene knockout method is CRISPRCs 9 technology, and the sgRNA expression cassette construction sequence of the knockout target is shown as SEQ ID NO: 2 to 5.

Technical Field

The invention relates to the technical field of plant breeding, in particular to application of an OsLAC20 gene in improving rice yield.

Background

The food problem is one of several problems faced in the world today. The search for genes affecting the rice yield-related characteristics has been the key research point for improving the grain yield. At present, a plurality of genes influencing the rice yield are discovered, and a theoretical basis is provided for improving the rice yield. However, in order to solve the contradiction between the growing population and the gradually reduced cultivated land area, the discovery of more genes for regulating the rice yield is still a big problem to be solved urgently.

The rice yield is a complex agronomic trait and is mainly determined by three factors of the effective spike number per unit area, the grain number per spike and the thousand grain weight. Wherein the number of grains per spike is also influenced by the setting rate. Therefore, the search for genes for regulating and controlling yield traits such as seed setting rate, thousand kernel weight, panicle type and the like is not only an important basic research, but also has important guiding significance for production. Several related protein-encoding genes have been identified. For example, PTB1 is a gene that regulates the setting rate of rice, which positively regulates the setting rate by affecting the growth of the pollen tube; GSD1 controls the maturing rate of rice by influencing the conductivity of plasmodesmata; THIS1 affects both seed set and rice plant row. Ghd7 and Ghd8 control the number of grains per ear of rice at the same time. Ghd7 encodes a CCT domain protein, Ghd8 encodes a subunit of a CCAAT-box binding protein complex. DEP1 is a key gene for controlling panicle type and panicle number, and the DEP1 mutant protein deleted at the C terminal can promote cell division, so that the panicle number is increased, and the rice yield is obviously improved. GW2 is a main effect QTL which is cloned earlier and controls grain width and grain weight, encodes an E3 ubiquitin ligase, and speculates that GW2 influences the development of rice grain glume and the development process of endosperm and regulates the size of rice grains by participating in the ubiquitination degradation process of proteins related to cell division. GS3 is a major QTL for controlling rice grain length and grain weight, and has little influence on rice grain width and grain thickness. However, no report is found about the application of OsLAC20 gene in rice yield.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, and provides application of the OsLAC20 gene in improving rice yield.

The above object of the present invention is achieved by the following technical solutions:

the research of the invention finds that the knockout of the OsLAC20 gene in the rice can obviously increase the maturing rate of the rice and increase the kernel and spike type of the rice, and the OsLAC20 gene is a negative regulation gene of the maturing rate and the yield of the rice. Therefore, the expression regulation of OsLAC20 can be used for cultivating high-yield rice, and a novel, simple and effective method is provided for cultivating high-maturing-rate or high-yield rice.

Therefore, the present invention claims the following uses of OsLAC20 gene:

the OsLAC20 gene is applied to improving the yield of rice.

The OsLAC20 gene is applied to increasing the maturing rate of rice.

The OsLAC20 gene is applied to increasing the grain and spike types of rice.

The OsLAC20 gene is applied to cultivating rice with high maturing rate and high yield.

The invention also provides application of the OsLAC20 gene in breeding high-maturing-rate and/or high-yield rice.

The high yield refers to larger grains, more grains per ear, heavier thousand grain weight and/or more effective grains per ear.

Wherein, the sequence of the OsLAC20 gene is disclosed as follows: the gene number of OsLAC20 in a rice genome database (http:// rice. plant biology. msu. edu/index. shtml) is: LOC _ Os11g42220, the nucleotide sequence of which is shown in SEQ ID NO: 1 is shown.

The invention also provides a method for cultivating high-maturing-rate and/or high-yield rice, and particularly relates to knocking out OsLAC20 gene in rice or silencing and/or inhibiting expression of OsLAC20 gene, so that high-maturing-rate and/or high-yield rice is obtained.

Preferably, the method for knocking out the OsLAC20 gene can be CRISPR-Cas9 technology.

As an alternative embodiment, the invention provides a method for cultivating high-maturing and/or high-yield rice, namely a method for constructing a rice mutant strain by knocking out OsLAC20 gene in rice by using CRISPR-Cas9 technology, which comprises the following steps:

s1, constructing an OsLAC20 knockout plasmid;

s2, transforming the rice callus by using the OsLAC20 knockout plasmid;

s3, screening and differentiating the transgenic rice positive callus.

The method for constructing the OsLAC20 knockout plasmid in the step S1 is as follows:

two target sites were designed on OsLAC20 using the in-line software CRISPR-P (http:// skl.scau.edu.cn/targettdign /). By primers 5'-gccgACACCCGTCAAGGATGTAC-3' and 3 '-ACACCCGTCAAGGATGTACgttt-5'; and primers 5'-gttgCCCACTATCACATGATCG-3' and 3 '-GCCCACTATCACATGATCGgttt-5' construct sgRNA expression cassettes of two target sites; the expression cassette is connected into pYLCRISPR/Cas9 vector with binary vector pCAMBIA-1300 as a framework and transformed into escherichia coli, and the construction of OsLAC20 knockout plasmid is completed.

After the detailed yield-related phenotype analysis is carried out on the mutant strain, the CRISPR-Cas9 technology is used for knocking out the grain length which is obviously longer than that of a wild type, and the thousand grain weight per ear is also obviously higher than that of the wild type. This indicates that OsLAC20 affects yield-related indices. Therefore, the OsLAC20 knockout technology can achieve the effect of increasing the yield, completely eliminate the transgenic trace through a genetic means, and is very suitable for being put into production and use.

In summary, it can be concluded that: the OsLAC20 gene is a negative regulation gene of rice yield, and the knock-out or knock-down of the OsLAC20 gene by various means can be used as a brand-new technical means for cultivating high-yield rice, so that the yield of the rice can be obviously improved. Particularly, when the OsLAC20 gene is knocked out by the CRISPR-Cas9 technology, transgenic traces can be screened out by a simple genetic means without affecting the effect of improving the rice yield, the method is safe and effective, can be popularized and used on a large scale, does not cause environmental pollution, and is safe to eat. Therefore, the expression regulation of OsLAC20 can be used for cultivating high-yield rice.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses application of OsLAC20 gene in improving rice yield for the first time. The research of the invention finds that the OsLAC20 knocked out by the CRISPR-Cas9 technology can obviously improve grain size and thousand kernel weight, and shows that the OsLAC20 gene is a negative regulation gene of rice yield, and expression regulation of the OsLAC20 can be used for cultivating high-yield rice, so that a brand-new, simple and effective method is provided for cultivating high-maturing rate or high-yield rice. Meanwhile, the high-yield rice is cultivated by a molecular biology technology, particularly, the OsLAC20 is knocked out by a CRISPR-Cas9 technology, transgenic traces can be completely eliminated by a genetic means, the yield of the rice is high, the environment cannot be polluted, and the rice is safe to eat.

Drawings

FIG. 1 shows the genotype detection of a CRISPR-Cas9 technical knockout OsLAC20 mutant strain.

Fig. 2 is a comparison of wild type and whole mutant, wherein wild type is on the left, and the mutant with OsLAC20 knocked out by CRISPR-Cas9 technology is on the right.

Fig. 3 is a comparison of ear sizes of wild type and mutant, wherein wild type is on the left and mutant with OsLAC20 knocked out by CRISPR-Cas9 technology is on the right.

FIG. 4 is a comparison of seed length of wild type and a CRISPR-Cas9 technology knockout OsLAC20 mutant. Wherein, the upper row is a wild type, and the lower row is a mutant strain of OsLAC20 knocked out by CRISPR-Cas9 technology.

FIG. 5 is a comparison of seed widths of wild type and CRISPR-Cas9 technology knockout OsLAC20 mutant. Wherein, the upper row is a wild type, and the lower row is a mutant strain of OsLAC20 knocked out by CRISPR-Cas9 technology.

FIG. 6 is a statistical comparison of thousand kernel weight of wild type and a CRISPR-Cas9 technology knockout OsLAC20 mutant. Grey columns indicate wild type, black columns indicate CRISPR-Cas9 technical knock-out OsLAC20 mutant.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.