CN110184280B

CN110184280B - GLW10 gene for controlling rice grain length and thousand grain weight, protein coded by same and application thereof

Info

Publication number: CN110184280B
Application number: CN201910531566.XA
Authority: CN
Inventors: 陈薇兰; 袁华; 钦鹏; 李仕贵; 刘艺; 邓朝杨; 涂斌; 王玉平; 马炳田; 陈郡雯
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2019-06-19
Filing date: 2019-06-19
Publication date: 2020-10-30
Anticipated expiration: 2039-06-19
Also published as: CN110184280A

Abstract

The invention discloses a GLW10 gene for controlling rice grain length and thousand seed weight, and a protein coded by the same and application thereof. The nucleotide sequence of the gene is shown as SEQ ID NO. 1; the amino acid sequence of the protein is shown as SEQ ID NO. 2. The gene has the function of positively regulating the rice grain length and thousand seed weight, and can be applied to increasing the thousand seed weight of rice and improving the rice yield.

Description

GLW10 gene for controlling rice grain length and thousand grain weight, protein coded by same and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and genetic breeding, and particularly relates to a GLW10 gene for controlling rice grain length and thousand seed weight, and a protein coded by the same and application thereof.

Background

Rice is one of the most important food crops in China, more than 65% of people in China use rice as staple food, and the rice consumption is extremely large. The rice is the first large grain crop in China, and the rice is placed at the head of the grain crop in planting area, total yield and single yield, and plays a decisive role in guaranteeing the national grain safety. However, in recent years, with the continuous increase of population in China, the continuous decrease of the number of agricultural labor, the annual decrease of arable area, the aggravation of environmental influences such as global warming and the like, the development of the rice industry in China faces various challenges. Therefore, the method further improves the rice yield and has very important strategic significance for ensuring the food safety and the agricultural sustainable development in China.

The rice yield belongs to complex agronomic traits and consists of the effective ear number of a single plant, the grain number of each ear, the setting rate and the thousand grain weight, and the traits are complex quantitative traits. Thousand kernel weight, one of the direct components of rice yield, is determined by grain type and grain fullness. The grain type mainly comprises 3 aspects of grain length, grain width and grain thickness. The grain type affects not only rice yield but also rice quality, especially appearance quality. Therefore, the excavation of the genetic basis of the grain type related genes is necessary for high-yield and high-quality breeding of rice, and a large number of grain type related genes or QTL sites are cloned at present, wherein the major QTL sites of grain length comprise: GS3, GL3.1, GS2, OsLG3, OsLG3b/LGY3, TGW3/GL3.3, etc.; the major QTL site of grain width includes: GW2, GS5, GW8, GW5/GSE5, GL7/GW7, OsOTUB1, and the like.

Although many grain type related genes have been reported at present, the research on the regulation mechanism of the genes is still only one-sidedly, especially the interaction relation among the genes, the regulation network and the like are not clear, and further excavation of the grain type and thousand seed weight related genes is urgently needed.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the GLW10 gene for controlling the grain length and the thousand seed weight of rice, and a protein coded by the gene and application of the gene.

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

a GLW10 gene for controlling the grain length and thousand seed weight of rice has the nucleotide sequence shown in SEQ ID No. 1.

Furthermore, the nucleotide sequence of the gene is the nucleotide sequence of the sequence shown in SEQ ID NO.1, which is subjected to substitution, deletion or addition of one or more nucleotides and can encode proteins with the same function.

The amino acid sequence of the protein coded by the GLW10 gene is shown in SEQ ID NO. 2.

Furthermore, the amino acid sequence of the protein is the amino acid sequence which is shown in SEQ ID NO.2 and is subjected to substitution, deletion or addition of one or more amino acids, and expresses the same function.

A plasmid comprising the above GLW10 gene.

A recombinant expression vector comprising the above GLW10 gene.

A transgenic cell line comprising the GLW10 gene described above.

An engineered bacterium comprising the GLW10 gene.

The GLW10 gene is applied to improving the grain length and thousand seed weight of rice and increasing the yield of rice.

The invention has the beneficial effects that:

the invention uses EMS mutagenic small-particle mutant GLW10 of Shuhui 498 as research material, uses BSA mixed pool sequencing, and combines a MutMap positioning method to position candidate GLW10 gene. A CRISPR/Cas9 system is used for editing GLW10 to obtain a knockout transgenic strain, compared with a wild type knockout strain, the grain length is obviously reduced, and the thousand grain weight is reduced. And the transgenic plant over expressing GLW10 shows that the grain length and the thousand kernel weight are obviously increased, which shows that GLW10 has the function of positively regulating and controlling the grain length and the thousand kernel weight of rice and can be applied to increasing the thousand kernel weight of the rice and improving the yield of the rice.

Drawings

FIG. 1 is a schematic structural diagram of a GLW10 gene knockout vector GLW10-BGK 03;

FIG. 2 is a schematic structural diagram of GLW10 gene overexpression vector pCAMBIA2300-35S-GLW 10-eGFP;

FIG. 3 is a schematic diagram of GLW10 gene knockout target sites and knockout plant mutation modes; "." indicates a deletion of the base at this position;

FIG. 4 shows the data of the grain type and thousand kernel weight of GLW10 gene knockout line; wherein ". x" indicates that the difference was significant at the 0.01 level;

FIG. 5 is a schematic diagram of the grain length and grain width of a GLW10 gene knockout strain; the scale is 3 mm;

FIG. 6 shows quantitative determination of GLW10 gene overexpression transgenic line; wherein ". x" indicates that the difference was significant at the 0.01 level;

FIG. 7 shows the grain type and thousand-grain weight test species data of GLW10 gene overexpression transgenic lines; wherein ". x" indicates that the difference was significant at the 0.01 level;

FIG. 8 is a schematic diagram of the grain length and grain width of a GLW10 gene overexpression transgenic line, and the scale is 3 mm.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the 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 it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

The invention unexpectedly discovers a small-particle mutant glw10 in an EMS (ethyl methanesulfonate) mutant library of Shuhui 498, and hybridizes glw10 with wild Shuhui 498 to construct F₂Separating and positioning a population, carrying out BSA pool-mixing sequencing on the extremely small-particle single strains in the population, and positioning to candidate GLW10 genes by using a MutMap positioning method; GLW10 is knocked out by using a CRISPR/Cas9 system, and the function of the gene in the aspect of regulating rice grain length and thousand kernel weight is discovered.

Example 1 CRISPR/Cas9 knockout vector construction of GLW10

The invention utilizes CRISPR/Cas vector construction kit of the Baige company to construct a knockout vector of GLW10, and the specific process is as follows:

(1) designing a knockout target site by using a targetDesign online tool of http:// skl.scau.edu.cn/website; the GLW10 gene nucleotide sequence (SEQ ID NO.2) is input into a website, and a 19bp gRNA target sequence T1 is generated and is as follows:

T1：5’-GGGTGCTTCCTGTCCAAGC-3’(SEQ ID NO.3)

(2) inputting the target point sequence T1 into an online design website of Oligo sequences of the Baige company (http://121.41.105.238/index/excrispr), selecting BGK03 vectors, and generating Oligo sequences corresponding to the kit, wherein the Oligo-F and Oligo-R sequences are as follows:

Oligo-F：5’-TGTGTGGGGTGCTTCCTGTCCAAGC-3’(SEQ ID NO.4)

Oligo-R：5’-AAACGCTTGGACAGGAAGCACCCCA-3’(SEQ ID NO.5)

the above Oligo-F and Oligo-R sequences were synthesized by Biotech, and then the synthesized Oligo-F and Oligo-R primers were dissolved in water to 10. mu.M.

(3) The following operations are carried out according to the instructions of the hundred-lattice CRISPR/Cas vector construction kit:

step 1: preparation of Oligo dimer

Preparing a PCR reaction system (18 mu.L Buffer Anneal, 1 mu.L Oligo-F, 1 mu.L Oligo-R), mixing uniformly, and performing the following reaction procedures by using a PCR instrument: heating at 95 ℃ for 3 minutes, then slowly reducing the temperature to 20 ℃ at the speed of 0.2 ℃/second, and cooling and renaturing to obtain the Oligo dimer.

Step 2: construction of the above Oligo dimer into CRISPR/Cas vector

The reaction system (2. mu.L CRISPR/Cas Vector, 1. mu.L Oligo dimer, 1. mu.L enzymeMix, finally water to 10. mu.L) was prepared on ice and mixed well, and then reacted at room temperature for 1 hour.

Step 3, transforming the reaction system into escherichia coli

Taking out Escherichia coli competent cell Trans1-T1 (Beijing Quanjin Biotechnology Co., Ltd.) from-80 deg.C, and thawing in ice water bath; then adding 5 mul of the reaction system prepared in the step 2, mixing the mixture evenly and gently, and incubating the mixture on ice for 30 minutes (without shaking); then placing the mixture in a metal bath at 42 ℃, immediately placing the mixture in an ice water bath after heat shock is carried out for 60 seconds, and standing for 2 minutes; then adding 500 mu L of non-resistant LB liquid culture medium into the centrifuge tube, and recovering for 10 minutes by metal bath at 37 ℃; then placing the mixture in a shaking table, and carrying out shaking culture at 200rpm and 37 ℃ for 1 hour to recover the thalli; appropriate volumes of the bacterial solution were aspirated, gently spread on LB plates containing kanamycin resistance using sterile spreading rods, and cultured overnight at 37 ℃ in an inverted manner.

And 4, step 4: and extracting plasmids to obtain a GLW10 knockout vector GLW10-BGK 03.

The plasmid extracted from the above LB plate by monoclonal shake culture (according to the OMEGA plasmid extraction kit), and the plasmid was sent to the Biotech company for sequencing to obtain correctly sequenced vector GLW10-BGK03 (see FIG. 1).

Example 2 GLW10 overexpression vector construction

Using a reverse transcription kit from Takara, 500ng of total RNA was used for the synthesis of cDNA by reverse transcription in accordance with the protocol. The synthesized cDNA was used as a template to amplify the sequence (SEQ ID NO.1) of the coding region of the GLW10 gene, and the amplification primers F and R have Kpn I and BamH I cleavage sites (sequences shown by underlining), respectively, and the sequences are shown below:

F：5’-CGGGGTACCATGGGGTGCTTCCTGTCCA-3’(SEQ ID NO.6)

R：5’-CGCGGATCCACCTCGCCAGCTATTTTG-3’(SEQ ID NO.7)

the GLW10 gene coding region sequence obtained by amplification is connected between KpnI and BamHI enzyme cutting sites of a plant expression vector pCAMBIA2300-35S-eGFP by using T4 ligase to obtain an over-expression vector pCAMBIA2300-35S-GLW10-eGFP of GLW10 (see figure 2).

Example 3 transformation of Rice Nipponbare with GLW10 knock-out vector and overexpression vector

(1) The plasmids of the constructed GLW10 knockout vector GLW10-BGK03 and the overexpression vector pCAMBIA2300-35S-GLW10-eGFP are respectively transformed into agrobacterium EHA105

The specific process of the agrobacterium transformation method is as follows: taking out EHA105 competent cells from a refrigerator at the temperature of-80 ℃, and quickly relieving the heart to thaw; add 1. mu.L plasmid into 1 tube of competent cells, stand for 30 minutes on ice; freezing in liquid nitrogen for 2 minutes; water bath at 37 deg.c for 5 min to dissolve cell; immediately adding 5 times of the volume of the non-resistant LB culture medium, and performing shake culture for 2-3 hours at the temperature of 28 ℃ and the rpm of 170; centrifuging at 7000rpm for 2 minutes, and resuspending the cells in LB medium with a volume of 100. mu.L; then spread on LB plate containing rifampicin and kanamycin resistance, blow-dried, and cultured at 28 ℃ for 2-3 days.

(2) And respectively transforming the knockout vector and the over-expression vector after transforming the agrobacterium into wild rice Nipponbare varieties by using an agrobacterium-mediated method to obtain knockout and over-expression transgenic plants of GLW 10.

Example 4 identification and phenotypic analysis of GLW10 knockout plants

After obtaining the GLW10 knockout transgenic plant in the embodiment 3, taking leaves to extract DNA, and determining the mutation mode of the knockout plant by utilizing detection primers KO-F and KO-R amplification sequencing; the specific sequences of primers KO-F and KO-R are as follows:

KO-F:5’-TGCTTCCCTACTACACACT-3’(SEQ ID NO.8)

KO-R:5’-GGAATGTACTAGCAGCAA-3’(SEQ ID NO.9)

a total of 3 knockout mutant lines with different mutation patterns were obtained and named KO1, KO2 and KO3 (see FIG. 3). The phenotype of the grain type of each line of the knockout mutant was examined and compared with that of wild type Nipponbare (WT), and the results are shown in FIGS. 4 and 5.

FIG. 4 shows rice grain type detection data of GLW10 knock-out mutant lines and wild type Nipponbare (WT); wherein A is the comparison result of the grain lengths of the knock-out mutant strains KO1, KO2, KO3 and wild type Nipponbare (WT); b is the grain width comparison result of the knock-out mutant strains KO1, KO2, KO3 and wild type Nipponbare (WT); c is the comparison of thousand kernel weight of the knock-out mutant strains KO1, KO2, KO3 and wild-type Nipponbare (WT).

FIG. 5 is a schematic representation of rice kernels of GLW10 knockout mutant lines and wild type Nipponbare (WT); wherein A is a schematic diagram of the grain length of knock-out mutant strains KO1, KO2 and KO3 and wild type Nipponbare (WT) rice; b is a schematic diagram of the grain widths of the knock-out mutant strains KO1, KO2 and KO3 and wild type Nipponbare (WT) rice; as shown in fig. 4 and 5, after GLW10 is knocked out, the grain length and thousand kernel weight of rice are obviously reduced, specifically, the grain length is obviously reduced by 10.4-12%, and the thousand kernel weight is obviously reduced by 17.9-21.5%; while the grain width has no obvious change.

Example 5: identification and phenotypic analysis of GLW10 overexpression plants

After obtaining the plants with GLW10 overexpression in example 3, total RNA was extracted from individual leaves (using a plant RNA extraction kit from OMEGA, according to the instructions). Using a reverse transcription kit from Takara, 500ng of total RNA was used for the synthesis of cDNA by reverse transcription in accordance with the protocol. Fluorescent quantitative PCR detection was performed with primer qGLW10, and ACTIN primer was used as internal control. The fluorescent quantitative detection primer sequence is as follows:

qGLW10-F：5’-GTATGGACCTTACAGCAGTA-3’(SEQ ID NO.10)

qGLW10-R：5’-GAGATGCTGAATACCAAGAAG-3’(SEQ ID NO.11)

ACTIN-F：5’-GACTCTGGTGATGGTGTCAGC-3’(SEQ ID NO.12)

ACTIN-R：5’-GGCTGGAAGAGGACCTCAGG-3’(SEQ ID NO.13)

the results of a grain type study of 3 independent transgenic lines OE1, OE2 and OE3 (see FIG. 6), which were quantitatively tested as overexpression, and compared with wild-type Nipponbare are shown in FIGS. 7 and 8.

FIG. 7 shows rice grain type detection data of GLW10 over-expressed plants OE1, OE2 and OE3 and wild-type Nipponbare (WT); wherein A is the grain length detection data of over-expression plants OE1, OE2 and OE3 and wild type Nipponbare (WT) grains; b is grain width detection data of over-expression plants OE1, OE2 and OE3 and wild type Nipponbare (WT) grains; c is the thousand kernel weight detection data of over-expressed plants OE1, OE2 and OE3 and wild type Nipponbare (WT) grains.

FIG. 8 is a schematic diagram of rice kernels of GLW10 overexpressing plants and wild type Nipponbare (WT); wherein, A is a schematic diagram of grain length detection of over-expression plants OE1, OE2 and OE3 and wild type Nipponbare (WT) grains; b is a schematic diagram of grain width detection of over-expressed plants OE1, OE2 and OE3 and wild type Nipponbare (WT) grains; as shown in the graphs in FIGS. 7 and 8, the GLW10 overexpression can improve the grain length and the thousand grain weight of rice, and is specifically shown in the way that the increase amplitude of the grain length is in positive correlation with the expression level, and finally, the thousand grain weight is remarkably increased by 8.5-12.6%, while the grain width is not remarkably different.

Therefore, in conclusion, the GLW10 gene can positively regulate the rice grain length and the thousand seed weight, can be applied to increasing the rice grain length and the thousand seed weight and improving the rice yield.

Sequence listing

<110> Sichuan university of agriculture

<120> GLW10 gene for controlling rice grain length and thousand seed weight, protein coded by same and application thereof

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Val Leu Pro Thr Ile Leu Ser Pro Leu Gly Lys Ala Cys Ser Gly Met

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Claims

1. A kind ofGLW10The application of the gene in controlling the grain length and thousand grain weight of rice and increasing the yield of rice is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. A device comprisesGLW10The application of the plasmid of the gene in controlling the grain length and thousand kernel weight of rice is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

3. A device comprisesGLW10The application of the recombinant expression vector of the gene in controlling the grain length and thousand seed weight of rice is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

4. A device comprisesGLW10The application of the genetic engineering bacteria in controlling the grain length and thousand grain weight of rice is characterized in that the genetic engineering bacteriaThe nucleotide sequence is shown as SEQ ID NO. 1.